I N I R D U C mODUCIION I N -...
Transcript of I N I R D U C mODUCIION I N -...
I~
I N I R o D U C I I o N
mODUCIION AND
lFffiVffiW OF ~
,,.-
't
,..j: II -
Chapter I Introduction & Review o(Literature
1.1. Introduction
Microorganisms constitute the oldest, vast and almost unexplored reservoir of natural
resources and are likely to provide innovative applications in challenging areas like food, energy and
climate change. They are vital for sustaining life on the biosphere directly or indirectly. The earth is
estimated to be nearly 4.6 billion years old (1 billion =109). The age of the oldest living systems has
been more difficult to establish, although many lines of evidence point to the occurrence of abundant
microbial life at least 3.5 billion years ago (Fenchel, 2002; Lazcano & Miller, 1994). There is a
general agreement that 80% of the natural history of life on Earth is exclusively a history of
microbial communities (Colwell et ai., 1996). The first line of evidence regarding the antiquity of
microbial life forms consists of ancient microbial fossils, which have been found at almost all places
where prehistoric environmental conditions support their preservation (Corsetti et ai., 2003). Flint
like siliceous rocks (cherts) may contain fossils of bacteria, particularly of large cyanobacterial cells
with specialized secondary structures embedded in silica (Kasting, 2001).
The microfossil research of Schopf (1996) and molecular chronometer studies of Doolittle et
aI., (1996) have established a framework for the divergent times of the major microbial groups: 3.5
Gy (1 Gy=1 billion year=109 years) for the cyanobacterial-like lineages, 2.1 Gy for the last ancestral
type (Progenote) common to all three domains, Archaea- Eukarya separation at 1.96 Gy, Gram
positive and Gram negative bacterial separation at 1.5 Gy, and the divergence of protists at 1.23 Gy.
The history of life on earth is unquestionably dominated by unicellular microorganisms and in terms
of relative biomass and physiological diversity, the contributions of microbes to global
environmental change far outweigh the contributions of complex multicellular macroorganisms.
During the first half of earth's evolutionary history, a set of metabolic processes that evolved
exclusively in microbes would come to alter the environments of the planet. They could adapt
themselves to live in diverse habitat because they were capable of exploiting a vast range of energy
sources (Hurst, 2001). When earth was turning to its modem shape for the initial period, prokaryotes
were the only life forms present (Mojzsis et aI., 1996) and they became the core biological machines
responsible for biogeochemical cycles that sustain other life forms and health of the planet.
A significant development in microbial ecology and evolution has been the realization that
microbial life, primarily prokaryotic life, is extremely hard, and can survive and indeed thrive in
environments previously thought uninhabitable on earth (Steven et aI., 2007). Both bacteria and
archaea (constituting prokaryotes) can live in extremes of conditions. All present day organisms in
biosphere depend on microbial activities (Pace, 1997). Their activities created environment
conducible for evolution of other organisms. Life continued to evolve giving rise to various life
forms with tremendous diversity and range of complexity as being observed on the present day earth.
Chapter 1 Introduction & Review o(Literature
Diversity of macroscopic life forms has been reasonably well documented over the centuries.
This was possible because of their large size and visibly distinguishing morphological features.
Prokaryotic diversity, on the other hand, did not draw as much attention because their existence came
to be known only with the discovery of microscope by Leeuwenhoek in 1673. Now it is known that
prokaryotes are the majority on this planet (4-6 x 103°) and their diversity is enormous and constitute
350-550 Pg of carbon (lPg = 1015g) contributing nearly as much carbon as plant (Whitman et aI.,
1998). They are incredible due to the fact that they communicate and interact among themselves as
well as with members of other domains which makes the diverse array of life possible on earth.
Bacteria provide foundation of our biosphere in the sense that they possess an unusual high
physiological and biochemical versatility. They can use reduced inorganic compounds as energy
source (electron donors) for chemolithotrophic metabolism or oxidized inorganic compounds as
electron acceptors for anaerobic respiration. Moreover, certain metabolic pathways, such as
fermentations, nitrogen fixation, methane formation and anoxic photosynthesis are only found among
prokaryotic microbes. This is also true for the biosynthesis of secondary metabolites such as certain
life saving antibiotics, various enzymes and toxins. The metabolic, physiological and genetic
diversity of prokaryotic microorganisms is far greater than that found in higher organisms. Earth gets
rid of accumulation of naturally occurring toxic substances, waste and xenobiotics by degradative
capabilities of microorganisms. Some of them obtain energy for growth by transferring electrons to a
wide range of harmful metals, such as uranium, chromium, arsenic and plutonium (Schleifer, 2004)
and other organisms in tum utilize the products of such degradative cycles making the cycle very
intricate and interdependent. Only a handful of microorganisms cause disease. On the other hand,
associations with microbes, particularly bacterial endosymbionts, are fundamental to the survival of
higher organisms. Symbionts carry out essential biochemical reactions for their eukaryotic hosts, e.g.
the biosynthesis of essential amino acids, vitamins, or degradation of certain macromolecules like
cellulose. Some marine worms use their sulfide oxidizing bacterial endo or ecto symbionts even as
sole feed source.
The benefits of improving the understanding of microbial diversity and ecology are economic
as well as social. The knowledge might provide new tools for bioremediation, biorestoration and
improved management of ecosystem as well as it offers the potential to identify new genes of
bacterial origin to be utilized for bioprospecting, biotransformations and in synthetic biology.
Although understanding the roles of bacteria in shaping the ecology and environment on earth are far
from complete, study of their diversity has become an important part in this direction.
As the existence of the microbial life was recognized only relatively recently in history (about
300 years ago), the knowledge gained is still rudimentary. Microorganisms still represent the largest
reservoir of undescribed biodiversity. The recent techniques such as ribosomal RNA gene
sequencing have helped to survey the biodiversity sufficiently faster and comprehensively. Till date
2
Chapter 1 Introduction & Review o(Literature
53 bacterial phyla are known in bacterial domain (27 phyla have culturable representatives and 26
phyla have no cultured representatives). About 13,700 bacterial species only have been formally
described and majority of them (~ 90%) lie within 4 of the 53 known bacterial phyla (Hugenholtz,
2002) and 23 out of the 53 bacterial phyla have only a few culturable representative (Keller &
Zenglar, 2004). Rest of the 26 bacterial phyla that do not have cultured representatives, their
presence is known from gene sequences only. Many lines of evidence indicate that less than 1 % of
the bacteria can be cultured so far. The term "the great plate count anomaly" was coined by Staley &
Konopka (1985) to describe the difference in orders of magnitude between the numbers of cells that
grow on nutrient media and the number countable by microscopic examination. There are different
explanations for this anomaly; growth state of cells in nature, dormancy, exceptionally high
concentration of nutrients, complex organic carbon in laboratory media and failure to provide
suitable conditions. The physiology and ecological role of these unknown "uncultured" bacterial
species in most cases can only be assessed after their isolation in pure culture or partly from
sequences of genes in culture independent approach. Thus one of the biggest challenges in
microbiology today is how to increase the percentage of cultivability or in other words how to culture
these so-called "unculturables".
Ecogenomics, direct environment shotgun sequencmg, genomics of cultured microbes and
functional genomics, together with different microbial ecological methods such as microarrays, real
time peR, FISH (Flourescent In Situ Hybridization), microautoradiography and new cultivation
techniques will contribute significantly to the understanding of hitherto uncultivated microbes of
various environment. However, attempts to develop and refine culturing techniques should continue,
as this is the only way to have a better knowledge about the biology of the microorganisms.
The biological diversity of the Indian subcontinent is one of the richest in the world owing to
its vast geographic area, varied topography and climate, and the juxtaposition of several bio
geographical regions. Because of its richness in overall plant and animal species diversity, India is
recognized as one of the 12-mega diversity regions of the world. By virtue of supporting and
sustaining rich biological, ethnic and landscape diversity, it manifests biodiversity at all levels and
spatial scales. It represents an example of conglomeration of diverse bio-climates influenced by
neighboring areas (particularly Mediterranean), the unique location, peninsular land mass, Gangetic
plains and the crown of complex chain of mountain systems - the Himalaya and Western Ghats.
India ranks seventh among the centres of diversity and origin of crop plants (Khoshoo, 1995). In
India, there are 148 endemic genera and 5,725 endemic species (Nair, 1986) that are mostly
distributed in 2 "hotspots" (Himalayas and Western Ghats along with Sri lanka) out of 25 recognized
in the world (Myers et aI., 2000).
As mentioned above, the Western Ghats is one of the two biodiversity hotspot present in India.
The Western Ghats are known to be tectonically active and an uplifted region. The high biodiversity
3
Chapter 1 Introduction & Review o(Literature
of this region may be due to large nutrients that volcanism brought in, the relatively higher thermal
gradients along this belt and widely varying elevations.
Some documentation of diversity of macroscopic life forms of India is available (Khoshoo,
1995; Krishnakumar et ai, 1998; Rajagopal & Bhat, 1998), and a few studies on fungal diversity in
the Western Ghats region have been reported (Natarajan et aI., 2005; Raviraja, 2005; Naik et aI.,
2008) unfortunately, except for a few fragmentary reports (Ghosh et ai., 2003; Bhatnagar &
Bhatnagar, 2005; Thajuddin & Subramanian, 2005, Chaudhuri & Thakur, 2006), a systematic study
of diversity of prokaryote from these and other regions of India is lacking. Most of the natural
habitats, especially aquatic ecosystems are nutritionally poor but rich in microbial flora. It is known
that majority of bacteria do not grow in standard laboratory conditions, bacteria from aquatic and
sediment habitats are notoriously difficult to obtain in culture (Kemp & Aller, 2004; Schleifer, 2004).
Study of bacterial diversity from such a habitat and their function is an interesting and challenging
area of research. In the present study, an attempt has been made to study total bacterial diversity of
aquatic sample, sediment sample and mangrove sediment sample of Western Ghats by both culture
dependent and culture independent approaches and their biotechnological potential was assessed by
screening them for various enzymes. A comparative study of the bacterial diversity in different
niches will help in understanding the niche specific occurrence, their interaction and pattern of
ubiquity and will also provide a rich bacterial resource for utilization of their biotechnological
potentials.
1.1.1. Objectives of the thesis:
India ranks tenth in the world for richness in flowering plants (17,000 species) and mammals
(372 species) (Kaveriappa & Shetty, 2001). Unfortunately, hardly any data is available for
prokaryotic diversity of India. A polyphasic approach to study culturable microorganisms and a
molecular approach to study total microbial diversity in selected niches of Western Ghats are
expected to throw some light on the richness of prokaryotes in this region. The present work is
proposed with the following objectives: -
• To study culturable bacterial diversity of selected soil/water sample(s) collected from
various niches of Western Ghats using a polyphasic approach .
• To study the total diversity of microbial community of selected sample(s) using molecular
phylogenetic approach .
• Screening of culturable microbes for important biomolecules.
The study is aimed at having some knowledge about niche specific bacterial diversity of
aquatic habitat (water as well as sediment sample) and ofa mangrove sediment in the Western Ghats.
To the best of my knowledge, the study is the first of its kind on bacterial diversity from this Western
4
Chapter 1 Introduction & Review o(Literature
Ghats region using both culture dependent and culture independent methods. Considering the impact
of bacteria on global processes that keeps earth properly functioning, the work is the beginning of
efforts to understand the bacterial diversity of various ecological niches of this region.
5
Chapter 1 Introduction & Review o(Literature
1.2. Review of literature
1.2.1. Biodiversity: what the term defines?
"By which one sees an un perishable entity in all beings and undivided among the
divided then the knowledge is pure. But if one merely sees the diversity of things with their
division and limitations, without the truth then that knowledge is merely ignorance" The
Bhagavad Gita, chapter XVIII (Hunter-Cevera, 1998).
The tenn "biological diversity" was introduced by Elliot Norse and colleagues (Harper &
Hawksworth, 1995) to define diversity at three levels of complexity: (i) genetic (intraspecies
diversity), (ii) species (numbers of species), and (iii) ecological (community diversity), but
subsequently the contracted expression" biodiversity" has become the common parlance (Wilson &
Frances, 1988). Many revisions have been proposed afterwards time to time. Each of the components
in the definition of biodiversity has a hierarchical structure spanning biomes to niches (ecological),
domains to populations (species and organismal), and population to nucleotide sequence (genetic).
The tenn biodiversity, according to Erwin (1991), is related to the number of species (species
richness), along with 'the richness of activity each species undergoes during its existence through
events in the life of its members, plus the nonphenotypic expression of its genome. Microbial
diversity includes the genetic composition of microorganisms, the environment or habitat where they
are found, and there ecological or functional role within the ecosystem. Biodiversity according to
Hunter- Cevera, (1998) is defined as "all hereditarily based variation at all levels of organization,
from the genes within a single local population or species, to the species composing all or part of a
local community and finally to the communities themselves that compose the living parts of the
multifarious ecosystem of the world". As the living world is mostly considered in tenns of species,
biological diversity commonly used as a synonym of species diversity, in particular of 'species
richness', which is the number of species in a site or habitat (Groombridge, 1992), as defined by
Erwin (1991).
Biodiversity explains the variety of life in all its fonns, levels and combinations. The 1992
United Nations Earth Summit in Rio de Janeiro defined biodiversity as "the variability among living
organisms from all sources, including 'interalia', terrestrial, and other aquatic ecosystems and the
ecological complexes of which they are part". This definition is adopted by United Nations
Convention on Biological Diversity (UNCB). Other definitions can also be found which are as
follows-
l. The range of significantly different types of organisms and their relative abundance in
an assemblage or community (Torsvik et al., 1998)
6
Chapter 1 Introduction & Review o(Literature
n. As per information theory, amount and distribution of information in an assemblage or
community (Atlas, 1984).
1.2.2. Estimating the scale:
Earth provides a large variety of habitats that supports huge array of life forms full extent of
which stilI remain unknown; especially of microorganisms. Twelve mega biodiversity regions have
been identified on earth which is divided into 25 hot-spot centers based on diversity of mainly plants
and vertebrate animal (Myers et ai., 2000). We have quit a fair idea of diversity of macroscopic life
forms but the extent of prokaryotic diversity is yet to be fully explored and so far we know only very
little of it. Bacteria, archaea (together constituting the prokaryotes), fungi, microscopic algae,
protozoa and viruses constitute the microbial world as already stated. Prokaryote cells outnumber
that of the eukaryotic cells on earth by several orders of magnitude. The number of microbial species
in nature is estimated to be in millions (Hong et ai., 2006). Even for samples obtained from
environments like soils, such estimates vary widely; from a few dozen (Hughes et ai., 2001) and
hundreds to tens of thousands (Kemp & Aller, 2004) to half a million (Dykhuizen, 1998). The
estimated amount of bound carbon, nitrogen and phosphorus in globally occurring prokaryotes are
shown in Table 1.1. An approximate estimation of diversity of biological world is summarized in
Table 1.2. Prokaryotes are very important component of biodiversity which is evident from the fact
that the recent estimate indicated that prokaryotes outdo eukaryotes both by number as well as by
biomass. According to Whitman et ai., (1998) about 500 billion tons of carbon are bound in
prokaryotes which constitute about half of the total carbon found in global biomass and in case of
nitrogen and phosphorus even 90% is estimated to be of prokaryotic origin.
Table 1.1. Estimated amounts of bound carbon, nitrogen and phosphorus in globally occurring prokaryotes. (Adopted from Schleifer, 2004)
Compound Amount of compounds Incorporation to amounts of
bound in tons compounds bound in plants
Carbon 3.5-5.5 x 1011 60-100%
Nitrogen 0.9-1.4 x 10" 10 times more
Phosphorus 0.9-1.4 x IO IU 10 times more
7
Chapter 1 Introduction & Review o(Literature
Table 1.2. Estimated numbers of species occurring on earth. (Adopted mainly from Schleifer, 2004)
= ... '" ~ Q,} Q,}
..Q .0:; Q e " = '"
Q,} .. ..:c " = Q,} Q. »
'" = .0:; '" OJ Group ... Q,} 0:
Q • 0:; "1:1 Q,} = ... ... Q,} Q,} Q. ~ = Q,} Q. - '" Q OJ
..Q '" 0: ... = OJ
e e Q ~ < = '= ~ ;Z; '" ~ 0
Microorganisms
Prokaryotes 5 >1000 <0.5 Very poor
Fungi 72 1500 4.8 Very poor
Protozoa 40 200 20 Moderate
Algae 40 400 10 Very poor
Viruses* 50 130 4 Very poor
Plants 270 320 84 Good
Animals
Nematodes 25 400 6 Poor
Crustaceans 40 150 26 Moderate
Insects 950 800 12 Moderate
Vertebrates 40 45 90 Good
Molluscs# 70 200 35 Moderate
Others# 115 250 46 Moderate
a In thousands,' adapted from Bull e/ al. 1992; # adapted from Bull & Stach, 2004
1.2.3. Microbial diversity extent- known:
The total number of prokaryotes on the earth is estimated to be 4 to 6 x 1030 and the amount of
carbon 3 to 5 X 1017 g contributed by prokaryotes corresponds to almost half of the total carbon found
in the global biomass (Whitman et at., 1998).
Prokaryotes are ubiquitous and even live in extreme habitats where a major part of other life
forms can not survive. Analysis of a range of representative habitats revealed that a major part of
prokaryotes are believed to live in oceanic sub surface followed by terrestrial, subsurface, soil and
aquatic habitat (Table 1.3). They are integral part of most of the animals including human and
performing functions vital to the survival of the host (Whitman, 1998). Several caveats must be
placed on these estimations including large variations resulting from sampling effort and
extrapolations and integrations from representative data. Nevertheless, these estimates carry some
striking inferences. The large popUlations imply that events that are rare in the laboratory may occur
frequently in nature, and they point to an enormous potential to accumulate mutations and thereby
acquire genetic diversity.
8
Chapter 1 Introduction & Review o(Literature
Table 1.3. Number and biomass of prokaryotes in the world. (Adopted from Whitman et al., 1998)
Environment Number of prokaryotic Biomass in prokaryotes cells xlO28
in Pg (lOISg)
Aquatic habitat 12 2.2
Oceanic subsurface 355 303
Soil 26 26
Terrestrial sub-surface 25-250 22-215
Total 415-640 353-546
Considering such a huge number and ubiquitousness, it is expected that prokaryotic species
diversity will be enormous. But in the laboratory conditions it has been observed frequently that only
small fractions of microbial community appear on variety of nutrient media (Staley & Konopka,
1985). One of the most important insights of microbial diversity came from reassociation kinetic
analyses of environmental DNA (Torsvik et al., 1990a). These authors also found that the numbers of
bacterial genomes present in soil and marine sediment was as great as 6,500 and 11,400,
respectively, representing an estimated 20,000 to 37,000 bacterial species (Torsvik, et ai., 2000).
