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CHAPTER 1
INTRODUCTION TO MICROBIAL . PIGMENTS
Pigments and Microbes
A number of different kinds of pigments are produced by
microorganisms. The pigments vary in color from red, yellow,
orange, purple etc. These pigments have been subjected to a lot of
research not only for their colorful nature, but also because they
play important roles in photosynthesis, photoprotection,
pathogenesis etc. Yet other pigments are interesting because their
functional role is unclear. One of this latter class of pigments, the
xanthomonadins, is the topic of this thesis. This chapter is a review
of the structure and function of the xanthomonadins and two other
groups of microbial pigments, the carotenoids and melanins. The
emphasis on these pigments is due to the possibility that they
might be involved in photoprotection.
Xanthomonas
The genus Xanthomonas is comprised almost entirely of plant
pathogens. The affected plants include representatives from
practically all major groups of higher plants. Xanthomonads are
gram-negative, aerobic, rod-shaped bacteria with a single polar
flagellum. A characteristic feature of this genus is the production
of copious amounts .. of extracellular polysaccharide. Another -.:..-----~--·-·- ··--·- -o•- --· - ---- ·-----·--·--
distinguishing feature is that most xanthomonads produce yellow
pigments called xanthomonadins which are distinct from the yellow
1
2 (carotenoid) pigments produced by Erwinia, Pseudomonas,
Flavobacterium and Corynebacterium strains (see Carotenoids). The
characteristic presence of xanthomonadin in members of the genus
Xanthomonas has led them to be used as chemo-taxonomic and
diagnostic markers (Starr, 1981 ). A few non-pigmented
. xanthomonads have been reported viz. Xanthomonas campestris
pathovar (pv.) manihotis (causal agent of bacterial blight in
Cassava; Dye and Lelliott, 1974).
Xanthomonadin Pigment
Xanthomonadin (from Xanthomonas juglandis, a walnut pathogen)
was reported to be localized at the cytoplasmic membrane by
Stephens and Starr (1963). The membrane-bound nature of the
pigment was determined based on the kinetics of release of the
pigment from cells of X. juglandis upon sonic ·treatment. It was
observed that the ·cells subjected to sonic or ballistic
disintegration released xanthomonadin at a rate identical to that of
a membrane component like reduced nicotinamide adenine
dinucleotide (NADH) oxidase. The structure of a xanthomonadin,
isolated from X. juglandis, was solved by Andrews et a/. (1976)
using heavy atom Patterson and Fourier crystallographic methods
(Figure 1 . 1 ). Xanthomonadins were then deduced as brominated aryl
polyene pigments. The presence of bromine in this molecule is
unique since xanthomonads are terrestrial microorganisms which as
a group rarely incorporate bromine in organic molecules. Very little
information is available about the biosynthesis of this pigment. It
has been postulated that the aromatic ring may be derived from the
B r
OMe
Figure 1.1: Structure of Xanthomonadin I
0
c~ ~e '\OCH2CH
I Me
Xanthomonadins are membrane bound, brominated aryl polyene
pigments that are characteristic of the genus Xanthomonas. The
structure of xanthomonadin I was described from X. juglandis (a
walnut pathogen; Andrews et a/. 1976).
3
Figure 1.2: Colonies of Xanthomonas oryzae pv. oryzae
The above photograph shows yellow colonies of Xanthomonas oryzae
pv. oryzae as seen under a stereo microscope. Colonies were
photographed after 4 days of incubation on Peptone Sucrose agar
medium (see Materials and Methods in Chapter 2) at 28°C.