This dramatic finding represents the first limitation for those wishing to compare microbial diversity
in different environments and samples, i.e. that tens of thousands of samples would be needed to
ensure complete coverage (not including the likelihood of catching the same species twice). Detailed
analysis of different soil samples by Torsvik et ai., (2002) using direct microscopic count and studies
based on community genomic complexities as determined by reassociation kinetics of total
community DNA (Table 1.4) revealed the fact that enormous diversity of bacterial species exists in a
sample which is not reflected by culture dependent approach. Extremes of habitats such as salt
crystallizing pond are expected to have less species diversity. Major taxonomic diversity also occurs
in the domain archaea, and culture independent surveys have revealed novel types in a wide variety
of habitats. Karner et ai., (2001) reported that one group of archaea, the pelagic Crenarchaeota, are
extremely abundant and the ocean contain over 1028 archaeal cells (Karner et ai., 2001).
Subsurface is a major habitat for prokaryotes, and the number of subsurface prokaryotes
probably exceeds the number found in other components of the biosphere (Table 1.3) out of the 3.8 x
1030 prokaryotes estimated to be in the oceanic and terrestrial subsurface, 97% or 3.7x 1030 occur at
depths shallower than 600 m.
9
Chapter 1 Introduction & Review o(Literature
Table 1.4. Prokaryote abundance as determined by fluorescence microscopy and total genomic diversity in prokaryotic communities calculated from the reassociation rate of DNA isolated from the community. Community genome complexity is described as numbers of base pairs (bp). Genome equivalents are given relative to the E. coli genome (4.1 x 109 bp) (Torsvik et al., 2002).
DNA source Abundance Community genome Genome
(cell/cm3) complexity (bp)
Equivalents
Forest soil 4.8x 1O~ 2.5xl01V 6000
Forest soil, cultivated prokaryotes 1.4 x 107 1.4 x 1O~ 35
Pasture soil 1.8 x lOw 1.5-3.5 X lOw 3500-3800
Arable soil 2.1 x lOw 5.7-14.0 x 10· 140-350
Pristine marine sediment 3.1 x 10~ 4.8 X 101V 11,400
Marine fish-farm sediment 7.7 x 10~ 2.0 X 10M 50
Salt crystallizing pond 6.0 x 10' 2.9 X 10' 7
1.2.4. Are microbes too diverse to count? The ability to measure bacterial diversity is a prerequisite for the systematic study of bacterial
biogeography and community assembly. It is therefore central to the ecology of surface waters, the
oceans and soils, waste treatment, agriculture, and global elemental cycles. However, the
experimental definition of bacterial diversity has never been undertaken for any naturally occurring
bacterial community anywhere, and the extent of prokaryotic diversity is widely held to be beyond
practical calculation (Wilson, 1994). Our understanding of bacterial biogeography and community
assembly is correspondingly vague, anecdotal, and controversial. For example, the global distribution
of some aquatic protozoa has been used to assert that the entire microbial world is composed of a
small number of ubiquitous organisms (Fenchel, et ai., 1997; Finlay & Clarke 1999) whereas the
apparently endemic distribution of some bacteria has been used to suggest the opposite (Cho &
Tiedje, 2000; Fulthorpe et ai., 1998). However, according to Curtis et ai., (2002) in estimating the
extent of microbial diversity, it is not necessary to count every single species or taxa in a sample. It is
sufficient to simply estimate the area under the bacterial species abundance curve for that
environment. They speculated that they could estimate the bacterial diversity on a small scale
(oceans 160 per ml; soil 6,400- 38,000 per g; sewage 70 per ml) and also diversity at a larger scale
(the entire bacterial diversity of the sea may be unlikely to exceed 2 x 106, while a ton of soil could
contain 4 x 106 different taxa).
In 9.ny community, the number of types of organisms observed increases with sampling effort
until all types are observed. The relationship between the number of types observed and sampling
effort gives information about the total diversity of the sampled community. The ideas that microbial
diversity cannot be estimated comes from the fact that many microbial accumulation curves are
10
Chapter 1 Introduction & Review o(Literature
linear or close to linear because of high diversity, small sample sizes, or both. At least for some
communities, microbiologists may be able to co-opt techniques that ecologists use to estimate and
compare the richness of macroorganisms. Plotting an accumulation (An accumulation curve is a plot
of the cumulative number of types observed versus sampling effort) or a rank-abundance curve helps
in this direction. Comparisons of accumulation curves and rank-abundance plots demonstrate that
some bacterial communities have been sampled equally well as some macroorganism communities
(Hughes et aI., 2001). Therefore, evaluating microbial diversity with statistical approaches available
for macroorganisms seems feasible. Figure 1.1 shows the accumulation curves for samples from five
communities: bacteria from a human mouth (Kroes, 1999), soil bacteria (Borneman & Triplett,
1997), tropical moths (Ricketts et at., 2001), tropical birds (Hughes, 2002), and temperate forests
(Hellmann, 1999). Differences in the richness and relative abundances of species in the sampled
communities underlie the differences in the shape of the curves. Because all communities contain a
finite number of species, if the surveyors continued to sample, the curves would eventually reach an
asymptote at the actual community richness (number of types). Thus, the curves contain information
about how well the communities have been sampled (i.e., what fraction of the species in the
community have been detected). The more concave-downward the curve, the better sampled the
community. Ultimately, microbes are too diverse to count exhaustively. While it would be useful to
know the actual diversity of different microbial communities, most diversity questions address how
diversity changes across biotic and abiotic gradients, such as disturbance, productivity, area, latitude,
and resource heterogeneity. The answers to these questions require knowing only relative diversities
among sites, over time, and under different treatment regimens.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Proportion of individuals sampled
1
Fig. 1.1. Accumulation curves for Michigan plants (x), Costa Rican birds (.), human oral bacteria (0), Costa Rican moths (_), and East Amazonian soil bacteria (e) (Hughes, 2001)
11
Chapter 1 Introduction & Review o(Literature
Some of the possible reasons which possibly explain the huge diversity found among
prokaryotes are as follows:
a) As they have evolved 3.5 billion years ago they have experience maximum changes in
earth's climate, geography and chemistry. These changes forced them to evolve differently
giving rise to such a huge diversity.
b) This huge population size implies that prokaryotes have an enormous potential to
accumulate genetic variability due to events such as mutations.
c) The rapid growth rate of prokaryotes implies that mutations and other rare genetic events
are more likely to occur in prokaryotes. The capacity for a large number of simultaneous
mutations distinguishes prokaryotic from eukaryotic evolution.
d) For essentially asexual, haploid organisms such as prokaryotes, mutations are a major
source of genetic diversity and one of the essential factors in the formation of novel
species.
1.2.5. Global pattern in microbial diversity:
Although microorganisms are perhaps the most diverse (Torsvik et aI., 2002; Venter et aI.,
2004) and abundant type of organism on earth, the distribution of microbial diversity at continental
scales is not well understood. Ecologists describing microbial biogeography typically invoke
Beijerinck (Beijerinck, 1913) from a century ago; "every thing is every where, the environment
selects". However, few studies have attempted to verify this statement or specify which
environmental factor exerts the strongest influences on microbial communities in nature (Papke &
Ward, 2004; Horner- Devine et aI., 2004). For centuries, biologists have studied patterns of plant and
animal diversity at continental scales. Until recently, similar studies were impossible for
microorganism, arguably the most diverse and abundant group of organisms on earth. Fierer &
Jackson, (2006) presented a continental scale description of soil bacterial communities and the
environmental factors influencing their biodiversity. They collected 98 soil samples from across
North America and South America and used an rDNA finger printing method to compare bacterial
community composition and diversity quantitatively across sites. Bacterial diversity was unrelated to
site temperature, latitude, and other variables that typically predict plant and animal diversity and
community composition was largely independent of geographic distance. The diversity and richness
of soil bacterial communities deferred by ecosystem type and these differences could largely be
explained by soil pH. Bacterial diversity was highest in neutral soils and lower in acidic soils, with
soils from the Peruvian Amazon the most acidic and least diverse in their study. A global picture of
microbial diversity has remained elusive, yet it is critical to understand microbial adaptation to
different environments and their function in those environments. Determining physical and chemical
factors, such as temperature, pH, or geography, that correlate with differences between diverse 12
Chapter 1 Introduction & Review o(Literature
microbial communities will reveal how easily microbes tolerate different kinds of environmental
change and will increase our understanding of microbial ecology and evolution. In addition,
determining the environment types that contain the most phylogenetic diversity will reveal where
new sequencing efforts to catalogue global bacterial diversity will be most efficient at uncovering
deep branching lineages. Lozupone & Knight, (2007) have reported the most comprehensive analysis
of the environmental distribution of bacteria to date, based on nearly twenty two thousand 16S rRNA
sequences compiled from III studies of diverse physical environments. They clustered the samples
based on similarities in the phylogenetic lineages that they contain and found that, surprisingly, the
major environmental determinant of microbial community composition is salinity rather than
extremes of temperature, pH, or other physical and chemical factors represented in the samples. They
also found that sediments are more phylogenetically diverse than any other environment type. Many
diversity studies so far done targeting soil bacteria have clearly revealed that bacterial diversity in
soil is enormous and that the composition and diversity of soil bacterial communities can be
influenced by wide range of biotic and abiotic factors. (Dunbar et aI., 2002; Tringe et aI., 2005).
Recently with the help of a new approach, the shotgun sequencing of entire communities without
the need to construct large insert clone libraries J. Craig Venter, made a significant attempt to sequence
and achieve the inventory of Sargasso Sea (Venter et al., 2004). In February and May 2003 researchers
took sea water samples from six marine research sites in the Sargasso Sea. Using a protocol in which
the water was filtered through decreasing size filters, from plankton net, through a 3.0 micron filter, a
0.8 micron filter, and a 0.1 micron filter in order to collect different sized single cell organisms. DNA
libraries were made and sequenced. Using this whole genome shotgun sequencing, 1.045 billion base
pairs of DNA sequence were produced. Using precise mathematical algorithms previously used to
assemble sequence results from single species, the researchers were able to assemble whole genomes
and major sections of genomes from the diverse microbial community found in the ocean. They
reported the presence of a minimum of 1,800 species (180 of them were novel) and 1,214,207 new
genes. One of the most important single discoveries from the Sargasso Sea environmental shotgun
sequencing study is the 782 new rhodopsin-like photoreceptor genes (Yooseph et aI., 2007). Only a
few dozen photoreceptors have been characterized in microorganisms to date and less than 200
photoreceptors have been discovered from all species, including human where they are responsible
for our vision. Therefore, this discovery represents a substantial increase in the total number of this
family of proteins.
Sorcerer II Global Ocean Sampling Expedition (GOS) lead by Ventor (Yooseph et aI., 2007;
Rusch et at., 2007) is a landmark effort in microbial diversity study. The group amassed 6.3 billion
bases and assembled them by computational tools as they developed to handle and analyse the
massive data set. In brief they found a great degree of diversity both within and between samples and
also highly abundant ribotypes (roughly equivalent to species). The study revealed the presence of
13
Chapter 1 Introduction & Review of Literature
novel genetic information in marine microorganism and novel metabolic process. Many of the open
reading frames (roughly equivalent to genes) are unlike known gene. The GOS analysis also nearly
doubles the number of previously known proteins. This enormous amount of data allowed the
researchers to better understand the genomic structure and evolution of microorganisms, as well as
the function of important protein families such as protein kinases, which are key regulators of
cellular function in all organisms.
1.2.6. Factors controlling microbial diversity:
The major underlying principle of diversity studies is probably the assumption that interactions
between populations in a habitat lead to an organized and stable community (Atlas, 1984). Marglef
(1968) stated that diversity and stability is inversely related to productivity (Ovreas, 2000). An
explanation of this view may be that the mature community needs less energy to maintain its
structure (Atlas, 1984). In a microbial community many different organisms will perform the same
processes and probably be found in the same niche. Diversity can vary with a number of factors-
1. Stress in one part can be rapidly amplified and spread to the whole system through positive
feedback links that tie the system together (Ovreas, 2000). Bacterial communities respond
to perturbation in the same manner as do communities of higher organisms, though much
faster. This is partly due to fact that microorganism have a much higher growth rate. In
addition the bacterial community may inhabit members and are ready to take advantage of
the new situation.
2. Chemical warfare between microbes promotes biodiversity (Czaran et ai., 2002). Several
studies have been concerned with diversity in response to stress such as heavy metals
(Baath et ai., 1998; Barkay, 1987; Dahlin et ai., 1997) herbicides (Ka et ai., 1994),
antibiotics (Belliveau et ai., 1991) and toxic chemical waste (Baya et ai., 1986; Burton et ai., 1982). It is evident now that the microbial communities have a high degree of
adaptability.
3. Plasmids, bacteriophages, and transposones are genetic elements with a continuous lineage
and their own evolutionary history. The influence of these factors in generating and
maintaining gene flux and in adding to the phenotype of their host, contribute to the
evolution of bacterial genomes. Bacterial genomes contain evidences for both vertical and
horizontal gene transfers (Campbell, 1981). Bacteria are products of an evolutionary
process that has occurred over thousands and millions of years.
4. Predator-prey interaction also influences biodiversity (Lebaron et ai., 1999). The system
controlling bacterial diversity seems to be a hierarchical system where lytic viruses,
predation and system nutrient content are closely linked together (Thingstad & Lignell,
14
Chapter 1 Introduction & Review o(Literature
1997). Bacterial diversity can be regulated by a combination of size selecting grazing and
host specific viral lysis (Bratbak et al., 1994).
1.2.7. Fundamental reasons for prokaryotic diversity studies- why
so important?
In comparison to almost 1 million of insect species there are currently only about ~ 9000
validly described species ofprokaryotes (www.bacterio.cict.fr/number). Considering that each insect
carries at least millions and even billions of prokaryotes, we know only a minor fraction of
prokaryotes present on Earth (Table 1.2). Apart from such huge abundance and biomass
representation, they have many unique properties and carries out very important functions (Schleifer,
2004). These are as follows:
1. Founder of our biosphere, "the prokaryotes" was the very first kind of living organisms that
appeared on earth at least 3.5 billion years ago. Therefore, Earth has been populated by
these microbes for most of its existence. The existence and evolution of other life forms
would not have proceeded without them. Those were prokaryotes only that created an
oxygenic environment which supported the life of higher organisms.
2. Global biosphere is mainly shaped by geochemical activities of prokaryotic life as they
maintain biogeochemical cycles. They affect all geochemical processes that occur at the
earth surface, as well as deep subsurfaces and in tum get affected by these activities also.
They complete nitrogen and sulfur cycle, oxidize and reduce metals and some of them in
tum obtain energy for growth by transferring electrons to a wide range of harmful metals,
such as uranium, chromium, arsenic and plutonium.
3. Prokaryotic microbes show an unusual high physiological and biochemical versatility.
They can use reduced inorganic compounds as energy for chemolithotrophic metabolism or
oxidized inorganic compounds as electron acceptors for anaerobic respiration. Moreover,
certain metabolic pathways, such as fermentations, nitrogen fixation, methane formation
and anoxygenic photosynthesis, are only found among prokaryotic microbes. This is also
true for the biosynthesis of secondary metabolites such as certain antibiotics and toxins.
Certain microorganisms carry out "anamox" reaction that refers to the anaerobic oxidation
of ammonia where nitrite is the terminal electron acceptor. The energetics of the reaction is
much more favorable than the oxic nitrification process. The bacteria that perform this
reaction, form a deeply branching monophyletic lineage within the phylum Planctomycetes
which till recently not been obtained in pure culture and were known only from culture
independent studies oftheir phylogenies and physiologies. Completely autotrophic nitrogen
removal over nitrite (CANON) where a combination of both aerobic ammonia oxidizers
15
Chapter 1 Introduction & Review o(Literature
and anammox bacteria are utilized to remove nitrogen from wastewaters (Jetten et al.,
2001) is an important application of anamox.
4. The metabolic, physiological and genetic diversity of prokaryotic microorganism is far
greater than that found in higher organisms. Some microorganisms are highly resistant to
radiation e.g. Deinococcus radiodurance and might be used for safe management of
radioactive waste.
5. Microbes are the frontiers of life because they are also determined by prokaryotic microbes.
They can survive and even thrive under the most extreme environmental conditions. They
can be found in all habitats where the physical and chemical circumstances allow the
existence of life. Some of them can grow in' hot spring at temperatures up to 121°C
(Kashefi & Lovley, 2003), others are found in frozen water in a fresh water lake beneath 4
kilometers of ice in central east Antarctica (Karl et aI., 1999). They exist and grow at low
pH as well as in saturated salt solution. Moreover, all natural occurring and also many man
made compounds will be degraded by microbes.
6. Microbial interactions among themselves in both positive and negative manner within a
single population. One population may benefit another in a one sided comonsal way, or two
different populations may interact synergistically. Such beneficial interactions are
facilitated by close physical proximity, as in biofilms and flocs. Such co operational
interactions have been shown for populations of Myxobacteria (Dworkin, 1996; Shimkets,
1990). Cooperation in a microbial population can function as protective mechanism against
hostile environmental factors. Microbial populations within a biofilm are orders of
magnitude more resistant to antimicrobial agents than suspended cells of the same
organisms (Shapiro, 1991). Resistance to antibiotics and heavy metals and the ability to
utilize unusual organic substrates are often genetically transmitted to other members of the
population (Hardy, 1981). Mutualism which is defined as a strong, specific, beneficial
interaction essential for survival of both partners is well studied in microbial world.
According to theory of serial symbiosis, some mutualistic endosymbiotic relationship had
key roles in the evolution of higher organisms (Margulis, 1971). Endosymbiotic
methanogens have been found in anaerobic ciliate protozoa living within the rumen. It is
likely that endosymbiotic methanogens can directly use molecular hydrogen produced by
the ciliate protozoan (Heckmann & Gortz, 1992). Syntrophism term is applied to the
interaction of two or more populations that supply each other's nutritional need.