4
5 shikimate pathway and the polyene chain from the polyketide~
pathway (Jenkins and Starr, 1982a). }
Recently, a 23 Kb gene cluster required for xanthomonadin
biosynthesis in Xanthomonas campestris pv. campestris (a pathogen
of crucifers like cabbage, radish etc.) was cloned by Poplawsky et
a I. 1993. Complementation analysis suggests that this region
includes seven complementation groups. RFLP and dot blot analysis,
using the xanthomonadin clone as a probe, suggested that these
genes are present in all xanthomonads irrespective of whether they
produce pigment or not. Sequences homologous to the xanthomonadin
gene cluster were not detected in non-xanthomonads and therefore
these genes could be used as diagnostic probes for the
identification of xanthomonads. A subsequent report indicated that
one of the genes (pig B) in this cluster enco_des~a_di.ftusible_factor
required for both _ptg.r:n_e_nt_ a.nd __ extcacelh,Jlac_Roly_~accharide
production (PoQiawsky and Chun, 1997). ---·--· .· -------~
Pigment deficient mutants of many Xanthomonads viz. X
campestris pv. campestris and Xanthomonas oryzae pv. oryzae
(causal agent of bacterial blight of rice) are fully proficient for
virulence when wound inoculated onto their respective hosts
suggesting that pigment is not essential for virulence of the
pathogen (Poplawsky et a/. 1993; Tsuchiya et a/. 1982; Durgapal,
1996). A pigment deficient mutant of X. juglandis was observed to
be more sensitive to photobiological damage when compared to the
pigmented wild type strain suggesting that xanthomonadins might '
serve to protect the bacterium from photodamage (Jenkins and
Starr, 1982b).
Xanthomonas oryzae pathovar oryzae
This organism is the subject of study in this thesis. It causes a
serious disease of rice called bacteri.al leaf blight. The symptoms
are characterized by yellowing ·and drying which begin at the tips
of rice leaves and spread down either one or both sides of the leaf
or through the mid vein (Figure 1.3). The pathogen gains entry into
the rice leaf either through wounds or natural openings called
'hydathodes' that are present on the edges of the rice leaf. The
bacterium multipt"ies inside the xylem vessels and travels through
them to more distant parts of the leaf and occasionally to other
parts of the plant. It also exits from the leaf in the form of ooze
droplets that collect on the surface of the leaf. Dispersal of the
pathogen Is achieved when these ooze droplets are splashed onto
neighboring uninfected plants by wind or rain. Dispersal of the
pathogen from one infected leaf to other leaves on the same adult
rice plant might also occur through ooze (Sridhar Dharmapuri and
Ramesh Sonti, unpublished data). During the off-season, the
pathogen is presumed to survive either on seeds, dried leaves and
stubble or on neighboring alternate host plants (like weeds) till the
next season (Alvarez et al. 1989; Figure 1.4 ).
The objective of this study is to determine the role of the
xanthomonadin pigment in the life cycle of X oryzae pv. oryzae. One
of the questions addressed is whether the pigment is required for
virulence following epiphytic entry of the pathogen into the plant
i.e. entry through the hydathodes after residence on the leaf surface. I
This is more akin to the natural mode of entry into the plant an"' is
6
A B
Figure 1.3: Disease symptoms of bacterial leaf blight of
rice
Xanthomonas oryzae pv. oryzae is a vascular pathogen that traverses
down the xylem vessels of the leaf. A, a healthy rice leaf. B, leaf
blight symptoms caused by Xanthomonas oryzae pv. oryzae. Infection
symptoms begin at the leaf tips and advance through the veins/mid
rib of the infected leaves.
7
Off season
Wild rices Stubble Seed (?)
.._Entry • Bacterial ooze
Plant to plant spread
leaf to leaf
Figure 1.4: Life cycle of Xanthomonas oryzae pv. oryzae
The pathogen exits from infected leaves in the form of bacterial ooze. Wind and rain
contribute to splash dispersal of the pathogen onto neighboring uninfected plants.
During the off season the pathogen is presumed to survive in the straw, stubble or on
alternate host plants. It has also been suggested that the pathogen might survive the off
season in seeds.
8
9 likely to result in exposure of the pathogen to air and light. These
are conditions under which pigment may enhance viability of the
pathogen (by providing photoprotection). Pigment deficient mutants
that were isolated after either EMS mutagenesis or marker
exchange of transposon insertions obtained in cloned pigment
biosynthetic genes were used in this study. Reporter gene fusions to
putative pigment promoters were obtained and their expression was
studied; specifically to determine whether these genes are
expressed when the pathogen is present within the rice plant.
During the process of cloning the X. oryzae pv. oryzae genes involved
in pigment biosynthesis, a number of methods for conducting
molecular genetics on an Indian isolate of X. oryzae pv. oryzae were
standardized and these are detailed in the following chapter.
Carotenoid Pigments
Carotenoids are isoprenoids containing a characteristic polyene
chain of conjugated double bonds and are located in chromatophores
(vesicular, tubular or lamellar membranous structures) which are
continuous with the cytoplasmic membrane. The two groups of
carotenoids known are the 'carotenes' or hydrocarbons and their
oxygenated derivatives called 'xanthophylls'. Unlike xanthomonadins
that are found exclusively in xanthomonads, carotenoids are present
not only in bacteria but also in photosynthetic algae, plants and
some fungi. About 600 carotenoids are known to occur naturally
with most of these being considerably similar to each other in
structure. Carotenoids usually contain 40 carbon atoms (C40) with
10 acyclic 'lycopene' (Figure 1.5A) considered as the basic compound
from which most, if not all, carotenoids are derived (Goodwin,
1973). The presence of carotenoids in bacteria and fungi is
discussed here.
Carotenoids. in photosynthetic bacteria
Approximately 60 different carotenoids have been identified
from photosynthetic bacteria and these have been tabulated by
Goodwin {1973). Photosynthetic bacteria are divided into two
classes i.e. the purple photosynthetic bacteria and the green
photosynthetic bacteria. All purple photosynthetic bacteria contain
acyclic carotenoids e.g. Spirilloxanthin (Figure 1 .58). In contrast,
the green photosynthetic bacteria are characterized by the presence
of aromatic carotenoids e.g. chlorobactene (Figure 1.5C). In
photosynthetic bacteria, two possible functions attributed to
carotenoids are that they act as accessory pigments in
photosynthesis and also function to provide protection against
photodamage.
The first study that provided evidence for photoprotection by
carotenoids was with a mutant of the photosynthetic purple
bacterium Rhodopseudomonas spheroides (Griffiths et a/. 1 955). It
was observed that the mutant strain (designated as 'blue-green')
accumulated a colorless carotenoid precursor called phytoene.
Bacteriochlorophyll was synthesized in the mutants but to lower
amounts when compared to the wild type. The growth of this mutant
strain proceeded normally under anaerobic conditions. However,
under aerobic conditions, the cells of 'blue-green' were rapidly
I I
A•
MeO
B_. eO
c ...
Figure 1 .5 A-C: Structures of lycopene (A), spirilloxanthin
(B) and chlorobactene (C).
Around 600 carotenoid structures have been reported. Most
carotenoids are derived from lycopene. An example of an acyclic
carotenoid i.e. spirilloxanthin and a cyclic carotenoid like
chlorobactene are shown here.
12 killed due to exposure to light and oxygen (in this case
bacteriochlorophyll acts as the photosensitizer). It was proposed
that the mechanism by which photoprotection occurs is that in the
presence of excess quanta, chlorophyll gets exCited to its triplet
state and chlorophyll in its triplet state could mediate abnormal
photosensitized reactions. Under these conditions, the carotenoid
pigments serve to quench the triplet state of chlorophyll. This
hypothesis is supported by the fact that carotenoid pigments can
interact with the triplet state of chlorophyll (Fujimori and
Livingston, 1957). It was also observed that carotenoids have the
ability to quench singlet oxygen (1 02) and that the ability of a
carotenoid to quench singlet oxygen in vitro is dependent on the
number of conjugated double bonds. The efficiency of
photoprotection by carotenoids was studied by a number of groups
and it was concluded that the least unsaturated carotenoid that
shows a protective effect was neurosporene with only 9 conjugated
double bonds and that with increased desaturation there was a
concomitant increase in photoprotection (Mathews-Roth et a/.
1974).