Syntrophism may allow microbial populations to perform activities, such as the synthesis
of a product that neither popUlation could perform alone. A classical example of such
syntrophism is exhibited by Enterococcus faecalis and Escherichia coli for conversion of
arginine to putrescine (Gale, 1940). Similarly cyclohexane is degraded by a mixed
16
Chapter 1 Introduction & Review o(Literature
population of a Nocardia species and a Pseudomonas species but not by either population
alone (Slater & Bull, 1978). Syntrophomonas sp. and Syntrophobacter sp. are hydrogen
producers and require the presence of the methanogen as hydrogen removers (Balows et
aI., 1992). In contrast to positive interaction, microorganisms produce substances toxic to
competing populations which is called amensalism. Production of ammonium by some
microbial populations is deleterious to other populations, some produce low molecular
weight alcohols, some produces antibiotics. Inhibitory substances produced by
microorganism may also act as preservatives for organic compounds in natural habitats e.g.
decomposition of cellulose in soil, organic acids are produced that prevent further
breakdown of cellulose metabolites in subsurface soil. Some examples are there for
parasitic bacteria too like Bdellovibrio that is parasitic on Gram-negative bacterial
populations (Starr & Seidler, 1971; Stolp & Starr, 1963).
7. Interactions of microbes and plants, e.g. association of nitrogen fixing bacteria and root of
leguminous plants, mutualistic relationship between Azolla and Anabaena sp. are
interesting and well studied phenomena. Rhizosphere and phyllosphere microbiota also
provide an open area for microbial diversity investigations. However, many bacteria are
known as plant pathogens too.
8. Most interactions between microbes and animals are beneficial. The mutualistic
relationship of microbial populations involves nutrient exchange and maintenance of
suitable habitat. Microbial, in particular bacterial endosymbionts are fundamental to the
survival of higher organisms. Without bacterial endosymbionts most animals would not
survive. Symbionts carryout essential biochemical reactions for their eukaryotic hosts, e.g.
the biosynthesis of essential amino acids, vitamins or the degradation of certain
macromolules. Some marine worms use their sulphide oxidizing bacterial endo or
ectosymbionts even as sole feed source. Most warm blooded animals contain extremely
complex microbial flora within their gastrointestinal tracts. In lower intestine, each gram of
feces contains approximately lOll microorganisms, belonging to up to 400 different species
(Lee, 1985). In some animals, such as pigs, and bovine animal the microbial populations of
the gastrointestinal tract contribute to the nutrition of the animals by fermenting
carbohydrates especially cellulose and animal can utilize the products of the cellulose
degradation (Kenworthy, 1973). In monogastric animals the main contribution to digestion
by intestinal microbial populations appears to be in the production of growth factors. In
some cases, microorganism supply required vitamins, to constitute an important barrier to
attack intestinal pathogen. The protozoa and bacterial population found within gut of lower
termites and wood eating cockroaches ferment cellulose anaerobically, producing CO2, H2,
and acetate. Some of the H2 and CO2 is converted to C~ by methanogenic archaea
17
Chapter 1 Introduction & Review o(Literature
(Leadbetter & Breznak, 1996). Some microbial population within termite gut fix
atmospheric nitrogen (Bonemann, 1973). The microbial populations which are maintained
within mycetomes supplement dietary deficiencies of the animal by producing growth
factors. Amphipods contain high proportions of chitinase producing Vibrio species that
partially degrade the chitin ingested by these animals. Mussels of family Mytilidae living in
deep sea thermal vent harbor methanotrophic bacteria with typical stalked internal
membrane structure. All aphids have cell clusters that are called mycetomes and the
individual cells mycetocytes. These cells harbor bacteria which upon elimination by
antibiotic treatment affect reproduction and the aphids eventually die (Baumann &
Bauman, 1994).
9. Both cultured and "uncultured" prokaryotes represent a huge genetical and
biotechnological potential and therefore an enormous source of new products and
processes. Diverse microorganisms have yielded important biological materials useful to
humans such as antibiotics (Crossley, 1986), drugs (Drews, 2000), enzymes (Cherry et ai.,
1999) and growth promoters (Parada et al., 1998).
10. Microorganisms can aid environmental restoration by oxidizing, binding, immobilizing,
volatilizing or otherwise transferring contaminants. Prokaryotic microbes are responsible
for degradation of natural products and man made harmful xenobiotic compounds. They
have capacity to remove many contaminants from environment by activity of enzymatic
processes. The most common type of bioremediation is oxidation of toxic, organic
contaminants to nontoxic products. Oxygen is the most common electron acceptor for
microbial respiration, and aerobic degradation of an extensive range of organic
contaminants, from aromatic hydrocarbons such as benzene to xenobiotics, such as
pesticides. Pseudomonas species and closely related organisms have been the most
intensively investigated for the degradation of aromatic contaminants. Microorganisms can
also anaerobically oxidize many contaminants with alternate electron acceptors such as
nitritates, sulphate and Fe (III) oxides. Geobacter species such as G. metallireduscens that
are highly enriched in subsurface environment can oxidize organic compounds with
reduction of Fe (III). Shewanella and Geothrix species release iron chelators which
solublise Fe (III) to Fe (III) oxides. Sulfate reducing bacteria such as Desulfobacula and
Desulfobacterium species, can oxidize hydrocarbons with sulfate as the electron acceptor
that are present in huge amounts in marine environments and serve as electron acceptor for
anaerobic degradation of contaminants. Microorganisms remove chlorides from
contaminant such as chlorinated solvents like tricholoroethane (TCE) is degraded by
Dehalococcoides ethanogenes. Many microbes capable of dehalogenetion are known
among which Dehalococoides species are particular important. Some can reduce inorganic
18
Chapter 1 Introduction & Review of Literature
contaminants such as nitrate percolate and selenite. Geobacter species can remove uranium
from contaminated water by conversion of U (VI) to U (IV). Microorganisms have been
found that can accumulate heavy metals like gallium (Bull, 1991). Chemolithotrophic
bacteria, like Thiobacillus ferroxidans, and T thiooxydans are increasingly being used in
mining for controlled bioleaching of metals (Rawligs & Silver, 1995).
11. They are responsible for certain diseases in animals, plants and humans.
Thus microbial diversity is fundamental to maintenance and conservation of global genetic
resources and represent by huge diversity in terms of their metabolic activities and their interactional
ability with other life forms (Schink, 1992). Diversity analysis is therefore important (Ovreas, 2000)
in order to-
• Increase knowledge of the diversity of genetic resources in a community.
• Understand patterns in relative distribution of organisms.
• increase the knowledge of fundamental role of diversity
• identify differences in diversity associated with management disturbing
• Understand the regulation of biodiversity.
• Understand the consequence of biodiversity (to what extant does the ecosystem functioning
and sustainability depend on maintaining a species level diversity).
Many have the ability to grow anaerobically and also to adapt to diverse environmental
conditions. Due to their huge impact on numerous interconnected life processes and their metabolic
capabilities that keeps the earth properly functioning and in order to best exploit microorganisms, it
is very important to know "what is there and what can be used" (Bull, 1991).
1.2.8. Current picture of microbial diversity:
1.2.8.1. The Black Matter in Microbial Space: 'diversity black box'
The number of species in all but the simplest communities can only be estimated statistically,
typically on the basis of a small subset of species (or their small rRNA sequences) observed directly,
even for samples obtained from similar environments (soils), such estimates vary widely; number of
bacteria exceeds far than what we know presently. For example, possibly 2-13 million species in
Atlantic forest tree canopy (Lambais et aI., 2006), 2 million species in the open sea (Curtis et al.,
2002) and about 40,000 species per gram of soil (Dykhuizen, 1998), from a few dozens and hundreds
(Hughes et al., 2001; Kemp & Aller, 2004). The quality of microbial richness predictions is an
important issue as they serve as the basis of all of the paradigms of biodiversity, its role, functions
and meaning. It is therefore of principal interest to know the true extent of microbial diversity,
19
Chapter 1 Introduction & Review o(Literature
starting from that in a single environmental sample. The number of microbial species in nature may
be in millions but most have never been observed or otherwise detected.
1.2.8.2. General limitations in studying microbial diversity:
There are problems associated with studying bacterial diversity. These arise not only from
methodological limitations but also from lack of taxonomic knowledge. It is difficult to study the
diversity of a group of organism when it is not understood how to characterize the species (Kirk et
aI., 2004). Main limitations for extensive diversity analysis are given below-
i. Spatial heterogeneity:
This is major problem in soil and sediment environments as very little is known about
spatial and temporal variability of microorganisms in these environments. Bacteria are
highly aggregated in soil existing in clumps or 'hot spots. Microbial communities may have
several nested levels of organization, and that they could be dependent on different soil
properties. Microbial communities exist on such a small scale, that possibly 1-5 gram of
soil could bias results and favor detection of dominant population.
ii. Limitation of molecular-based methods:
Limitations of culture based methods have, to some extent, been overcome by usmg
molecular techniques; however, they are not without their own limitations. If method of
DNA extraction used is too gentle, Gram negative, but not Gram-positive bacterial cells
would be lysed. If the method is too harsh, both gram negative and gram-positive cells may
be lysed but their DNA may become sheared (Von Wintzingerode et ai., 1997). This
variation may lead to biases in molecular based studies. With environmental samples, it is
necessary to remove inhibitory substances such as humic acids, which can interfere with
subsequent PCR analysis. Subsequent purification steps can lead to loss of DNA or RNA,
again potentially biasing molecular diversity analysis. Differential amplification of target
genes due to different affinities of primers to templates, different copy numbers of target
genes, hybridization efficiency and primer specificity are also the causes. Although above
discussion sets forth some limitations of molecular based study which can influence the
analysis and interpretation of molecular based microbial community analysis yet these
methods provide valuable information about the microbial community as opposed to only
culture based techniques.
iii. Taxonomic ambiguity: 'species concept in prokaryotes'
Another problem associated with measuring microbial diversity in soil is the problem of
defining microbial species. There is no official definition of bacterial species (Colwell et
ai., 1995). There are many opinions and suggestions as to how to define a bacterial species.
20
Chapter 1 Introduction & Review o(Literature
For practical purposes a consensus definition was adopted. According to this view a
bacterial species is a collection of strains showing 70% or more genomic relatedness
(DNA-DNA hybridization) and ~Tm of 5°C or less of the hybrid molecules (Wayne et aI.,
1987). It has been observed that at this level of genomic relatedness the 16S rRNA gene
sequence similarity is usually 97% or more (Stackebrandt & Goebel., 1994).
iv. Inability to culture:
Many lines of evidence indicate that only a small fraction of naturally occurring
prokaryotes is culturable by standard techniques (Amann et al., 1995). As stated above
very little about their identity and possible functions is known. The total number of
described prokaryotic species known at present is about 9,000 only (www.bacterio.cict.fr)
which is less than 1 percent of the currently estimated bacterial species diversity. The
existence of these species is only known from their 16S rRNA gene sequences and
predicted from statistical analysis. Since conventional recognition of a prokaryotic species
requires its cultivation but only a minor fraction can be cultured in the laboratory. This
means that majority of the species are not known to the scientific community and remains
to be discovered as supported by many observations:
1). "Great plate count anomaly" the term, first introduced by Staley & Konopka (1985) is
based on the observation that number of organisms present in a sample seen directly
under a microscope is 10 to 100 times more than the number of colonies they form on
conventional laboratory media (Staley & Konopka, 1985). Some methods which do
not require culturing of microbes for their detection and enumeration are:
epiflourescence microscopy with stains such as acridine orange, 4', 6' -diamino 2-
phenylindol (DAPI), direct imunoflourescence, epiflourescence microscopy, and
direct viability count by nalidixic acid. Microautoradiography and stable isotope
probing have revealed that up to 36% (Zimmermann et aI., 1978) and in some cases
staining by SYTO 9 even 90 % (Janssen et ai., 2002) uncultured majority may be
metabolically active. Autoradiography combined with direct microscopic observation
(in which bacteria incubated with radiolabelled substrate, such as tritiated glucose, are
subsequently collected on a bacteriological filter placed on a glass slide, coated with
a photographic emulsion) indicated that 2.3-56.2 % of the total bacteria are
metabolically active (Meyer-Reil, 1978). Some of the reasons for their inability to
grow in the laboratory are:
• These ostensibly "uncultured" cells have permanently lost culturabilty i.e.
S-rr·S TH-17112 ~y{qJ . ~4--{ ~
21
579.3 84697 St 1111111111111111 i 11111111111111
TH17112
TH
Chapter 1 Introduction & Review o(Literature
effectively had gone to the stage of "viable but non-culturable". They are in
dormant stage from which recovery method is still unknown (Barer &
Harwood, 1999).
• They are simply unable to grow on standard isolation media.
• Cell damage caused by oxidative stress preventing growth until repair (for
instance by SOS response mechanism) has been completed.
• Inhibition by high concentrations of substrates in the media (substrate-
accelerated death).
• The induction of lysogenic phages during cultivation.
• Lack of cell-to-cell communication in laboratory media (Kaeberlein et aI.,
2002).
2). Second supporting evidence comes from measurements of the genetic diversity of
bacteria in soil or marine sediment using the reassociation kinetics of their genomic
DNA that indicated the presence of 4000 to 13000 different bacterial genomes
(Torsvik et aI., 1990a; Torsvik et al., 1994; Torsvik et al., 1993), exceeding the
number of bacterial species currently known. Such a huge number of bacterial species
were never isolated on culture media. The number of different cultured species from
the same sample was calculated to be 21 from 206 isolated morphotypes when
determined by renaturation kinetics of the cultured isolates. Table 1.5 shows ratio of
cultivable to microscopically detectable prokaryotes in different niches.
3). Further support for a high diversity of so far 'uncultured" bacterial species came from
rRNA gene based culture independent molecular phylogenetic studies. The database
consisting of these gene sequences contains many phylotypes with no close
cultivatable relatives. Some of these distantly related sequences have also been
proposed to belong to new candidate divisions consisting solely of gene sequences
whose presence has been indicated by the culture independent approach (Hugenholtz
et aI., 1998a; Rappe & Giovannoni, 2003). There are currently 53 phyla (Fig. 1.3) in
bacteria, 26 of which have got no cultured representatives (Keller & Zengler, 2004)
and continuous from the detection of novel 16S rRNA gene sequences in
environmental samples.
22
Chapter 1 Introduction & Review o(Literature
Table 1.5. Ratio of cultivable to microscopically detectable prokaryotes in percentage in various niches (data taken from Bull & Stach, 2004).
Habitat Culturability (%)
Sea water 0.001-0.1
Fresh water 0.25
Mesotrophic lake 0.1-1
Unpolluted estuarine 0.1-3 waters
Activated sludge 1-15
Sediments 0.25
Soil 0.3
0.05
~. Verrucomicrobia 7
JI
IL
~ 'l
OP3
WS3 BRC1
NKB19
OP9 wsz
vadinBE97 Chlamydiae 13
Planctomycetes 9
Firmicutes 1205
Cyanobacteria 4 Fusobacteria 25
OP10 SC4
Actinobacteria 1367 NC10
~ Bacteroidetes 220
Chiorobi8
~ -t
-t
Ir-l
t 1c!
Marine Group A Caldl!hnx 1
OS·K
C..emmatimonadetes 1 Fibrobacteres Z
Proreobacteria 1808 Oeterribacteres 7 Chrysiogenes arsenatis 1
SBR1093
SC3
Acidobacteria 3 OPB
Nitrosptae 6 Termite Group 1
TM6 SynergiSieS 1
OP5 Spirochaetes 91
B01-5 group
WS6 TM7
WS5 Guaymas 1
ABY1
Chbrofiexi 11 Deinococcus· Thef/17'Js 24
Thermodesulfobacteria 2 OP1 4 ---.l ThemlOtogae 25 - 4'-------.. -... Coprothermobacter 2
1----11 .... fJctyoglomi 2
{::~~~::~::::~~AqWftae13 ""'I Oesu!furobacterium 1
OP11
Fig 1.2. Phylogenetic tree of the domain Bacteria based on 16S rRNAgene sequences (Keller & Zengler, 2004). Bacterial phyla with cultivated representatives are shown in blue and green. Bacterial phyla with no cultured representative are shown in red Numbers in red represent the published species within a phylum.
23
Chapter 1 Introduction & Review o(Literature
1.2.8.3. Cultivating the uncultivated:
Cultivation of microorganisms has been viewed as fundamental for understanding of microbial
physiology and metabolism. It is well known that conventional cultivation approaches access only a
tiny subset of the wide diversity of microorganisms inferred to be present in any given environment.
Methods for cultivation that are based on modified traditional approaches have resulted in the
isolation of some previously uncultured, phylogenetically distinct microorganisms. Various methods
employed in improving cultivation of uncultured bacteria from environment sample by doing some
manipulations in traditional culturing methods. Some of them are as follows-
(i). Using low-nutrient media and increased incubation times, addition of pyruvate or
catalase to reduce oxidative stress, addition of carbon substrates at only low
concentrations Janssen and co-workers were able to obtain from soil, pure culture
representatives of several subdivisions of the recently recognized bacterial phyla
Verrucomicrobia and Acidobacteria (Bartscht et al., 1999; Brewer et aI., 1977;
Janssen et al., 2002; Sa it et al., 2002).
(ii). Stevenson et aI., (2004) changed various culturing conditions (oxygen concentration,
nutrient level, addition of humics and signaling molecules) to increase the
Culturability of microorganisms in soil.
(iii). Signaling compounds added to media have also been reported to aid in culturing
aquatic microorganisms (Bruns et aI., 2002; Bruns et aI., 2003).
(iv). Simulating natural environment (for example by placing microorganisms in a
diffusing chamber and incubating the chamber in an aquarium as has been done for
cultivating aquatic bacteria, simulating the organism's natural settings) (Kaeberlein et
al., 2002). Up to 40% inoculated organisms formed micro colonies, but the majority
of these did not grow after passage to Petri dishes. The one that did not continue to
grow on passage appeared to be mixed culture, and author invoked specific signaling
mechanisms to explain this behavior.
(v). Culturing under elevated CO2 concentration and/or limited O2 concentration,
incorporating detoxifying reactive oxygen species in plating media (Stevenson,
2004).
(vi). Use of gellan-gum instead of agar as solidifying agent (Tamaki et aI., 2005)
24
Chapter 1 Introduction & Review o(Literature
Some recently developed innovative culturing techniques are also employed,
(i). Single cell manipulation techniques such as optical tweezers and Laser micro
dissection, also hold promise for targeted isolation of microorganisms (Frohlich &
Konig, 2000)
(ii). Oligonucleotide probes in combination with optical tweezers to track and separate a
novel hyperthermophilic archaeon from an Obsidian Pool community (Huber et ai.,
1995).
(iii). Use of 16S rRNA-directed probes to track the progress of isolation attempts in serial
dilutions without previous knowledge of the physiology of the organisms (Rappe et
al.,2002).
(iv). High throughput methods to grow encapsulated single cells under environmentally
relevant conditions(Zengler et aI., 2002).