Carotenoids in non-photosynthetic bacteria
A number of unique C30, C45 and C50 carotenoids are found in
non-photosynthetic bacteria. Photoprotection offered by
carotenoids in non-photosynthetic bacteria was first demonstrated
by Mathews and Sistrom ( 1 9 59) in Sarcina /utea. They observed that
a carotene-less mutant of S. lutea was rapidly killed upon exposure
to light, air and a photosensitizer like toluidine blue whereas the
13 pigmented strain was unaffected. A photoprotective effect offered
by carotenoids was also observed in Corynebacterium poinsettiae
where unlike the pigmented strain, the colorless mutant succumbed
rapidly to light and air in the, presence of an exogenous
photosensitizer (Kunizawa and Stanier, 1958). T~is photosensitive
effect on the mutant strain was not seen in an atmosphere of
nitrogen. Similar experiments without the use of an exogenous
photosensitizer were performed with Mycobacterium marinum. This
bacterium produces carotenoids only in the presence of light;
therefore, dark-grown pigmentless bacteria were compared with
light grown pigmented bacteria for survival under bright light in the
presence of air. The results showed that the non-pigmented bacteria
succumbed more rapidly to the photodamage than the pigmented
cells (Wright and Rilling, 1963).
Fungal Carotenoids
Many fungal species are characterized by the presence of
carotenoid pigments. Both carotenes and xanthophylls have been
reported. They can be found among Phycomycetes, Ascomycetes,
Basidiomycetes and Myxomycetes. Two fungi, Sporidiobolus
johnsonii and Dacryopinax spathularia, showed an increase in
sensitivity to light if the pigment was absent indicating a role for
the carotenoid pigment in photoprotection (Margalith, 1992).
Melanin Pigments
The black pigments often observed in microbes are considered to
be melanin or melanin-like pigments. Melanins are brownish or
14 black pigments obtained by the oxidation of aromatic amino acids,
principally tyrosine, accompanied by polymerization of the
resulting derivatives. Melanins are produced by a number of bacteria
for e.g. the marine bacterium Shewanella colwanella; Proteus
mirabilis as well as Rhizobium spp. Some Ps_eudomonas aeruginosa
strains were shown to produce melanin-like pigments under
specific growth conditions (Margalith, 1992). A number of other
microbes like Serratia marcescens and Streptomycetes have been
shown to produce melanin or melanin-like pigments in the presence
of tyrosine (Trias et a/. 1989; Margalith, 1992). A few Xanthomonas
spp. have also been observed to produce a brown diffusible pigment
e.g. Xanthomonas ampe/ina (causes canker disease on grapevine) and
Xanthomonas phaseoli pv. fuscans (which causes a disease on bean)
{Dye and Lelliott, 1974}. However, it is not known whether this
brown pigment is melanin or a melanin-like pigment.
In fungi, melanin production is observed in structures such as
hyphae, conidia etc. (Margalith, 1992). A number of phytopathogenic
fungi produce melanin (Margalith, 1992; Bell and Wheeler, 1986).
One such interesting example is Magnaporthe grisea which causes
the 'rice blast disease'. The fungus penetrates the host cell wall by
increasing the hydrostatic pressure on its hyphae. Howard and
Ferrari ( 1989) showed that melanins in the cell walls of
appressoria, or swollen hyphal tips, of this pathogen imparts
differential permeability leading to a high internal solute
concentration. This creates a turgor pressure which facilitates the
penetration into the host tissue. Melanin deficient mutants of
Magnaporthe grisea were observed to be non-pathogenic on rice
15 (Chumley and Valent, 1990). Melanin biosynthetic inhibitors like
'tricyclazole' have been useful in prevention of penetration of
appressoria into host tissues and are· therefore being used to
control the rice blast fungus (Woloshuk et · a/. 1983; Margalith,
1992). The human pathogenic fungus, Cryptococcus neoformans,
produces melanin and a polysaccharide coat. Both these phenotypes
have been associated with virulence of the pathogen (Nosanchuk and
Casadevall, 1997).