(v). Using the MicroDrop micro dispenser system (Bruns et aI., 2003).
(vi). Dilution to extinction, to recover oligotrophic bacteria (Button et aI., 2001) and gave
some important massages-
1. Long incubation times are likely to be required to allow maximum recovery.
2. A large proportion of the isolates were first representatives of novel lineages in the
division Acidobacteria, Actinobacteria, Proteobacteria and Verrucomicrobia.
(vii). Filter acclimatization method which employs a filtration step, which removes most of
the readily cultivable bacteria and than acclimatization procedure to from low to high
substrate concentration of standard microbial media (Hahn et aI., 2004).
(viii). Agar nodule based in situ cultivation system (Koch et aI., 2006).
(ix). Microfluidic strategy for microbe isolation and genome amplification (Marcy et aI.,
2007)
Ammonia oxidizing planctomycete so called "missing lithotroph" was isolated by density
gradient centrifugation following careful electron microscope scrutiny of multiple biofilm (Kuypers
et ai., 2003). Isolation of Sphingomonas aisakensis RB2256 (Vancanneyt et aI., 2001) ,
Cycloclasticus oligotrophus (Button, 1998), Leptospirillum Jerrodiazotrophum , a member of
Leptospirillum group III within the Nitrospira (Tyson et aI., 2005), Acidobacteria and SAR 11
phylum, that was previously described only by environmental 16S rRNA gene sequences, were
possible by using these innovative approaches and also provided unique genetic information about
oral representatives of the uncultured phylum TM7. Use of dilution culture has produced significant
increase in the culturability of planktonic lake bacteria (Bussmann et ai., 2001) and of soil bacteria
25
Chapter I Introduction & Review o(Literature
(Janssen et al., 2002). Although these methods contributed significantly and opened up a way to
study the "as yet" uncultured but due to these requires enough labor, time and sophisticated
fabrication work which is a bottle neck that is why still there is a need to find more practical ways to
tapping into microbial diversity.
1.2.8.4. Opening the diversity black box:
Application of molecular phylogenetic methods in the 1980s (Pace et al., 1986; Stahl et af.,
1984) based on rRNA gene sequences founded the way of accessing diversity by culture
independent approach that brought revolution as prokaryotes could now be studied without culturing
them. It also confirmed that only small proportion of total popUlations was being cultivated as
observed by direct microscopic observations and plate counts. The use of techniques e.g. analysis of
cloned 16S rRNA gene analyses, PCR-based Denaturant Gradient Gel Electrophoresis (DGGE),
Single Strand Conformation Polymorphism (SSCP) have revealed spectacular patterns of diversity
even in previously well studied habitats. As a result, the use of 16S rRNA gene based approaches
found wide acceptance in determining community structure and change in community profiles of
prokaryotes from many environmental habitats. In recent years molecular detection methods have
evolved for more extensive characterization of uncultured natural diversity. Various methods like
fluorescent in situ hybridization, metagenomics, and oligonucleotide microarray enabled
simultaneous detection of different organisms, activity in addition to the presence of organisms, in a
habitat to be determined, and biochemical pathways of uncultured organisms to be reconstructed.
However, in number of instances these methods have yet to be rigorously tested and validated with
environmental samples (Bull et af., 2004).
1.2.8.5 Reservoir of phylogenetic information: rRNA genes:
Since Woese and Fox (1977) first proposed the 16S rRNA gene as a phylogenetic tool to
describe the evolutionary relationships among organisms and Pace et al. (1986) described its use for
classifying unculturables microorganisms in the environment, a huge repertoire of 16S rRNA gene
sequences are now available in GenBank and new ones are being added at a rapid rate (Benson et af.,
2008; and RDP Release 10, Update 5; http://rdp.cme.msu.edulmisc/news. jsp#oct3008). The
development of rRNA based methods for phylogenetic analyses and bacterial identification in
combination with special databases may undoubtly be regarded as one of the milestones in the
history of microbiology. Consequently a comprehensive sequence dataset is available in generally
accessible databases in plain or processed format and number of entries is permanently increasing. A
reasonable fraction of validly published bacterial species is represented by 16S rRNA sequences. The
phylogenetic analysis of these data provides the basis for an ongoing evaluation and restructuring of
the current bacterial systematics accompanied by emendations and renaming of bacterial taxa. It is
also widely accepted to apply the rRNA technology as an integrated part of polyphasic approach for
26
Chapter 1 Introduction & Review o(Literature
description of new species and higher taxa. The rRNA genes are organized in the form of an operon
(Fig. 1.3)
16S rRNA tRNA 23SrRNA 5S rRNA tRNA
Fig. 1.3. Organization of rRNA operon in E. coli.
The copy number of this rRNA operon varies from bacteria to bacteria and starting from one to
as many as 15 has been reported (Acinas et ai., 2004). Although, protein were considered first for
these kinds of studies but later on was proved to be less efficient than to rRNAs. Different
approaches were taken into consideration. One approach in which rRNA cataloguing was done
followed by sequencing and in second approach DNA-rRNA hybridization studies were done for
phylogenetic studies (Woese, 1992) for analyzing natural population of microbes, in which unknown
diversity is anticipated, there are several reasons to focus on the rRNA molecule (Olsen et aI., 1986).
These are as follows:
1. The rRNA, is an integrated component of the protein synthesizing machinery and
functionally and evolutionary homologous in all organisms.
ii. The rRNAs are ancient molecules and are extremely conserved in overall structure.
Thus, the homologous rRNAs are readily identifiable by their sizes.
Ill. Nucleotide sequences are also conserved. Some sequence stretches are conserved
across the primary kingdoms while others vary. The highly conserved regions also
provide convenient hybridization targets for cloning the rRNA genes and for primer
directed sequencing techniques.
IV. The rRNA constitutes a significant component of cellular mass, and they are readily
recovered from all types of organisms for accumulation of a data base of reference
sequences
v. The rRNA provides sufficient sequence information to permit statistically significant
comparIsons.
VI. The rRNA genes seem to lack artifacts of lateral gene transfer between
contemporaneous organisms. Thus relationships between rRNAs reflect evolutionary
relationship of the organisms.
27
Chapter 1 Introduction & Review o(Literature
Of the three generic rRNAs, 5S rRNA, 16SrRNA, and 23S rRNA, the 5S rRNA and 16S
rRNA received most attention. The 5S rRNA, because of its relatively small size, was amenable to
sequence analyses by the late 1960s. However, 5S rRNA is still too small to provide meaningful
phylogenetic inferences.
The 16S rRNA, in contrast, is of reasonable size (~1600 bp) and contains a wealth of useful
phylogenetic information. The 23S rRNA (~3000 bp) is almost twice the size of 16S rRNA (Brosius
et ai., 1980) and is not practical for large scale sequencing.
1.2.8.6. Protein coding genes as molecular marker for phylogeny prediction:
Several characteristics of the 16S rRNA gene, such as it is present in all prokaryotes,
indispensable, mostly conserved but with some variable region have allowed it to become the most
commonly used molecular marker in microbial diversity studies. Inferences on bacterial phylogenetic
relationship based on a single molecule may have limitations. Recently attempts are being made to
use essential housekeeping genes, such as recA, RNA polymerase ~ (rpoB) , pyruvate kinase (pyk),
alanine dehydrogenase (aid), for phylogenetic analysis. Criteria of such selection were, their wide
distribution, they are unique without any paralogues, long enough for sequence information but short
enough to be economical and finally the gene(s) reflect whole genome relatedness (Zeigler, 2003).
Alternatively core house keeping gene, such as the RNA polymerase ~ (rpoB) for differentiating
closely related organisms (Case et ai., 2007), gene encoding pyruvate kinase(pyk) gene encoding
alanine dehydrogenase (aid) to distinguish Bacillus giobisporus and Bacillus psychrophilus (Palys et
ai., 2000), catalytic subunit of proton trans locating ATPase (FIF213- subunit in particular), recA
protein and RNA polymerase (13 and 13' for bacteria and B, B' and B" for archaea) (Ludwig & Klenk,
2001). Recently, as many as 30 genes have been proposed that can be used for the determination of
genome relatedness.
Some other genes that have been used for better taxonomic resolution are as follows:
i. DNA gyrase B subunit (gyrB) (Hatano et ai., 2003; Le Roux et ai., 2005; Yamamoto et ai.,
1999).
ii. DNA fragment coding for sigma factor 70 (Yamamoto & Harayama, 1998).
iii. Gene sequences coding for heat shock proteins (Griffiths & Gupta, 2001).
iv. Genes coding for elongation factor TU and F-ATPase-l3-subunit(Paradis et ai., 2005).
v. Gene encoding translation initiation factor 2 (Hedegaard et aI., 2001).
28
Chapter 1 Introduction & Review o(Literature
1.2.8.7. "Twin track approach"; combining the two approaches:
Culture-independent molecular approaches are tending to replace culture-based methods for
comparing the composition, diversity, and structure of microbial communities. Investigations based
on these approaches have led to the conclusion that traditional methods of culturing natural
populations have seriously underestimated archaeal and bacterial diversity. Culture independent
approach has shown that most of the bacteria in that grow on conventional media, are not the most
abundant in natural habitat. The molecular approaches provide a new perspective on the diversity of
prokaryotes in nature but do not provide the organisms as such in culturable form. This means that
valuable functional traits can, at best, only be inferred from phylogenetic affinities. It is assumed
with certain level of uncertainty that organisms related in phylogeny are also related to the function
in natural habitats. However, phylogenetic coherence need not correlate with physiology (Hahn et
aI., 2004; Jaspers & Overmann, 2004). This means that the pure culture approach is required more
than ever to understand the metabolic diversity of bacteria. Relatively few studies have involved a
"Twin-track" approach whereby both cultivation and direct recovery of bacterial 16S rRNA gene
sequences have been used to gain insight into the microbial diversity of natural bacterial
communities (Dunbar et aI., 1999; Hengstmann et aI., 1999; Schut et aI., 1993). Samples of DNA
extracted from seawater, soil, and cyanobacterial mats of hot springs appear to represent predominant
populations in these ecosystems, while the species that grow on culture plates are numerically
unimportant in intact natural communities. Comparative studies such as these have shown that both
plating and 16S rDNA cloning suffer from biases that can distort community composition, richness,
and structure if applied alone. Two major conclusions were drawn from these studies. (i) for the most
part, direct enrichment techniques select for populations which are more fit under the chosen
enrichment conditions and may not be numerically significant, and (ii) the growth of numerically
dominant populations may be favored by using inoculums diluted to extinction, especially in growth
medium which reflects the conditions in the habitat under study (Bull et ai., 2000). A somewhat
mixed picture emerges from comparative studies of natural microbial ecosystems. The two
approaches some times provide different assessments of relative community diversity, the
discrepancies may be because of sampling different subset of the microbial communities and to
limitations inherent in each of the two approaches for e.g. biases are involved in molecular methods
starting from nucleic acid extraction, PCR, cloning etc (Ellis et aI., 2003; Von Wintzingerode et aI.,
1997). The culturable fraction of bacteria may not be the dominant population (Hugenholtz, 2002)
and thus are less likely to be detected by sequence based molecular phylogenetic approach. In
addition, highlighting consistent relationship between environments based on dual approaches may
be highly habitat dependent- due to limited ability of a single cultural method to survey the full
extant of the bacterial communities and the influence of bacterial physiology in situ on the success of
cultivation in the laboratory.
29
Chapter 1 Introduction & Review o(Literature
It is concluded that both innovative culture dependent and culture independent methods have a
role to play in unraveling the full extent of microbial diversity in natural habitats. Such combinatorial
approaches helps in making strategies about culturing a still uncultured bacteria if the latter is in
close phylogenetic proximity of a well-defined cultured bacterial strain. It is expected that as more
attempts are made in such combinatorial approaches, our knowledge about the diversity will increase
from the point of view of both structural diversity and functional diversity.
1.2.9. Approaches to study prokaryotic diversity:
The major microbial processes of importance to global ecosystem functions and its
sustainability are the result of microbial cell metabolism, growth, death, or enzymatic function of
non-growing cells. Methods that reveal the composition of microbial communities can be applied
over time and space in response to different environmental conditions to understand the linkages
between key populations and processes. Once tools for community analysis are available they can be
applied to almost any question addressing the soil microbial state and function. Methods that are used
to open the microbial "black box" can be grouped into those that measure the members present
(structure) and those that provide some measure of functionality in natural habitat. These methods
can be positioned according to level of taxonomic hierarchy at which they resolve differences. Some
questions are adequately addressed at a coarse level of resolution, while others require a fine scale of
resolution. The coarse scale usually samples the entire community while methods for fine scale
resolution often require analysis of target populations only in order to achieve the fine -scale
resolution. A complete list of bacterial taxonomic methods and their level of taxonomic resolution
have been suggested (Vandamme et aI., 1996). It is now widely accepted that methods used to
analyze prokaryotic diversity of any niche should take into account both culture based as well as
culture independent approaches to get a maximum unbiased idea about the diversity and the
ecosystem function. Techniques used to analyze prokaryotic diversity can be divided into two types.
I. Approaches evaluating community structure
1. Culture dependent approach.
2. Culture independent approach. This includes two kinds of techniques:
a. Biochemical based approaches
b. Molecular based approaches (based mainly on 16Sr RNA gene)
II. approaches evaluating community structure and function.
1.2.9.1. Approaches evaluating community structure:
30
Chapter 1 Introduction & Review o(Literature
1.2.9.1.1. Culture dependent approach:
To gain a comprehensive understanding of microbial physiology or to access metabolic
pathways containing genes dispersed throughout the genome, cultivation of microorganisms is
required. Only 26 out of 53 bacterial phyla contain previously cultivated microorganisms, with many
phyla represented by only a few isolates and some phyla containing only one described species
(Keller & Zenglar, 2004). So far, only five phyla - Actinobacteria, Bacteroidetes, Cyanobacteria,
Firmicutes, and Proteobacteria - include species that produce bioactive molecules and represent
95% of all cultivated and published species. The rest of the phyla with cultivated members (21 phyla)
represent only 5% of all published species. Conventional cultivation of microorganisms, however, is
selective and is biased towards the growth of specific microorganisms (Eilers et ai., 2000; Ferguson
et ai., 1984). The growth of at least 105 cells in a colony on plate-count medium is required for
visualizing colonies by eye, and growth media in common use selects for microorganisms that are
fast-growing, grow to high density, are resistant to high concentrations of nutrients and are able to
grow in isolation. It could be argued that these traditional cultivation strategies use conditions that
are completely different to the natural environment of many microorganisms and are an important
contributing factor to the failure to cultivate most microorganisms in pure culture. New cultivation
methods have been developed to increase the number of culturable bacterial species. Despite the
spectacular advances in the molecular detection and circumscriptions of microorganisms and
functional genomics, organisms in culture are essential for providing an understanding of microbial
interactions, pathogenesis, phenotypic variability, and for biotechnological innovations.
1.2.9.1.1.1. Various strategies of culture dependent approach:
To study prokaryotic diversity choice of media and method of isolation of microbes depend
upon factors like the nature of target organisms intended to be cultured, the environmental niche
from where isolation needs to be made, number of samples. Choice of an appropriate medium is
crucial when culturing bacteria in nature. Some of these techniques are listed below:
1). Plating based methods
Traditionally, diversity was accessed using selective plating and direct viable counts. These
methods are fast, inexpensive and can provide information on the active, heterotrophic
component of the population. In many cases nutritionally poor media like R2A and diluted
standard media like TSBA (Tryptic Soya Agar) and NA (Nutrient Agar) gives higher viable
count than plating the environmental sample on normal standard media. The culturability of
bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at
11100 of its normal concentration (dilute nutrient broth [DNBD as the growth
medium(Janssen et ai., 2002). Suzuki et ai., (1997) compared collection of isolates from
R2A plates with 16S rDNA sequences from clone library of sea water sample. They found
31
Chapter 1 Introduction & Review o(Literature
that most of the cultured bacteria were novel (Suzuki et al., 1997). The success of low
nutrient media at isolating some abundant bacteria has been confirmed in bulk soil from
Australian pasture, nutrient broth diluted 100 times and solidified with gellan gum resulted
in 14% of the total microscopic counts. Plating has also been used to isolate new, abundant
bacteria from some extreme environments. For example, close relatives of the recently
discovered extremely halophilic bacterium, Salinibacterium rubber Bacteroidetes phylum,
make up to 25% of the total prokaryotic community in Spanish saltier ponds (Anton et ai.,
2002). Direct plating on low-nutrient plates has been successful in isolating the most
abundant seawater bacteria in some other cases also. Gonzalez & Moran (1997) found that
up to 40 % of the colonies isolated from coastal water in the US were members of the
abundant Roseobacter clade from the Alphaproteobacteria (16% of all the marine rDNA
clones)(Gonzalez & Moran, 1997; Rappe et ai., 2000). Plating has also been used to isolate
new, abundant bacteria from some extreme environments(Anton et ai., 2002).
2). Enrichment
One way of isolating abundant natural bacteria by enrichment is to examine bacteria
growing in highest-dilution tubes during MPN enumeration experiments. This method has
been applied successfully to grow bacteria revealed as numerically abundant by 16S rDNA
cloning methods. Chin et ai. (1999) used this method to investigate anoxic rice paddy field
soil. Several isolates constituted more than 5% of the total direct count. These isolates
included members of Verrucomicrobia, Bacteroidetes and Gram-positive bacteria (Chin et
al., 1999). A similar approach has been used to culture lake water bacteria (Bartscht et ai.,
1999) in which a new synthetic medium was used to mimic natural lake water. When
compared with direct microscopy, MPN counts gave up to 7% culturability. This approach
was extended by addition of 10J.lg cAMP to the MPN tubes using artificial brackish
seawater. This gave culturability averaging about 15% (range (2-100%). From Baltic sea
(Bruns et al., 2002). Enrichment techniques have been particularly successful for isolation
of naturally abundant thermophiles in the order Aquificales from a hot-subsurface aquifer
from a gold mine Takai et ai., (2002) tested 900 enrichment media and showed that three of
them were successful for isolation of the target organisms.
3). Micromanipulation
Micromanipulation can also be a valuable aid form isolating bacteria, especially when
fluorescent in situ hybridization (FISH) with phylogenetic probes is used to visualize the
target bacteria. Huber et ai., (1995) used a strongly focused infrared laser to separate a new
hyperthermophilic archaea from a hat pool in Yellowstone National Park. After separation
by micromanipulation, the aggregates were successfully grown in pure culture (Huber et
32
Chapter 1 Introduction & Review of Literature
aI., 1995). Morphology without FISH probes is sometimes enough to target isolation.
Kaempfer, (1997) has isolated morphologically distinct filamentous bacteria, common in
activated sludge wastewater treatment systems(Kaempfer, 1997).
4). Extinction culture
Button et al. (1993) described this method (also called as dilution culture/dilution to
extinction) in which the total bacteria present in the sample is calculated microscopically
and then diluted with filter-sterilized water until only a few bacteria remain and then
growing the cells in either the unamended water or 'by adding small amounts of organic
substrates to culture them. In the original trials of the method with seawater from
Resurrections Bay, Gulf of Alaska, it was observed that almost all the marine bacteria
retained their viability (about 60 % of the direct counts)(Button et aI., 1993). Schut et al.
(1993) isolated 37 strains from Resurrection Bay and the North Sea using this approach.
Both seawater salts agar and Zobell 2214E agar gave counts that were 80 % of the total
counts. Seven of these isolates have been assigned to the new species Sphingomonas
alaskensis with isolate RB2256 as type strain. The SARI clade of Alphaproteobacteria is
one of the most abundant bacterial groups in the oceans, accounting for 26% of all rRNA
sequences isolated from sea water (Geovannoni & Rappe, 2002). Rappe et al. (2002)
reported the isolation of 11 cultures of SAR-l1 clade using extinction culture. These are
one of the smallest bacteria in culture and have been named "Candidatus Pelagibacter
ubique"(Rappe et aI., 2002).
1.2.9.1.2. Culture independent study of prokaryotic diversity:
Many culture independent approaches in which molecular biology has found their
application were developed to characterize microbial communities that are able to generate a
fingerprint of diversity as well as methods that use the conventional techniques can be used to
decipher microbial diversity such as community level physiological profiling (CLPP) and
Phospholipid Fatty Acids profiling (PFLA). The advantages of applying both of these approaches do
not require culturing the microbes thus eliminating the bias associated culture dependent method.
The application of these tools by microbial ecologists has rapidly enhanced our knowledge of
prokaryote abundance, diversity and their function in their habitat. Each of these methods measures a
different aspect of the community (diversity, in situ detection, and community dynamics). Molecular
detection methods involve (i) direct lysis of bacterial cell and (ii) The analysis the extraction of the
nucleic acids from the matrix and (iii) the analysis of targeted sequences or the whole body of
genetic information. Two main types of molecular technique are available to study bacterial
communities using DNA directly extracted from natural environments (Ranjard et aI., 2000).
33
(
Chapter 1 Introduction & Review o(Literature
1. Molecular approaches which usually investigate parts of this information by focusing on
genome sequences which are targeted and amplified by PCR that are called "partial
community DNA analysis".
2. Molecular approaches which try to investigate all the genetic information in the extracted
DNA and are called "whole community DNA analysis". Each of these methods are
described below-
1.2.9.1.2.1. Biochemical based approaches:
1). Community level physiological profiling (CLPP)
This technique was developed by Garland & Mills (1991) for a rapid and community level
physiological profiling (CLPP). This technique is widely used to characterize microbial
communities (Garland & Mills, 1991; Lehman et al., 1995). The response or CLPP
involves (1) the overall rate of color development by tetrazolium dye which gets reduced
when a substrate gets oxidized producing a visible color development. (2) The richness and
evenness of the response among well (or diversity), and the pattern, or relative rate of
substrate oxidation among well. The major strength of this approach are its (1) low man
power requirements, which enables intensive sampling across temporal and special scales
and (2) reliance on metabolic traits that could lead to functionally relevant characterization
of change in microbial communities (Garland, 1997). The technique uses a commercially
available 96-well microtitre plate to assess the potential functional diversity of the bacterial
population through sole carbon source utilization (SSCU) patterns. Gram-negative (GN)
and gram-positive (GP) plates are available from Biolog (Hayward CA, USA,
www.biolog.com) and each contains 95 different carbon sources and one control well
without a substrate. Subsequently, Biolog introduced an Eco-plate containing 3 replicates
of 31 different environmentally relevant carbon sources and one control well per replicate
(Choi & Dobbs, 1999). Inoculated popUlations are monitored over time for their ability to
utilize substrates and the speed at which these substrates are utilized. Multivariate analysis
is applied to the data and relative differences between soil functional diversity can be
assessed. This method has been used in arctic soils (Derry et al., 1999), soil treated with
herbicides (EIFantroussi et al., 1999) or inoculation of microorganisms (Bej et al., 1991).
The method has also been used successfully to assess potential metabolic diversity of
microbial communities in contaminated sites(Derry et al., 1998; Konopka et al., 1998),
plant rhizospheres (Ellis et aI., 1995) (Garrity, 1996; Grayston & Campbell, 1996), Similar
in principal to the Biolog system is the API system (Merieux, France). There are a number
of API strips available with various carbon sources that can be used to measure functional
diversity (Torsvik et al., 1990b).
34
Chapter 1 Introduction & Review o(Literature
2). Fatty acid methyl ester (FAME) analysis
A biochemical method that does not rely on culturing of microorganisms is fatty acid
methyl ester (FAME analysis). This method provides information on the microbial
community composition based on the groupings of fatty acids(Ibekwe & Kennedy, 1998;
Zelles, 1999). Fatty acids make up a relatively constant proportion of the cell biomass.
Signature fatty acids that are integral part of cell membrane can differentiate major
taxonomic groups within a community. It has been used to study microbial community
composition and population changes due to chemical contaminants (Kelly et ai., 1999;
Siciliano et ai., 2003) and agricultural practices (Bossio et ai., 1998; Ibekwe & Kennedy,
1998). Therefore, a change in fatty acid profile would represent a change in the microbial
community. For FAME analysis, fatty acids are extracted directly from soil, methylated
and analyzed by gas chromatography (Ibekwe & Kennedy, 1998). FAME profiles of
different soils can be compared using multivariate analysis. Ibekwe & Kennedy, (1998)
used phospholipids fatty acid analysis (PLFA) and CLPP to study microbial communities
in the rhizosphere of plants from the field and from green house pots and was able to
demonstrate a clear difference between microbial communities of these ecosystems.
Cellular fatty acid composition can be influenced by factors such as temperature and
nutrition(Graham et ai., 1995).
1.2.9.1.2.2. Molecular approaches:
1). Nucleic acid reassociation
DNA reassociation based on the principle that two strands of DNA which have a minimum
level of similarity will reanneal appropriate conditions are provided. It gives a measure of
genomic complexity (types of DNA molecules) of the microbial community and has been
used to estimate diversity (Torsvik et ai., 1990a; Torsvik et aI., 1996). In this method total
DNA is extracted from environmental samples, purified, denatured and allowed to
reanneal. The rate of reassociation will depend on the similarity and concentration of
sequences present. As the complexity or diversity of DNA sequences increases, the rate at
which DNA reassociates will decrease (Theron & Cloete, 2000). Time taken needed for
half of the DNA to reassociate (the half association value Cot1/2, where Co is the molar
concentration of nucleotides in single stranded DNA at the beginning of the reassociation,
and tl/2 the time in seconds for 50 % reassociation) can be used as a diversity index. It
predicts both the amount and distribution of DNA (Torsvik et ai., 1998).
2). Guanine plus cytosine (G+C) content
35
Chapter 1 Introduction & Review o(Literature
Analysis of the guanine plus cytocine (G+C) content of DNA is useful when a coarse level
of resolution is meaningful. This technique is utilizes the variation of amount of nucleotide
residues. prokaryotic DNA varies in G+C content from 24%-76% G+C versus A+T, and
that particular taxonomic groups only include organisms that vary in G+C content by no
more than 3-5% (Goodfellow & O'donnell, 1993); Vandamme et al., 1996). Hence G+C
can be related to taxonomy and can be used to detect changes in community structure
(Nusslein & Tiedje, 1999). It requires an ultracentrifuge to separate the G+C fraction. Base
composition separation is based on the principle that bisbenzimidazole binds to adenine
and thymine which altogether changes the buoyant density of DNA in proportion to its T
(hence G+ C) content. A gradient of DNA fragments of different G+C contents is then
established using CsCI density gradient centrifugation. The different fractions are then
collected using a fraction collector. The DNA in each fraction is quantified by
spectrophotometry and its G+C content is established by using a standard curve. G+C
content together with ARDRA and rDNA sequence analysis was used by Nusslein and
Teidje (1999) to study the differences in microbial diversity between a vegetative cover of
forest and a pasture in Hawaiian soil and concluded that all the three methods worked well
to detected the changes in microbial community revealing that plants have a strong
influence on microbial community composition.
3). Nucleic acid hybridization and Fluorescent In Situ Hybridization (FISH)
In the field of molecular ecology hybridization techniques has been proved to be one ofthe
most important qualitative and quantitative tool in molecular bacterial ecology (Clegg et
aI., 2000; Griffiths et aI., 1999; Guo et aI., 1997; Schramm et aI., 1996; Theron & Cloete,
2000). This technique takes in account the use of specific probes that binds to Nucleic acid
at a very specific region. Various studies it has been shown that DNA as well as RNA can
be targeted by using oligonucleotide or polynucleotide probes that has been designed from
known sequences and ranging in specificity from domain to species. These probes are
generally labeled with markers at the 5' -end (Theron & Cloete, 2000). Among many
fluorescent markers commonly used, derivatives of fluorescein and rhodamine have been
used extensively and have become most popular. To measure spatial distribution and
relative abundance of certain groups of microorganisms in environmental samples,
quantitative dot-blot hybridization in which the sample is lysed to release all nucleic acids
than ribosomal DNA sequences of interest are quantified relative to total rDNA by dot-blot
hybridization with specific and universal oligonucleotide probes. At cellular levels this
method can be applied in situ in which samples are fixed cells are permeabilized and
fluorescently labeled probes are added to the sample after addition of the probe it is
allowed to hybridize. After incubation is over the cells are visualized (Head et aI., 1998). 36
Chapter 1 Introduction & Review o(Literature
Schramm and coworkers applied this method to study spatial distribution of bacteria in
biofilms (Schramm et aI., 1996).
4). CARD-FISH
Combining FISH with catalyzed reporter deposition (CARD-FISH) has been demonstrated
to substantially enhance bacterial cell detection in situ (Schonhuber et aI., 1999). CARD
FISH has recently been applied for the identification of pelagic marine Bacteria (Pernthaler
et aI., 2002), Cyanobacteria (Schonhuber et al., 1999), Actinobacteria (Sekar et aI., 2003),
and sedimentary, marine Archaea and Bacteria (Ishii et aI., 2004), with all studies reporting
superior cell detection in comparison to conventional FISH. Tujula et al., (2006)
demonstrated the first application of CARD-FISH to study the bacterial community on the
surfaces of marine macroalgae Ulva lactuCG, Delisea pulchra, Corallina officinalis,
Amphiroa anceps, Porphyra sp. and Sargassum linearifolium (Tujula et aI., 2006). CARD
FISH involves a number of pre-treatment steps including embedding, permeabilisation and
inactivation of endogenous peroxidase followed by hybridizations with HRP probes
(Thermo Electron, Germany) targeting Bacteria (EUB338 i-iii) (Daims et aI., 1999» and
then visualized Cells were visualized with a Leica TCS-SP confocal laser scanning
microscope (CLSM).
5). PCR-based approaches
The 16S rONA has been targeted extensively to study prokaryote diversity. It allows
identification of prokaryotes and predicts phylogenetic relationships as well. Although this
technique mainly involves cloning of target genes and their sequencing of the clones, they
may sometimes becomes thousands in number to reach any conclusion making this
technique time consuming as well as expensive (Muyzer & Small a, 1998; Tiedje et al.,
1999). Due to this reason many other techniques discussed below have been developed to
assess prokaryotic diversity of natural environments.
i). Denaturing gradient gel electrophoresis DGGE)ffemperature gradient gel
electrophoresis (TGGE)
PCR -amplified 16S rRNA gene were analyzed by applying DGGE. Primers
annealing to conserved region of the rONA were used to amplify the variable V3
region flanked by primers. Different base composition in this variable region of
rDNA from the community, gave PCR products with different melting points.
The PCR products were separated according to their melting point in
polyacrylamide gels with 15-55% denaturing gradient (100% denaturant
comprised 7M urea and 40% formamide). The gels were run at 60 C. by applying
group specific probes the affiliation of the DGGE bands to main phylogenetic 37
Chapter 1 Introduction & Review o(Literature
subclasses of bacteria, was determined .furthermore, this approach allowed
identification of bands from the putative numerically dominant bacteria. The
strongest band were punched out from the gel, reanalyzed by PCR-DGGE to
ensure that they are consisted of a single sequence, and subjected to sequencing
the sequence were aligned to those obtained from various databases such as
NCBI by BLAST program in order to assign them to bacterial subclass and
probe designing. TGGE uses the same principle as DGGE except that here the
denaturing conditions are maintained using a temperature gradient. Advantages
of this technique are that it is reliable, reproducible, and rapid, mUltiple samples
can be compared simultaneously for their fingerprints and it is possible to
monitor changes in microbial populations over a spatial and temporal gradient.
Limitations include PCR biases, laborious sample handling, and variable DNA
extraction efficiency. Moreover, DNA fragments of different sequences can have
same mobilities and a single species can give mUltiple bands due to heterogeneity
in its 16S rRNA gene. Holben et al. (2004) used DGGE to assess community
diversity and to detect minority populations of bacteria in the digestive tracts of
chickens. Some researcher's have also used DGGE analyses to target catabolic
genes such as methane monooxygenase (Fjeilbirkeland et al., 2001; Knief et al.,
2003). This provides information regarding specific group of microbes.
ii). Single strand conformation polymorphism (SSCP)
Another technique that relies on electrophoretic separation based on differences
in DNA sequences is single stranded conformation polymorphism (SSCP) was
also developed to detect known or novel polymorphism or point mutations in
DNA (Orita et aI., 1989). On a polyacrylamide gel, differences in mobilities in
single stranded DNA of same sizes are caused by their folded secondary structure
(Lee et aI., 1996) resulting in a pattern that reflects number of phylotypes present
in the sample. Folding and therefore mobility of DNA fragments will be
dependent on the DNA sequences when DNA fragments are of equal size and no
denaturant is present. SSCP has all the limitations of DGGE. Also, some single
stranded DNA can form more than one stable conformation. Therefore, one
sequence may be represented by more than one band on gel. It does not require a
GC clamp or the construction of gradient gels and has been used to study
bacterial and fungal community diversity (Peters et al., 1999; Stach et aI., 2001).
This method has been also been applied to study succession of bacterial
communities (Peters et aI., 2000), rhizosphere communities (Schweiger and
Tebbe, 1998), anaerobic bioreactor (Zumstein et aI., 2000). 38
Chapter 1 Introduction & Review o(Literature
iii). Restriction fragment length polymorphism (RFLP)/Amplified ribosomal DNA restriction analysis (ARDRA)
The method is based on DNA polymorphism and involves digestion of genomic
DNA/ PCR amplified 16S rRNA gene sequences using restriction enzymes
(usually a four-cutter). Fragments are then separated on agarose or
polyacrylamide to generate a profile of microbial community. This method has
been used most frequently to screen clones (Pace, 1996) or to measure bacterial
community structure (Massol-Deya et ai., 1995; Smit et ai., 1997). Although this
method cannot be used for quantifying diversity or following specific ribotypes
but it has found its application for detecting structural changes in the microbial
community. A single species can give many restriction fragments thus sometimes
complicating study in complex communities when analysed by RFLP.
iv). Terminal restriction fragment length polymorphism (T -RFLP)
With the help of this method a community profile is generated for complex
communities as well as it provides information on diversity as each visible band
in t-RFLP represents a single operational taxonomic unit or ribotypes (Tiedje et
ai., 1999). In this method amplified product is digested with appropriate
restriction enzyme as done in ARDRA but the PCR primer is labeled with a
fluorescent dye, such as CY3 or 6-FAM (phosphoramidite fluorochrome 5-
carboxyfluorescein). Because this allows detection of only the labeled terminal
restriction fragment, it simplifies the banding pattern (Liu et ai., 1997). By doing
a profile to profile comparison a large number of bacterial species can be
identified in a profile as a data bank has been produced on the sizes of the 16S
rDNA terminal restriction fragment for a given enzyme on a large set of bacterial
species. This procedure has now been automated by use of automated sequencers
to allow sampling and analysis of large number of samples (Osborn et ai., 2000).
By using sequencing systems for T-RFLP (for separation of T-RFLP fragments)
resolution and sensitivity has been remarkably improved. Also interpretation of
peak height and area can be made, which can be further interpreted in terms of
number, and abundance of OTU's. Functional genes such as the mercury resistant
determinants (mer genes) have been studied by T-RFLP.
v). Ribosomal intergenic spacer analysis (RISA)/Automated ribosomal intergenic spacer analysis (ARISA)
The region between 16S rRNA and 23S rRNA is termed as intergenic spacer
region (lOS) and has been targeted to interpret microbial diversity in soil and
rhizosphere of plants (Borneman & Triplett, 1997), in contaminated soil (Ranjard
39
Chapter I Introduction & Review o(Literature
et al., 2000) and in response to inoculation (Yu & Mohn, 2001). The method
involves the analysis of the length polymorphism of the intergenic spacer region
(IGS) between the small (16S) and large (23S) rRNA genes whose size varies
from 50 bp to more than 1.5 kb depending on the species. The primers target
regions within the adjacent genes and can be defined so that part of the 16S
rRNA gene is co-amplified and directly separated on polyacrylamide gels on the
basis of size. Further sequencing of the 16S rRNA gene can provide a finer
taxonomic identification of the bands. In RISA the sequence polymorphisms are
detected using silver stain while in ARISA the forward primer is fluorescently
labeled and is automatically detected (Fisher & Triplett, 1999). ARISA increases
the sensitivity and reduces time.
vi). 16S rRNA library construction and sequencing
In this approach target genes are amplified from community DNA and then
cloning is done to separate sequences. These sequences can be characterized
individually using ARDRA and/or by sequencing. Sequencing allows a fine
identification of uncultured bacteria as well as an estimation of their relatedness
to known culturable species. There are some limitations and drawbacks of the
PeR-cloning approach as there exists bias due to the peR (i.e. choice of primers,
annealing temperature, inhibition of the enzyme by humic substances, formation
of chimeric peR products), in addition it is also time-consuming and
cumbersome since it is necessary to sample a large number of clones in order to
obtain a good diversity estimation of the amplified sequences, requires expensive
equipment (sequencer) and the cloning strategy used. Moreover, the substantial
richness found within the soil bacterial community and the number of clones per
library (usually about 100 clones) precludes the application of diversity
measurements in terms of species evenness. However, such a sampling size can
be informative for estimating diversity, both richness and evenness, by
considering higher level phylogenetic groups (i.e., subdivisions within
Proteobacteria, Firmicutes, Actinobacteria etc.). This approach has been used to
decipher prokaryotic diversity of soil, marine, freshwater and subsurface
environments.