In several fungi, melanins have been shown to provide protection
to fungal spores against ultraviolet and solar radiation (Margalith,
1992). Survival of conidia and sclerotia over prolonged periods is
correlated with the occurrence of melanins or melanin-like
pigments. It was observed that melanized sclerotia or cell walls
are resistant to attack by microorganisms unlike non-melanized
portions of the two. Melanins have also been proposed to trap free
radicals (hydroxyl radical, OH0) and protect cells from oxidizing
conditions (Goodchild et a/. 1981; Margalith, 1992)
The genes responsible for melanin biosynthesis· have been
described in Streptomyces lividans, Streptomyces antibioticus and
from a number of Rhizobium species. Melanin biosynthetic genes
from Rhizobium are reported to be present on a plasmid (Cubo et a/.
1988). Melanin may be required for detoxification of polyphenolic
compounds which may accumulate in senescing nodules of legumes
(Margalith, 1992).
A great many other pigments, not discussed in this chapter, are
produced by microbes for e.g. bacteriochlorophyll produced by
photosyn,thetic bacteria, phenazine antibiotic pigments produced by
16 certain Pseudomonas aeruginosa strains, riboflavins produced by
fungi, bacterial indigo produced by Pseudomonas indoloxidans to
name a few. The function of some of these pigments is well
established while that of others is largely unknown. The pigments
whose functions are unknown are likely to be more than just
secondary metabolites. Their physiological functions may become
more apparent with further studies.
17 References
Alvarez, A. M., Teng, P. S., and Benedict, A. A. 1989. Methods for
epidemiological research on bacterial blight of rice. In: Bacterial
blight of rice, International Rice Research Institute, P. 0. Box 933,
Manila, the Philippines. pp 99-11 0.
Andrews, A. G., Jenkins, C. L., Starr, M. P., and Hope, H. 1976.
Structure of xanthomonadin I, a novel dibrominated aryl-polyene
pigment produced by the bacterium Xanthomonas juglandis.
Tetrahedron Letters 45: 4023-4024.
Bell, A. A., and Wheeler, M. H. 1986. Biosynthesis and functions of
fungal melanins. Ann. Rev. Phytopathol. 24: 411-451.
Cerda-Olmedo, E. 1985. Carotene mutants of Phycomyces. Methods in
Enzymol. 11 0: 220-243.
Chumley, F. G., and Valent, B. 1990. Genetic analysis of melanin
deficient, nonpathogenic mutants of Magnaporthe grisea. Mol. Plant
Microbe Interact. 3: 135-143.
Cubo, M. T., Buendia-Ciaveria, A. M., Beringer, J. E., and Ruiz-Sainz, J.
E. 1988. Melanin production by Rhizobium strains. Appl. Environ.
Microbial. 54: 181 2-1 81 7.
18 Durgapal, J. C. 1996. Albino mutation in rice bacterial blight
pathogen. Curr. Science 70: 15.
Dye, D. W., and Lelliott, R. A. 1974. Xanthomonas In: Bergey's Manual
of Determinate Bacteriology (Edited by Buchanan, R. E., and Gibbons,
N. E.) The Williams and Wilkins Company, Baltimore. U.S.A. pp 243-
253.
Fujimori, E., and Livingston, R. 1957. Interactions of chlorophyll in
its triplet state with oxygen, carotene, etc. Nature 180: 1036-1038.
Gantotti, B. V., and Beer, S. V. 1982. Plasmid borne determinants of
pigmentation and thiamine prototrophy in Erwinia herbicola. J.
Bacteriol. 1 51: 1627-1629.
Goodchild, N. T., Kwock, L., and Lin, P. S. 1981. Melanin, a possible
cellular superoxide scavenger. In: Oxygen and Oxy-radicals in
Chemistry and Biology (Edited by Rodgers, M.A. J., and Powers, E. L.)
Academic Press, London. pp 645-648.
Goodwin, T. W. 1973. Microbial carotenoids In: Handbook of
Microbiology (Edited by Laskin, I. A., and Lechevalier, H. A.) CRC
Press, Cleveland, Ohio, U. S. A. Vol 3, pp 75-83.
Griffiths, M., Sistrom, W. R., Cohen-Bazire, G., and Stanier, R. Y.