1.2.9.2. Approaches evaluating community structure with function:
1) Microautoradiography-FISH (FISH -MAR)
40
Chapter 1 Introduction & Review o(Literature
This technique has been designed to detect metabolically active microbes. A metabolically
active microbe takes up specific radiolabelled substrates and can be further detected by
using pre-designed; specific 16S rRNA targeted FISH probes. Environmental samples are
first incubated for a short-term incubation with specific radio labelled substrates like 3H_
acetate, 14C-pyruvate, 14C-butyrate then thin sections of these samples are fixed on to the
glass slides and subsequent analysis by FISH and inverse confocal laser scanning
microscopy is done. This technique allows the detection of bacteria that are biologically
active in taking up a specific radiolabelled substrate. Daims and coworkers (2001) analyzed
nitrifiers and denitrifiers complex microbial communities using this technique.
2) Radioisotope array
In this method, an environmental sample is incubated with a 14C-Iabelled substrate first,
than environmental sample is subjected to RNA extraction followed by labeling with a
fluorophore further analysis with an oligonucleotide array containing DNA probe
sequences specific for the 16S rRNA genes of the bacteria of interest from the sample.
Community members that have incorporated the 14C isotope into their RNA are determined
by scanning for fluorescence and incorporation of the radioactive isotope. By using this
technique incorporation of 14C-bicarbonate by autotrophic ammonia oxidizers in activated
sludge was determined (Adamczyk et ai., 2003).
3) Stable isotope probing (SIP)
Radajewski et aI., (2000) used this technique for studying methylotrophs in a forest soil
and discovered that Acidobacterium and Beijerinckia species previously not known to be
methylotrophic were involved in the process. This technique involves analysis of the
labelled biomarker upon exposure of environmental sample to a stable -isotope (such as
i3C and 15N) enriched substrate for which utilization and assimilation has to be determined
for specific group of bacteria. In DNA-SIP identity of microorganism is linked to its
function by analyzing DNA which gets labeled upon exposure to -isotope enriched
substrate. Methodology of this technique is as follows:
a). Sample is incubated with labeled substrate ( i3CH30H or J3CH4),
b). DNA is isolated from the sample and subjected to CsCI density gradient
centrifugation. Separated heavier DNA (labelled) from lighter DNA (unlabeled)
represents the combined genomes of those bacteria, which are actively metabolizing
the labeled substrate.
c). The labeled DNA can then used as a template in PCR with primers targeting the l6S
rRNA genes or any other functional gene.
41
Chapter 1 Introduction & Review o(Literature
4) DNA microarrays
More recently DNA-DNA hybridization has been used together with DNA microarrays to
depict the microbial diversity. In DNA microarray denatured sequence fragments (single
genes or whole genomes) are attached to a solid support (in the form of an array, known as
a chip). Further steps involve random labeling of the total community DNA, hybridization
to the array and then detection and analysis of the individual dot hybridization data to
interpret the diversity of the sample. The microarray can contain specific target genes such
as nitrate reductase, nitrogenase or naphthalene dioxygenase to provide functional diversity
information or can contain a sample of environmental standards (DNA samples with less
than 70 % hybridization) representing different species found in the environmental sample
(Greene & Voordouw, 2003). The latter approach called as Reverse Sample Genome
Probing (RSGP) is used to analyze the most dominant culturable species. An internal
standard should be included both in the labelled probe and in the spotted array. This
method has been used to analyze microbial communities in oil fields (Voordouw et al.,
1991, 1992, 1993) and in contaminated soils (Shen et al., 1998; Hubert et al., 1999; Greene
et al., 2000). It is a useful technique when diversity is low; this method has got advantages
over PCR based diversity studies when diversity is comparatively low. The only limitation
associated to this technique is the problem of cross hybridization and possible detection of
only most abundant species (Greene & Voordouw, 2003).
5) Metagenomics approach
New tools like 'Metagenomics' (Environmental genomics, Community genomics) are
accessing microbial diversity to provide novel genes and biosynthetic pathways of those
bacteria, which are uncultured till date. Metagenomics is the culture-independent genomic
analysis of microbial communities. Each organism in an environment has a unique set of
genes in its genome; the combined genomes of all the community members make up the
"metagenome". Metagenome technology (metagenomics) has led to the accumulation of
DNA sequences and these sequences are exploited for novel biotechnological applications
(Ferrer et aI., 2005). Due to the overwhelming majority of non-culturable microbes in soil,
metagenome searches will always result in identification of hitherto unknown genes and
proteins. Thus, the probability of uncovering hitherto unknown sequence makes this
approach more favorable than searches in already cultured microbes. The term is derived
from the statistical concept of meta-analysis (the process of statistically combining separate
analyses) and genomics (the comprehensive analysis of an organism's genetic material)
(Rondon et aI., 2000). The method involves-
42
Chapter 1
I).
Introduction & Review o(Literature
Isolation of high quality environmental or total community DNA. The DNA isolation
methods can be divided into two categories: Direct method that involves lyses of cells
contained in the sample matrix followed by separation of the DNA from the matrix
and cell debris and indirect method wherein cells are first separated from the soil
matrix followed by cell lyses. The crude DNA recovered by both the methods is
purified by standard procedures.
II). Restriction digestion of the extracted DNA is done to cut DNA into desired size by
using either restriction endonucleases or by mechanical means such as nebulization,
French press and air nozzles. In a classical approach, small inserts libraries «lOKb)
were constructed in a standard vector [pBluescript SK (1)] and transformed in
Escherichia coli DH5a as a host strain (Henne et ai., 1999). However, small insert
libraries do not allow detection of large gene clusters or operons. To circumvent this
limitation researchers have been employing large insert libraries, such as cosmid
DNA libraries (mostly in the pWE15 vector) with insert sizes ranging from 25-35kb
(Entcheva et ai., 2001) and bacterial artificial chromosome (BAC) libraries with insert
sizes up to almost 200 kb (Beja et ai., 2000; Rondon et ai., 2000). Construction of
fosmid libraries with inserts of 40 kb of foreign DNA has been reported (Beja et ai.,
2002). Genes for antibiotic synthesis have been cloned successfully from soil in the
BAC vector, pBeloBAC 11, and the cosmid superCos 1 (Brady et ai., 2001; MacNeil et
ai., 2001; Gillespie et ai., 2002). The host for the initial construction and maintenance
of almost all the published libraries is E. coli (Daniel, 2005). Shuttle cosmids or BAC
vectors can be used to transfer libraries that are produced in E. coli to other hosts such
as Streptomyces or Pseudomonas species. The choice of the vector system depends on
the quality of isolated DNA, the desired average insert size of the library, the copy
number required, the host and the screening strategy that will be used, all of which
depend on the aim of the study. Small-insert libraries are useful for isolation of single
genes or small operons encoding new metabolic functions. Large-insert libraries are
more appropriate to recover complex pathways that are encoded by large gene
clusters or large DNA fragments for the characterization of genomes of uncultured
microorganisms.
Ill). The final step IS the screening of metagenomic libraries. For screening of
metagenomic libraries three methods are normally employed-
1. Functional screening: based either on metabolic activity (function-driven
approach). Interestingly, searches in metagenome-derived DNA libraries have
mainly focused on a rather small group of enzymes. Among these are lipases
and esterases (Voget et ai., 2003; Henne et ai., 2000). They usually display
43
Chapter 1 Introduction & Review o(Literature
exquisite chemo-, regio-, and stereoslectivities and they do not require
cofactors (Jaeger et aI., 1999). Oxidoreductases are another example of useful
catalysts with a high enantioselectivity that have been identified in
metagenome searches (Knietsch et aI., 2003). Of particular interest,
nicotinamide-dependent alcohol reductases are employed for the preparation of
deuterium or tritium labeled compounds, production of dihydroxyacetone and
as tools for enzymatic analysis of serum lipids (Hummel, 1999). Similarly,
polysaccharide-modifying enzymes such as the starch modifying enzymes are
of considerable interest to industry. Therefore, a significant number of
metagenome searches have identified polysaccharide-modifying genes (Healy
et aI., 1995; Richardson et ai., 2002). Further, the isolation of enzymes useful
for the production of bulk chemicals (Knietsch et aI., 2003), proteases (Santosa,
2001) and nitrilases (DeSantis et aI., 2002) has been reported. Metagenome
searches have also focused on the isolation of genes involved in vitamin
biosynthesis. Interest in 2,5-diketo-D-gluconic acid synthesis is related to a
new biotechnological process for the production of vitamin C (ascorbic acid)
using glucose as a substrate (Eschenfeldt et aI., 2001); and interest in biotin
biosynthesis genes is linked to the construction of biotin overproducing
bacteria for large-scale fermentation of this vitamin (Streit et aI., 2003).
Finally, the isolation of genes encoding for novel therapeutic molecules is a
very valuable area of research (Gillespie et aI., 2002).
II. Sequence based approach: Sequence analysis of large insert libraries with
environmental DNA combined with genetic and functional analysis has the
potential to provide significant insight into the genomic potential and
ecological roles of cultured and uncultured microbes. The importance of this
potential for understanding complex environments can be estimated by the
following examples.
a. Recent analysis of genome fragments recovered directly from marine
bacterioplankton suggested the presence of a new bacterial rhodopsin,
proteorhodopsin. Adaptation of proteorhodopsin proteins to different
habitats combined with the genetic and biophysical data indicate that
proteorhodopsin-based bacterial phototrophy is a globally significant
oceanic microbial process (Beja et ai, 2000).
b. Culture-independent partial or nearly complete recovery of microbial
genomes from an environmental sample by an extended random
shotgun-sequencing approach offers a highly intriguing approach to
44
Chapter 1 Introduction & Review o(Liferafure
study natural microbial communities. A recent example gave significant
insights into the community structure and the metabolism of a natural
acidophilic biofilm growing on the surface of a flowing acid mine
drainage. This was mainly possible through reconstruction of the
microbial genomes present in this niche. For this purpose the near
complete genomes of Leptospirillum group II and Ferropiasma type II
were reconstructed.
c. Analysis of the complex metagenome of model biofilms growing on
rubber-coated valves within drinking water networks using the
cultivation independent approach. This analysis revealed significant
insights into phylogenetic, catabolic and metabolic abilities of the
analyzed microbial community. Large-scale sequencing projects such as
the one initiated by Craig Venter for the metagenome of the Sargasso
Sea (Venter et ai., 2004) resulted not only in the identification of
numerous novel genes but also 180 novel bacterial species. This is a very
significant achievement of sequence based metagenome analyses.
Similar approaches have been initiated by European laboratories to
sequence complete or partial metagenomes of a phylogenetically highly
diverse biofilm (Schmeisser et ai., 2003). Complementary to this, Tyson
et ai., (2004) described the nearly complete sequence analyses of the
metagenome of an acidophilic biofilm.
d. Analysis of metagenomic DNA has been proposed as a strategy for
evaluating numbers of soil microorganisms (Miller et ai., 1999).
Aoshima et ai. (2006) reported a slow-stirring method for the isolation of
metagenomic DNA from various kinds of soil with minimal shearing.
They have obtained a linear proportional relationship between soil
bacterial biomass and the amount of DNA isolated by this method.
Therefore, the bacterial biomass could be evaluated by quantifying levels
of environmental DNA. However, co extraction of extracellular DNA
should be considered, which may lead to the overestimation of number
of living bacteria.
e. Deutschbauer et ai., 2006 have demonstrated tracking of recombinant
microorganisms by using metagenomic approach. The possibility of
genetically engineered microorganisms to be used in environmental
applications requires an understanding of the fate of recombinant DNA
introduced into the environment. To study the fate of a geneticallY
45
Chapter 1 Introduction & Review o(Literature
engineered orgamsm introduced into a soil environment, direct
extraction of DNA followed by a specific quantitative detection based on
PCR or hybridization are used to determine the persistence of the
recombinant gene.
f. An exciting extension of the metagenomics is the high-throughput
analysis of the "mobilome" or mobile metagenome, the genomics of
mobile elements from uncultured organisms (au et at., 2007). Genes
present on mobile genetic elements (MGE) that populate soil ecosystems
constitutes the mobilome of the environment. MGE include plasmids,
transposons, insertion sequences and integrons, which may move
between bacterial cells in a population or mobilize into a new host
species and introd~ce new genetic material (Jones & Marchesi, 2007).
g. As described earlier in section 1.2.5, Venter and his colleagues reported
the results of a Global Ocean sampling expedition (Rusch et at., 2007),
an environmental metagenomics project that aimed to shed light on the
role of marine microbes by sequencing their DNA without the need to
cultivate them. The analysis has been the biggest application of
metagenomics in lieu of the number of samples collected and analyzed.
A total of 41 different surface water samples (mostly marine) were
collected over a distance of 8000 km from the North Atlantic through the
Panama Canal and ended in South Pacific. Total DNA was extracted
from each sample and libraries were constructed and analysed by
shotgun sequencing approach. An extensive dataset consisting of 7.7
million reads (6.3 billion bp) of sequence data that amounted to a total of
~ 5.9 Gbp of nonredundant sequence was obtained. A total of 4, 125
distinct full length or partial 16S rRNA genes were identified. Out of
these 811 were identified as distinct ribotypes and more than half of the
ribotypes were found to be novel « 97 % similarity). They found that
relatives of SAR 11 and SAR 86 appear to be ubiquitously abundant in
all the samples.
1.2.9.3. Other molecular tools for taxonomy that require cultivation:
(i). rep-peR
Probably the most popular fine scale method to resolve differences in populations between
different sites is the use ofrep-PCR (repetitive extragenic palindromic-PCR) (Versalovik et
aI., 1994). Many organisms, both prokaryotic and eukaryotic, contain highly repetitive
46
Chapter 1 Introduction & Review of Literature
short DNA sequences that are 1-10 base pair long repeated throughout their genomes (Zeze
et al., 1996;Longato & Bonfante, 1997) depending upon the rate of evolution, these
sequences may be diagnostic and allow differentiation down to the species or strain level
and has been used for the identification of bacteria since it provides a genomic fingerprints
of chromosome structure, and chromosomal structure is considered to be variable between
strains (Tiedje, 1999). These highly repetitive sequences are also referred as microsatellite
regions. However this method requires an isolate. It is often used as the first level screen to
indicate how closely related the isolates are. This method can be used directly on small
amounts of cells, e.g. a colony, without prior DNA extraction, thus making it possible to
analyze 60-100 strains overnight. The method is rapid, reproducible, the data were suitable
for storage in searchable database, and provides the highest level of taxonomic resolution
of any current peR based method. There are three primer sets that have been found to work
in most bacteria; these are known as REP, BOX and ERIC. It has been successfully used to
differentiate organisms like Rhizobium (Vinuesa, et al., 1998), plant pathogenic
Xanthomonas (Louws et al., 1994). Teidje et aI., (1995) evaluated the degree of endemism
in soil populations.
(ii). Rihotyping
More accurate method for genotype determination is that of the molecular biological
approach of ribotyping by comparing similarities in the rRNA gene sequences. A more
recent ribotyping technique is the patented method called riboprinting. The Qual icon
(DuPont) RiboPrinter is an automated system that takes a purified bacterial suspension,
lyses the cells, extracts the DNA, restriction endonuclease digests the DNA, separates the
digest on a gel, transfers the DNA bands to a membrane, probes the bands with non
radioisotope-labelled, 5S-16S-23S rRNA-specific probes (Southern hybridization),
photographs the membrane, and finally compares the bar code-like pattern to databases in
order to identify the genus and species(Bruce et aI., 1995). The entire process takes
approximately 8 h and requires only a small amount of growth sample from a Petri dish.
Although this method and instrument were originally developed to identify the food-borne
pathogens, Listeria, Salmonella, and Staphylococcus spp. and E. coli, it has since been used
for many applications, including the identification of spoilage bacteria in the brewing
industry (Funuahashi et aI., 1998; Leisner et al., 1999). The manufacturer has demonstrated
with food-borne pathogens that the method is close to 100% accurate at identifying genus
and species and often has the ability to differentiate at the subspecies level. This, in tum,
has utility in epidemiological studies for tracking isolates. Based on a historical database of
isolates encountered in manufacturing facilities, riboprinting may also be useful for
predicting the pathogenicity, spoilage capability, or other phenotypic expressions of the
47
Chapter 1 Introduction & Review o(Literature
organisms (Barney et ai., 2001) 16S ribosomal DNA (rDNA) sequencing of a number of
isolates showed that Pediococus damnosus isolates with distinctively different RiboPrinter
patterns had identical sequences, except for 1 bp in one strain. The sequence analysis
method was very good at identifying the organisms by genus and species but did not
differentiate at the subspecies level. The riboprinting method, on the other hand, gave the
correct genus and species and also allowed the sub speciation of many strains. It was
particularly useful in identifying an acid-resistant isolate (Barney et aI., 2001).
(iii).MALDI TOF
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF
MS) fingerprinting is a fast and reliable method for the classification and identification of
microorganisms, with applications in clinical diagnostics, environmental and taxonomical
research, or food-processing quality control. The general use of MALDI for the
characterization of large biomolecules led directly to obvious applications involving the
analysis of isolated bacterial proteins. More surprising was the observation that MALDI
TOF mass spectrometry could be applied directly to crude cellular fractions or cellular
suspensions and that the resulting data from such complex mixtures could provide evidence
for chemotaxonomic classification. Most simple analysis of a sample starts by applying a
small amount of biological material directly onto the MALDI target plate. The starting
material can be a single colony or a centrifuged portion of a liquid culture. The thin
microbial film is overlaid with matrix (oc.-cyano-4-hydroxycinnamic acid; HCCA; Jackson,
2001).
1.2.10. Bacterial Systematics: 'a roadmap to diversity'
The profounding complexities in microbial world are:
1. Large Microbial diversity
2. Heterogeneously distribute diversity over environment, space and time
3. Diversity of viable but nonculturable
4. Diversity of gene expression
5. Lateral gene transfer
Taxonomy, the principles and practice of classification of organisms that make up scattered
and unparallel diversity enables their detection and identification. The extant to which a general,
phylogenetically based taxonomy can be predictive depends on the relative contributions of
horizontally and vertically transmitted genes. Taxonomy is key to this understanding (Bull et al., 2004).
48
Chapter 1 Introduction & Review o(Literature
Bacterial systematics began in much the same way as the systematics of animals and plants.
Systematists of both microbes and macrobes began with the observation that organisms fall into
clusters of very similar organisms, and they demarcated and named these clusters. These species
demarcations were originally based entirely on phenotype, principally on morphology in the case of
animals and plants, in the case of bacteria it was solely based on and on metabolism. However, the
practices of microbial and macrobial systematics diverged in the 1940s and 1950s when a theory
based concept for species was brought into the systematics of animals (Mayr 1942) and plants
(Stebbins 1957). First, with Mayr's biological species concept (Mayr 1942), and later with many
alternative concepts of species (de Queiroz 1998). Systematists of animals and plants sought to make
their species more than just clusters and aimed to identify species that represented the fundamental
units of ecology and evolution. Despite the plethora of modem species concepts used today, nearly
all these concepts share certain standard attributes, which are as follows: species are cohesive i.e.
some force acts to constrain divergence within species (Meglitsch, 1954) they are irreversibly
separate because there is no force of cohesion that constrains divergence between species (Wiley,
1978); they are ecologically distinct and thus able to coexist within a community (Valen, 1976) and
they are monophyletic i.e. each species is invented only once. It has long been understood that
diversity within the highly sexual animal and plant species is constrained by a powerful force of
genetic exchange. It has also been observed that bacterial species, which recombine sexually at a low
rate, can also be defined so as to meet the criterion of genetic exchange, as well as the other attributes
of species (Cohan et aI., 2001).
The taxonomy of prokaryotesarrived much later than other kingdom. Carl von Linne classified
microscopic organisms in the genus "chaos" and in 1874 Theodor Billroth (1829-1894) believed that
there was only one bacterial species, Coccobacteria septica, which could occur in any form
depending on growth conditions. Initial studies on the taxonomy of prokaryotes and protozoa were
based on higher eukaryote systematics, as exemplified by Ehrenberg (1795-1876) who classified
them into several genera, and Ferdinad Cohn, a trained botanist an adherent of Ehrenberg, who was
one of the first to hold that bacteria could be arranged into genera and species that had a high degree
of constancy although his views did not begin to be accepted until the isolation of pure cultures. The
most widely accepted classification, strictly based on morphological criteria and summarizing all the
species described by the end ofthe 19th century, was that ofMigula (1897).
Classification of prokaryoted which is data dependent (Goodfellow & O'Donnell, 1993)
changes its shape with each influx of new technology and new data. Technological developments
such as the development and application of chemotaxonomy (Goodfellow & O'Donnell, 1994),
Numerical taxonomy (Sneath & Sokal, 1973), small subunit (SSU) rRNA (SSU rRNA=16S rRNA in
prokaryotes) sequencing (Woese, 1987), DNA: DNA pairing (Grimomt, 1988), and molecular
fingerprinting techniques (Stackebrandt & Goodfellow, 1991; Mougel et ai, 2002) have led to
49
Chapter 1 Introduction & Review o(Literature
fundamental establishment in systematics. For the past three decades, whole-genome DNA-DNA
hybridization has allowed quantification of the fraction of genome that is not shared across individual
organisms. Early on, systematists determined a criterion of DNA-DNA hybridization that frequently
corresponded to the established, phenotype-based species demarcations. Annealing of 70% or less
genome became a 'gold standard' for demarcating organisms into different species (Johnson 1973;
Wayne et al. 1987).
The integrated application of many techniques (polyphasic taxonomy) and as described by
Wayne et al., (1987), has its profound impact in prokaryotic systematics for the past 20 years
(Vandamme et al., 1996; Priest & Goodfellow, 2000). Use of the 16S rRNA as a universal tool has
revealed fundamental taxonomic relationships from domains (Woese, 1987) to the diversity of
prokaryotes in ecological niches (HugenhoItz et al., 1998a; Ward, 1998). The 16S rRNA sequencing,
in prokaryotes is an essential component of modern polyphasic taxonomy (Stackebrandt et ai, 2002).
Systematics and taxonomy, like the rest of biology are currently in the throes of new
technological revolution. Researches in this direction have all resulted in ~9000 validly described
prokaryotic species (J.P. Euzeby, http://www.bacterio.cict.fr) and 7,06,103 prokaryotic 16SrRNA
sequences in the ribosomal database project, the RDPII data base (Maidak et al., 2001,
http://rdp.cme.msu.edu/misc/html). Whole genome sequencing and developments in high
throughput sequencing technology like pyrosequencing are providing unprecedented amounts of
data. Prokaryotic systematics is undoubtedly facing the imbalance between high throughput
sequencing and the concept of polyphasic taxonomy (Stackebrandt et. al., 2002). Currently,
taxonomy is reliable only at the level of broad phylogenetic groups (well delineated by even partial
16S sequences) and at the species level within certain well studied taxa such as the genus
Mycobacterium (Goodfellow & Magee, 1998). For many genera, identification of species remains a
major problem, as exemplified by the genera Nocardia (Goodfellow et al., 1999) and Rhodococcus
(Goodfellow et al., 1998).
1.2.10.1. Polyphasic taxonomy versus genomic taxonomy:
With the development in chemistry, molecular biology, and computer science prokaryotic
systematics has changed drastically .a relatively large set of techniques are being used routinely for
prokaryote classification. However it is of primary importance to understand at which level these
methods carry information. The kind of information that each technique retrieves is directly related
to its resolving power. Currently prokaryote taxonomists agree that a reliable classification can be
achieved only by applying wide range of techniques and the approach is termed as 'polyphasic
approach'. Polyphasictaxonomy, the term first coined by Colwell (1970), is now considered as a
standard approach for characterization of a prokaryotic taxon. This approach implies that the all
genotypic, phenotypic and phylogenetic information must be simultaneously investigated as
50
Chapter 1 Introduction & Review of Literature
extensively as possible genomic parameters are gained from all data that can be retrieved from
nucleic acids either directly through sequencing or indirectly through parameters such as G+C mol%
DNA-DNA similarity, 16S rRNA gene sequence analysis, or DNA-based typing methods such as
restriction fragment length polymorphism, low frequency restriction pattern analysis, randomly
amplified polymorphic DNA, Amplified fragment length polymorphism, rep -PCR , and amplified
ribosomal DNA restriction analysis.
Phenotypic refers to the way in which the genotype is expressed, the visible or otherwise
measurable physical and biochemical characteristics of an organism. Phenotype information is
retrieved by the use of classical phenotype analysis including chemotaxonomic studies incorporated
in polyphasic taxonomy. Phenotypic information is derived from a wide range of morphological
observations of cells and colonies (shape, endospore, flagella, inclusion bodies) and physiological
and biochemical features (temperature growth range, salt concentrations, resistances to antimicrobial
agents, enzyme activities, metabolism of compounds) among others. Chemotaxonomy markers refers
to those chemical constituents of the cells that are useful to characterize prokaryotes, including
components such as cell wall composition, cellular fatty acids, isoprenoide quinines and polyamines.
Techniques such as serotyping, electrophoretic profiles (whole cell protein profile,
lipopolysaccharide profiles, multilocus enzyme electrophoresis), and spectroscopy (Fourier transform
infrared spectroscopy, UV resonance Raman spectroscopy) provide strain- unique patterns that might
be useful for identification and discrimination purposes.
Each of these methods has varying levels of capacity to resolve of different hierarchical levels
in bacterial classification (Vandamme et ai., 1996). The polyphasic approach has been considered a
consensus approach in describing novel taxa and drawing relationships among various taxa in
prokaryote taxonomy (Abraham et ai., 1999; Anderson & Wellington, 2001).
In the last few years due to significant development in sequencing techniques and
bioinformatic analysis in prokaryotic systematics has resulted in sequencing of a large number of
prokaryotic genomes over the last few years. At the time of writing, genomes of 720 bacteria and 53
archaea have been completely sequenced and those of 1253 bacteria and 37 archaea are in different
stages of completion (www.ncbi.nlm,nih.gov/genomesllproks.cgi and www.genomes.org). These
data are increasingly being used to compare the entire genomes from organisms to deduce their
relationships and phylogeny. Few novel approaches for assessing taxonomic relationship based on
whole genomes are as follows (reviewed by Coenye et ai., 2005).
1. Comparison of overall gene content and order.
2. Comparative sequence analysis of conserved macromolecules (Santos & Ochman, 2004;
Zeigler, 2003).
51
Chapter 1 Introduction & Review o(Literature
3. Genome Blast Distant phylogeny (GBDP) that includes phylogenetic inference on the basis
of whole genome sequence information.
4. Comparison of dinucleotide relative abundance between organism and its relation with 16S
rRNA gene sequence and DNA-DNA-hybridization (Karlin et ai., 1997).
5. Comparison of presence or absence of certain specific molecular features in the genome
(Fitz-Gibbon & House, 1999; Wolf et ai., 1999).
6. Comparison of overall metabolic reactions or pathways (Podani et ai., 2001; Hong et ai.,
2004).
In addition to these methods, DNA microarray and subtractive hybridization based methods are
emerging as techniques to study the overall genome difference of related microorganisms.
Microarray technique for example are now available for identification of specific bacteria (Hamels et
ai., 2001; Volokhov et ai., 2003) especially those which are of clinical significance. Ribosomal RNA
based phylogenetic DNA microarrays (called 'phylochips') that consists of collection of
oligonucleotide probes that detect the target microorganisms at multiple taxonomic levels of
specificity are now increasingly being developed and applied for rapid identification purposes in
diagnostic and environmental microbiology (Guschin et ai., 1997; Wilson et ai., 2002). Multilocus
sequence typing is also becoming a strong tool to differentiate closely related microorganisms
(Maiden et ai., 1998; Helgason et ai., 2004). Although it seems too early to speculate on how the
different new genomic data will be used in the developing genomic taxonomy but at least the road
map towards a genomic taxonomy of prokaryotes is now under construction" (Coenye et ai., 2005).
1.2.10.2. Bacterial species concept:
The ultimate unit of classification is the species. The foundation of species concept was laid
down after the establishment of Linnaean system of classification in the 18th century on the basis of
metazoan diversity. In the beginning of 20th century, the biological species concept (BSC) was
developed by Ernst Mayr, which defines species as "groups of actually or potentially interbreeding
natural populations which are reproductively isolated from other such groups". BSC has been shown
to be successful for animal world, particularly for insects but for animals reproducing
parthenogenetically, algae, plants, fungi and prokaryotes there was difficulty in applying this
concept. Early definitions of bacterial species were often based on monothetic groups described by
subjectively selected sets of phenotypic properties (Goodfellow, 1997) had severe limitations as, for
example, strains which varied in key characters could not be identified as a member of an already
classified taxon. Additionally, these original classifications were produced simultaneously by
different microbiologists that applied different criteria to the classification of the same group of
organisms. The number of species in a genus was influenced by the aims of the taxonomist, the
52
Chapter 1 Introduction & Review o(Literature
extent to which the taxon had been studied, the criteria adopted to define the species and the ease by
which the strains could be brought into pure culture. Some classifications were defined unevenly, for
example when members of environmentally and medically important genera had been under
classified and those in industrially significant taxa over classified (Goodfellow, 1997). Moreover,
this practice often leads to nomenclatural confusions, where a single species could be simultaneously
classified under several different names (Van Niel, 1952). Until the discovery of DNA as an
information-containing molecule, prokaryote classification was based solely on phenotypic
characteristics. The development of numerical taxonomy (Sneath, 200 1), in which the individuals are
treated as operational taxonomic units that are polythetic (they can be defined only in terms of
statistically co varying characteristics), resulted in a more objective circumscription of prokaryotic
units. The discovery of genetic information gave a new dimension to the species concept for
microorganisms. Parameters like G+C content and overall DNA-DNA similarity have additionally
given insight into phylogenetic relationships. Thus, the species concept for prokaryotes evolved into
a mostly phenetic or polythetic. This means that species are defined by a combination of
independent, covarying characters, each of which may occur also outside the given class thus not
being exclusive of the class (Van Regenmortel, 1997). There is no official definition of a species in
microbiology. However, from a microbiologist's point of view "a microbial species is a concept
represented by a group of strains, that contains freshly isolated strains, maintained in vitro for
varying periods of time, and their variants (strains not identical with their parents in all
characteristics), which have in common a set or pattern of correlating stable properties that separates
the group from other groups of strains" (Gordon, 1978). This definition only applies to prokaryotes
which have been isolated in pure culture (essential for the classification of new prokaryotic species),
and excludes uncultured organisms which constitute the largest proportion of living prokaryotes.
However, a prokaryote species is generally considered to be "a group of strains that show a high
degree of overall similarity and differ considerably from related strain groups with respect to many
independent characteristics", or a collection of strains showing a high degree of overall similarity,
compared to other, related groups of strains" (Colwell, 1995). There are, in the literature, at least
three different species definitions that, to date, tend to disappear due to the unification of criteria: (i)
taxospecies, defined as a group of organisms (strains, isolates) with mutually high phenotypic
similarity that form an independent phenotypic cluster, (ii) genomic species as a group showing high
DNA- DNA hybridization values, and (iii) nomenspecies as a group that bears a binomial name
(Colwell, 1995).
The species concept in prokaryotes is not theory based rather it is more arbitrary,
anthropocentric and is made for practical purposes. There are two main problems in species concept
of prokaryotes. First, it is difficult to compare species unit of prokaryote with its counterpart in
eukaryote and secondly, the use of the devised unit may not be satisfactory to a given scientist who is
53
Chapter 1 Introduction & Review o(Literature
working in the very same field due to reductionistic, monistic or plurastic use of taxonomy
(RossellO-Mora,2003).
By 1970, the concept of polyphasic taxonomy was developed and was subsequently applied in
description as well as delineation of prokaryotic species. According to this, the genotypic and
phenotypic characters of the strains should be evaluated thoroughly so as come to a conclusion
(Colwell, 1970; Vandamme et ai, 1996). By the end of 1980s following this concept, the species
definition was based on overall comparison of the genomes of the strains. The over all genome
relatedness was measured by DNA-DNA hybridization (DDH) methods and difference in melting
temperature (~Tm) of the heteroduplexes versus homoduplex of the DNA strands. This led to a
definition of bacterial species in 1987 as described earlier "species generally would include strains
with approximately 70 % or greater DNA-DNA relatedness and with 5 °C or less ~Tm" (Wayne et
ai., 1987). The DDH data is very much consistent with recently available complete genome sequence
data. However, DDH is time consuming, cannot be applied for uncultivated bacteria and cannot be
compared with greater ease like a gene sequence in a database (Gevers et ai., 2005). By the early
1990s the availability of 16S rRNA gene sequence data was increasing very rapidly. Around middle
of 1990 a correlation between the 16S rRNA gene sequence similarity and overall genome
relatedness was made. It was observed that, for strains that showed less than 97% 16S rRNA gene
sequence similarity among them, overall genomic relatedness was less than 70% (Stackebrandt &
Goebel, 1994). Although, 16S rRNA gene loses its resolving power beyond 97% sequence similarity
it proved very useful for delineating bacterial taxa or candidate taxa in both culture dependent study
as well as culture independent study. But, such overdependence of organismal evolution and
demarcating point using only one-gene sequences has several drawbacks. Therefore to have better
resolution a combination of different innovative methods like multi locus sequence typing (MLST),
analysis of bacterial cell with FTIR or MALDI-TOF, application of typing methods (AFLP, RAPD,
ARDRA etc) in addition to 16S rRNA gene sequence data and DNA-DNA relatedness data (if
required) has been suggested for taxonomic conclusion (Stackebrandt et ai., 2002). It is worth
mentioning that the delineation at species level (or any hierarchy) should also be supported by
phenotypic, chemotaxonomic and other as many evidence as possible (Stackebrandt et ai., 2002).
1.2.11. Prokaryotic phyla: known so far:
1.2.11.1. Current picture in domain bacteria:
Molecular techniques involved in surveying the uncultured majority in natural environment
have uncovered profound knowledge of microbial diversity over the past decade. The number of
estimated microbial phyla over the past two decades, expanding from 11 in 1987 to 36 in 1998
(Hugenholtz, 2002) to 53 phyla (Keller & Zengler, 2004) proposed at present (Raymond, 2008),
54
Chapter 1 Introduction & Review o(Literature
1. Phyla with cultured representatives
Carl Woese (1987) published a benchmark paper in microbial biology, the first comprehensive
synthesis of bacterial evolution placed in the context of all life forms. From the 16S rRNA sequences
and catalogs available through 1987, Woese and colleagues were able to delineate 11 major groups
or lineages, which since have variously been referred to at the taxonomic rank of kingdom, phylum,
class, order, and division. The 11 original phyla recognized in 1987 has been divided into 12 phyla as
the Gram-positive bacteria are now recognized as two separate phyla, the Firmicutes (low G+C) and
Actinobacteria (high G+C). The 12 major bacterial divisions identified still represent most of the
taxa that can be readily cultivated and characterized by using cultivation methods, these are
Firmicutes (low G+C) Actinobacteria (high G+C), Proteobacteria (classical Gram-negative bacteria)
which on the basis of cultivation-dependent and -independent approaches, are generally recognized
as one of the most successful microbial groups on the planet which includes two of the most studied
genera of microorganisms, Escherichia and Pseudomonas. Other phyla included in original phyla are
Cyanobacteria, Thermotogae, Chlorojlexi, and Bacteroidetes. Cyanobacteria are oxygenic
photosynthetic bacteria, Thermotogae that are sheathed, obligatory anaerobic, fermentative
heterotrophs. Chlorojlexi (green non-sulfur), as originally defined, included the thermophilic
phototroph Chlorojlexus, the mesophilic, gliding chemoheterotroph Herpetosiphon, and the
hyperthermophilic chemoheterotroph Thermomicrobium. These microbes exhibit divergent metabolic
strategies. Cytophaga, Bacteroidetes, and Flavobacterium form a major lineage, known now as the
Bacteroidetes phylum commonly known as (CFB group; Cytophaga, Flavobacterium and
Bacteroidetes). Five other bacterial phyla were also included namely Chlamydiae, Planctomycetes,
Spirochaetes, Chlorobi and Deinococcus-Thermus. The only phyla of bacteria known to have cell
walls that are not composed of peptidoglycan are Planctomycetes and Chlamydia. They have a
number of other odd features as well, particularly the presence of an intracellular compartment in
some species that contains the cell's DNA. The first members of the phylum Planctomycetes were
isolated in the 1970s as heterotrophs growing in dilute media. With the discovery that the
Planctomycetes responsible for anaerobic ammonia oxidation (the anamox reaction), physiological
diversity within the phylum expanded further. Four genera have been described: Pirellula,
Planctomyces, /sosphaera, and Gemmata under the phylum Planctomycetes. At a relatively early
stage in the application of environmental gene cloning methods, it became apparent that the
Planctomycetes were far more prevalent in the environment than would have been suspected from
their scant presence in culture collections (DeLong et al., 1993; Ehrich et al., 1995). It is now
apparent that they are common in soils and sediments, as well as the fresh, marine and hot spring
environments with which they were originally associated.