1 9 55. Function of carotenoids in photosynthesis. Nature 1 7 6: 1 211-
1214.
19
Howard, R. J., and Ferrari, M. A. 1989. Role of melanin in
appresorium function. Exp. Mycol. 13: 403-418.
Jenkins, C. L., and Starr, M. P. 1982a. The pigment of Xanthomonas
populi is a nonbrominated ary!-heptaene belonging to xanthomonadin
pigment group II. Curr. Microbial. 7:195-1,98.
Jenkins, C. L., and Starr, M. P. 1982b. The brominated aryl polyene
(xanthomonadin) pigments of X. juglandis protect against
photobiological damage. Curr. Microbial. 7: 323-326.
Johnson, E. A., and Schroeder, W. A. 1995. Microbial carotenoids In:
·Advances in Biochemical Engineering I Biotechnology (Edited by
Fiechter, A.) Springer-Verlag, Berlin. Vol 53: 120-178.
Kunizawa, R., and Stanier, R. Y. 1958. Studies on the role of
carotenoid pigments in a chemoheterotrophic bacterium
Corynebacterium poinsettiae. Arch. Microbial. 31: 146-1 56.
Margalith, P. Z. 1992. Pigment Microbiology. Chapman & Hall, London.
pp 1-157.
Mathews, M. M., and Sistrom, W. R. 1959. Function of carotenoid
pigments in non-photosynthetic bacteria. Nature 184: 1892-1893.
I'"
20 Mathews-Roth, M. M., Wilson, T., Fujimori, E., and Krinsky, N. I. 1974.
Carotenoid chromophore length and protection against
photosensitization. Photochem. and Photobiol. 22: 59-61.
Nosanchuk, J. D., and. Casadevall, A. 1997. Cellular change of
Cryptococcus neoformans : contributions from the capsular
polysaccharide, melanin and monoclonal antibody binding. Infect.
lmmun. 65: 1836-1841.
Poplawsky, A. R., Kawalek, M. D., and Schadd, N. W. 1993. A
xanthomonadin-encoding gene cluster for the Identification of
pathovars of Xanthomonas campestris. Mol. Plant-Microbe Interact.
6: 545-552.
Poplawsky, A. R., and Chun, W. 1997. pigS determines a diffusible
factor needed for extracellular polysaccharide slime and
xanthomonadin production in Xanthomonas campestris pv.
campestris. J. Bacteriol. 179: 439-444.
Starr, M. P. 1981. The genus Xanthomonas. In: The Prokaryotes.
(Edited by Starr, M. P ., Stolp, H., Truper, G. H., Balows, A. and
Schlegel, G. H.), Springer-Verlag, Berlin. Vol. 1, pp 742-763.
Stephens, W. L., and Starr, M. P. 1963. Localization of carotenoid
pigment in the cytoplasmic membrane of Xanthomonas juglandis. J.
Bacteriol. 86: 1070-1074.
21 Spurgeon, S. L., and Porter, J. W. 1980. Carotenoids In: The
Biochemistry of Plants (Edited by Stumpf P. K., and Conn. E. E.).
Academic Press Inc. N. Y. Vol4, pp 419-483.
Tabata, K., Ishida, S., Nakahara, T., and Hoshino, T. 1994. A
carotenogenic gene cluster exists on a large plasmid in Thermus
thermophilus. FEBS letters 341: 251-255.
Trias, J., Vinas, M., Guinea, J., and Loren, J. G. 1989. Brown pigment
in Serratia marcescens cultures associated with tyrosine
metabolism. Can. J. Microbial. 35: 1 037-1 042.
Tsuchiya, K., Mew, T. W., and Wakimoto, S. 1982. Bacterial and
pathological characteristics of wild types and induced mutants of
Xanthomonas campestris pv. oryzae. Phytopathology 72: 43-46.
Woloshuk, P. M., Sisler, M. D., and Vigil, E. L. 1983. Action of the
antipenetrant, tricyclazole, on appresoria of Pyricularia oryzae.