Since 1987, 14 bacterial phyla such as Verrucomicrobia, Fusobacteria, Caldithrix,
Gemmatimonadetes, Fibrobacteres, Defferibacteres, Acidobacteria, Nitrospira, Synergistes,
55
Chapter I Introduction & Review o(Literature
Thermodesulfobacteria, Coprothermobacter, Dictyogiomi, Aquificae and Desulfurobacteria
(Hugenholtz, 2002; Hugenholtz et ai., 1998a; Pace, 1997) were also included. Included in this group
are phyla of predominantly thermophilic and chemolithoautotrophic microorganisms. Some members
of the phylum Aquificae can oxidize hydrogen gas as an energy source for chemolithotrophic growth
(Huber, et al., 1992). The Desulfurobacteria grow by sulfur reduction anaerobically (sulfate reducing
bacteria), and Thermodesulfobacterium hydrogenophilum grows by sulfate reduction.
Phylum Acidobacteria is apparently ubiquitous and abundant in nature, as based on results
from cultivation-independent molecular ecology studies, especially in soils (Buckley & Schmidt,
2002; Barns et al., 1999). This phylum includes three species of divergent physiology
(Acidobacterium capsuiatum, Hoiophaga foetida, and 'Geothrix fermentans'), thus making it difficult
to predict many characteristics of the Acidobacteria-related microorganisms detected in
environmental samples (Barns et al., 1999). The phylum Nitrospira is also noteworthy in that it
includes obligately chemolithotrophic, nitrite-oxidizing genus Nitrospira, and the obligately
chemolithotrophic, ferrous iron-oxidizing genus Leptospirillum. Members of this phylum are also
apparently ubiquitous in the natural environment, judging by the large number of Nitrospira rRNA
gene clones that have been reported in the past few years. Under the phyla which are under
represented in culture are Verrucomicrobia, Chioroflexi, and Gemmatimonadetes including
Pianctomycetes.
Since 1995 the phylum Verrucomicrobia has been recognized as a separate phylum of
Bacteria, but currently counts only a small number of cultivated microorganisms as members. This
phylum was known from two genera and five species of prosthecate, aerobic heterotrophs isolated
from freshwater environments which are Verrucomicrobium vinosum, Prosthecobacter fusiformis, P.
debontii, P. vanneervenii, and P. dejongeii. Although an obligately anaerobic, heterotrophic genus
(Opitutus terrae) from paddy field soil has recently been isolated and characterized. In addition to the
named microorganisms, three taxonomically uncharacterized isolates of "ultramicrobacteria" from
paddy field soil with 16S rRNA gene sequences closely related to 0. terrae have also been described.
A rich diversity of microorganisms awaits exploration within this phylum, which includes major
clusters of rRNA gene clones that are ubiquitous in natural freshwater and soil microbial
communities (Buckley & Schmidt, 2002; Zwart et al., 2002).
The phylum Chioroflexi (green non-sulfur bacteria), deep-branching lineage of the domain
Bacteria, was one of the initial group of phyla recognized early in the application of small subunit
rRNA sequencing to taxonomic questions within the Bacteria. Chioroflexi rRNA genes appear
frequently in clone libraries constructed from subsurface oceanic bacterioplankton (Bano &
Hollibaugh, 2002; Giovannoni & Rappe, 2000), freshwater bacterioplankton (Urbach et al., 2001),
soils (Chandler et al., 1998), sediments (Coolen et ai., 1998) and geothermal hot springs (Hugenholtz
et al., 1998a). Thus cultivation-independent investigations of the diversity present in a wide variety
56
Chapter 1 Introduction & Review o(Literature
of microbial communities have found members of this phylum to be ubiquitous in the natural
environment. The limited number of cultivated members of this phylum is an interesting group of
diverse phenotypes, which include gliding, filamentous isolates that contain some form of
bacteriochlorophyll (Chloroflexus, Oscillochloris, Chloronema, and Heliothrix); filamentous,
mesophilic, strict aerobic chemoheterotrophs (Herpetosiphon) which do show gliding motility; a
hyperthermophilic, irregular rod-shaped, nonmotile aerobic chemoheterotroph (Thermomicrobium
roseum); and an irregular cocci-shaped isolate able to reductively dechlorinate tetrachloroethene
('Dehalococcoides ethanogenes').
Identified first as a candidate phylum in 2001, the phylum Gemmatimonadetes has been
recognized as a main line of descent within the Bacteria. It has since been proposed as a phylum,
only cultivated representative of the genus is Gemmatimonas aurantiaca (Zhang et aI., 2003). It has
become a diverse assemblage of environmental rRNA gene clone sequences. At least four subgroups
can be clearly delineated within this phylum. Zhang and coworkers hypothesized that one identifying
feature of this phylum may be that its members possess a gram-negative cell envelope lacking
diaminopimelic acid in their peptidoglycan similar to one possessed by G. aurantiaca (Zhang et aI.,
2003).
2. Candidate Phyla of Uncultured Microorganisms
Extensive use of 16S rRNA gene cloning and sequencing tools to identify microorganisms in
natural samples has revealed an enormous diversity within bacterial phyla. It is also apparent that
some of the recovered clone sequences did not appear to belong to any of the known bacterial phyla
(Fuhrman et al., 1993; Liesack & Stackebrandt, 1992). It was later discovered that a portion of these
"unaffiliated" environmental gene clone sequences were providing scientists with the first evidence
of such ubiquitous, but not yet recognized, phyla as the Verrucomicrobia and Acidobacteria. Many
artifacts (e.g., chimeric gene clones, peR errors, sequencing errors) and methodological errors (e.g.,
improper reference or outgroup taxon selection, inadequate quantity of sequence information,
improper alignment, use of an inappropriate alignment mask) can cause the misplacement of gene
clone sequences in phylogenetic trees. Unaffiliated clones from different studies frequently clustered
together in further analyses to form monophyletic groups supported the conclusion that many of
these sequences were in fact real and formed major lines of descent within the bacterial domain
which did not contain cultivated relatives. These lineages have since become known as candidate
divisions or phyla, with the term candidate implying that no cultures yet exist to represent the group
(Hugenholtz et aI., 1998a; Hugenholtz et aI., 1998b). Phrase "candidate phylum" has been given for
these deeply diverging clusters of sequences that are phylogenetically equivalent to phyla of cultured
microorganisms as delineated in Bergey's Manual of Systematic Bacteriology.
57
Chapter 1 Introduction & Review o(Literature
Before 1998, the OS-K group (named after a 16S rRNA gene clone recovered from a
microbial mat of thermal Octopus Spring); Marine Group A (named after gene clones recovered from
Pacific Ocean bacterioplankton samples); and Termite Group 1 (named after gene clones recovered
from the intestine of the termite Reticulitermes speratus), were three groups of sequences composed
solely of environmental gene clones were generally thought to form main lines of descent within the
domain Bacteria. Hugenholtz and coworkers defined a candidate phylum as "an unaffiliated lineage
in multiple analyses of datasets with varying types and number of taxa and having <85% identity to
reported sequences, indicating its potential to represent a new bacterial division [phylum]"
(Hugenholtz et at., 1998b) and "as a lineage consisting of two or more 16S rRNA sequences that are
reproducibly monophyletic and unaffiliated with all other division [phylum] level relatedness groups
that constitute the bacterial domain" (Hugenholtz et aI., 1998a; Dalevi et aI., 2001).
Hugenholtz and coworkers (Hugenholtz et aI., 1998b) described 12 lineages that potentially
represented candidate phyla from a Yellowstone hot spring (designated as OPI-12). Recently, a
novel thermophilic chemoheterotrophic filamentous bacterium was obtained from a hot spring in
Japan that was enriched through various isolation procedures that belongs to the phylogenetic group
termed OP5 (Mori et at., 2008). In that same year, Dojka and coworkers (Dojka et aI., 1998)
described 6 lineages four of them formed candidate phyla from a hydrocarbon- and chlorinated
solvent-contaminated aquifer (WS 2, 3, 5 and 6). Afterwards eight candidate phyla have emerged
from the expanding public databases of 16S rRNA gene sequences these are SC3 and SC4 identified
from arid soil, NC 10, BRCl, identified from bulk soil and rice roots of flooded rice microcosms,
Guaymasl, identified from hydrothermally active marine sediments, NKB 19, identified from deep
sea sediments and activated sludge, and SBRI093, identified from activated sludge. Poribacteria
were detected in 2004 in marine demosponges and, to date, remain unknown from any other
environmental niche (Fieseler et aI., 2004). No pure culture of Poribacteria is available but,
according to specific fluorescence in situ hybridization analysis, they can occur in high numbers in
these sponges. Some uncertainty exists in determining the total number of candidate phyla; however
this number currently at about 26.
Result of cultivation-independent molecular surveys has revealed that the bacterial domain
consists of many more divisions, with few or no cultured representatives. Currently there are 53
phylogenetically well-resolved bacterial divisions are present (Pace, 1997; Hugenholtz et aI., 1998a).
In the domain Bacteria, phyla with no cultivated representatives, demonstrate that the microbial
species in culture collection centres provide only a limited and incomplete picture of microbial
diversity.
58
Chapter 1 Introduction & Review o(Literature
1.2.11.2. Current picture of domain Archaea:
From 16S rRNA phylogeny two main line of decent (phyla) has been delineated within archaea
"Crenarchaeota" and "Euryarchaeota". Euryarchaeota shows the greatest phenotypic diversity among
known cultivable species comprised of halophiles, methanogens, thermoacidophiles and some
hyperthermophiles. In contrast the phenotypic diversity of cultivable Crenarchaeota is much more
limited with only the hyperthermophiles. The total number of phylum-level lineages in the archaeal
tree is 18, of which 8 (44%) have cultivated representatives and 10 (56%) have none. A higher tally of
23 phyla is arrived at if lineages not meeting the selection criteria are included in the estimate. These
include "Methanopyri" currently represented by a single sequence, and environmental group C3,
which has no full-length representatives. Most archaeal research has concentrated on the cultivated
methanogenic (such as Methanococci) and thermophilic (such as Thermoprotei and Thermococci)
lineages. As is the case with the Bacteria, most candidate Archaeal phyla are completely
uncharacterized at this point. However, some environmental Archaeal 16S rRNA sequences detected
by PCR indicate the presence of new lineages that branch between Crenarchaeota and Euryarchaeota.
Barns and coworkers have suggested a tentative third phylum, the Korarchaeota (Barns et a!., 1996),
which was further shown to be firmly inside the Crenarchaeota (Robertson et al., 2005) but
representative of this lineage have not yet been isolated in pure culture. Sequences that branch even
deeper than Korarchaeota in the archaeal16S rRNA tree were also reported (Takai et al., 2001a, b). A
new phylum Nanoarchaea was proposed by Huber et al. (2002) based on the discovery of a parasitic
archaea of reduced size (Nanoarchaeum aquitans). It possesses the smallest cellular genome known to
date (less than 0.5 Mbp) and lives in parasitic association witha hyperthermophilic Crenarchaeote of
the genus Jgnicoccus and branches deeply in the Crenarchaeote tree. More phyla are very likely have
to be defined in Archaea and this could produce an archaeal classification more similar to the bacterial.
1.2.12. An introduction to Western Ghats:
The Western Ghats, identified as one of the biodiversity hotspots of the world, is a 1600 km long
chain of mountain ranges running parallel to the western coast of the Indian peninsula (Praveen &
Nameer, 2009). It has a land area of3702 Sq. Kms and a coast line of 104 Kms. They extend from the
mount of the river Tapti (21°N) to the South of India (about 8°N), the only gap in the chain being
Palghat Gap (Radhakrishna, 1993). Based on the topography and geology the Western Ghats can be
divided into three major regions (Praveen & Nameer, 2009):
North Western Ghats (Surat to Goa): This region consists of the most homogeneous part of
the Ghats, hugging the coast for almost 600 km. It corresponds to the western edge of the vast plateau
formed by the basaltic outpourings of the Deccan trap. Its elevation is generally between 700 to 1000
m and with some peaks even higher for example Kalusbai (1646 m) and Mahabaleshwar (1438 m).
59
Chapter 1 Introduction & Review o(Literature
Central Western Ghats (Goa to Nilgiris): The basaltic outpourings cease to the north of Goa.
However, towards the south, the Ghats consists of complex pre-Cambrian rocks. In the central Western
Ghats, the rocks are predominantly of Dharwar system (among the oldest in India) and Peninsular
Gneiss. The elevation generally range between 600 m to 1000 m up to 13° 30' N (except Kodachadri:
1343m). From Kudremuch (1892 m) and up to Wynad, the edge of the plateau is very often higher
than 1000 m and with several peaks ranging between 1713 and 2339 m. Towards 11° 30' N, the
Western Ghats rise abruptly in the Nilgiris horst which is made of Charnokites rock. The Nilgiri
Mountains constitute an elevated plateau attaining a maximum height of2637 m at Dodda Betta.
Southern Western Ghats (South of the Palghat Gap): This region is mostly formed of
Charnokites rocks. The Ghats which are interrupted by a gap (Palghat Gap) of about 30 km wide
reappear abruptly as the Anamalai-Palani block whose high plateau attain a height of 2695m in the
Anaimudi peak, the highest point in South India. They end almost at the southern tip of India, about 20
km before Kanniyakumari. This last part, which is very rugged, culminates at 1869 m in the
Agastyamalai peak.
1.2.12.1. Climate and soil type:
The climate of the Western Ghats shows rainfall gradients and a temperature gradient. The
western slopes of the Ghats are subject to direct influence of rain-bearing winds of the south-west
monsoon. They receive 2000 to 7500 mm of rainfall. These totals decay rapidly to <800 mm towards
the east within a distance of 7 to 60 km. The second, north-south gradient is determined by the time of
arrival and withdrawal of the monsoon. The temperature gradient is mostly related to increase in
altitude. However, it is not uniform throughout the Ghats because of variability in the relief from south
to north. In general, the mean temperature of the coldest month varies from 23°C at sea level to 12°C
at 2300 m. Soil type is laterite and lateritic clayey-loamy soil.
As already stated, Western Ghats is one of the 25 hot spot for the diversity in the world due to its
unique position in the South Asia. Studies on exploration of animal species, plant species, and fungi
have been started. In the Western Ghats, based on the ecological factors and floristic composition, four
major forests and 23 floristic types have been distinguished. These types are closely correlated with the
temperature and rainfall regimes. Wet evergreen, dry evergreen, moist deciduous and dry deciduous
forests are clearly distinguished by the mean annual rainfall, whereas low, medium and high elevation
wet evergreen types are distinguished by the decrease in minimum temperature with increasing
altitude. In addition to forests, high altitude grasslands are another unique ecosystem in the Western
Ghats.
60
Chapter 1 Introduction & Review o(Literature
1.2.12.2. Floral diversity:
Four thousand species of flowering plants are known from the Western Ghats. The gymnosperm
flora is represented by Cycas circinalis (Cycadales), Decussocarpus wallichianus (Coniferales) and
Gnetum ula and G. contractum (Gnetales). Amongst the lower plants around 150 species of
pteridophytes, 200 species of bryophytes, 200-300 species of algae and 800 species of lichens are
known. There are 600 species of fungi known from the Western Ghats.
Fifty-six genera of flowering plants are considered endemic to the Western Ghats (Nayar, 1996).
The validity of endemism at generic and higher taxonomic levels is however subject to systematic
revisions. Study survey for endemic plants of Western Ghats and West Coast distributed in Goa was
carried out which resulted in the collection of 113 endemic species. Although the exact number
keeps varying with the author and time, what is of interest is that nearly 38% of all species of
flowering plants in the Western Ghats are endemic. Further it is to be noted that 63% of India's
evergreen woody plants are endemic to the Western Ghats. Nearly 650 species of plants in the Western
Ghats are trees. The Nilgiri Mountain is considered as the most important centre of speciation of
flowering plants in the Western Ghats. Among which 82 species are endemic to these hills. These
mountains are also unique in having a mosaic of mountain forests and savannas often referred to as the
'shola-grassland' complex.
1.2.12.3 Faunal diversity
Scientific research on the invertebrates of the Western Ghats has largely been restricted to a few
groups of organisms. As with any other tropical region, the invertebrate diversity of Western Ghats is
best known by the butterflies. Out of 300 species 37 species are endemic. The biodiversity study on
insects of Western Ghats has been done extensively. A total of 15, 260 individuals belonging to 12
order, 59 families and 94 genera were collected in 293 sampling sessions from 39 localities in the
Western Ghats (Subramanian, 2003). There are around 218 species of primary and secondary
freshwater fishes in the Western Ghats. Fifty three percent of all fish species (116 species in 51 genera)
in the Western Ghats are endemic (Talwar & Jhingran, 1991; Jayaram, 1999; Menon, 1999; Daniels,
1991). One hundred and twenty one species of amphibians are known from the Western Ghats
(Daniels, 1991). Of these, 94 species are endemic. The phytodiversity of Western Ghats is explored,
identified and documented by the Southern and Western Circles of BSI located at Coimbatore and
Pune, respectively. This documentation has been published in the form of District and State Floras
such as Flora of Karnataka: Analysis (VoU), Flora of Tamilnadu: Analysis (Vol. 1-3), Flora of
Maharashtra: Monocotyledons (Vol. I), Flora of Goa (two volumes), Flora of Kerala (Grasses), Flora
of Cannanore, Flora of Thiruvananthapuram, Flora of Palaghat, Flora of Nasik and Flora of
Mahabaleshwar. Faunal surveys of Western Ghats are being conducted by ZSI through its Regional
61
Chapter 1 Introduction & Review o(Literature
Stations in Pune, Chennai and Kozhikode. A document on faunal diversity of Nilgiri Biosphere
Reserve has been published.
Diversity of plants and animals are reasonably documented and recently two reports on the
fungal diversity were published (Natarajan et ai., 2005; Raviraja, 2005; Manoharachary et ai., 2005).
There is almost no report which can throw some light on the prokaryotic world of such a pristine area
in India i. e. The Western Ghats. This work therefore was initiated with the aim to investigate
prokaryotic diversity of a few selected ecological niches ofthis region.
62