Physiol. Plant Pathol. 23: 245-259.
Wright, L., and Rilling, H. C. 1963. The function of carotenoids in a
photochromogenic bacterium. Photochem. and Photobiol. 2: 339-342.
Yo, K. Y., Lai, E. M., Lee, L. Y., Lin, T. P., Hung, C. H., Chen, C. L., Chang,
Y. S., and Liu, S. T. 1994. Analysis of the gene cluster encoding
carotenoid biosynthesis in Erwinia herbicola Eho13. Microbiology
140: 331-339.
22 CHAPTER II
DEVELOPMENT OF METHODOLOGY FOR CONDUCTING MOLECULAR
GENETIC STUDIES ON AN INDIAN ISOLATE OF XANTHOMONAS
ORYZAE PATHOVAR ORYZAE
Abstract
Xanthomonas oryzae pv. oryzae causes one of the serious
diseases of rice. This chapter describes the standardization of
methods for conducting molecular genetic studies on our laboratory
wild type strain of X. oryzae pv. oryzae, which belongs to the
dominant pathotype of this pathogen in India. Conditions for gene
transfer by electroporation as well as by conjugation with donor E.
coli cells were standardized. A genomic library of this strain, with
an average insert size of 30 Kb, was constructed in the broad host
range cosmid vector pUFR034. Restriction digestion and Southern
hybridization of two randomly selected clones from the library
suggests that cloned DNA is not rearranged when reintroduced into X
oryzae pv. oryzae. The results obtained from colony hybridizations,
using previously' identified sequences from a Philippine isolate of X
oryzae pv. oryzae as probes, indicate that the genomic library
constructed is an adequate representation of the X. oryzae pv .. oryzae
genome. Conditions were also standardized for performing
transposon mutagenesis of cloned X. oryzae pv. oryzae DNA in E. coli
using a mini-TnSgus transposon and marker exchange of these
insertions into the X. oryzae pv. oryzae chromosome.
23 Introduction
Xanthomonas oryzae pv. oryzae is the causal agent of bacterial
leaf blight, a serious disease of rice (Starr, 1981 ). To date most of
the research on this pathogen has been conducted using Philippine
isolates. Owing to quarantine reasons, these strains could not be
used in this study. This chapter describes the standardization of
some methods for conducting molecular genetic studies on our
laboratory wild type strain (called BXO 1 ) of X. oryzae pv. oryzae.
This strain belongs to the dominant pathotype of X. oryzae pv. oryzae
in India (Reddy and Reddy, 1989; Yashitola et a/. 1997). It was
provided to us by Dr. A. P. K. Reddy of the Directorate of Rice
Research, Hyderabad where it was isolated from an infected leaf
sample collected at Chinsuria, West Bengal.
Conditions for gene transfer into BXO 1 by conjugation with donor
E. coli strains as well as by electroporation were standardized. A
genomic library of X. oryzae pv. oryzae was constructed in the broad
host range cosmid vector pUFR034 (DeFeyter et a/. 1990) using
Esherichia coli DHSa, as the host. Plasm ids were isolated from ten
random clones of the genomic library, to estimate the average insert
size. In order to confirm that the library included a complete
representation of the X. oryzae pv. oryzae genome, two previously
identified clones from a Philippine isolate of X. oryzae pv. oryzae
(Kelemu and Leach, 1990; Hopkins et a/. 1992) were used as probes
in colony hybridizations.
A previous report on Xanthomonas campestris pv. malvacearum (a
cotton pathogen) indicated that DNA cloned in E. coli can be
subjected to substantial rearrangement when reintroduced back into
24 X. campestris pv. malvacearum (DeFeyter et a/. 1990). If this were
the case in X. oryzae pv. oryzae, it would interfere with subsequent
genetic analysis of the cloned DNA. Therefore, two randomly
selected clones from the genomic library were mobilized into X
oryzae pv. oryzae. The plasmids were isolated from the
transconjugants and subjected to Southern analysis to assess their
structure in X. oryzae pv. oryzae.
In addition, procedures were standardized to obtain transposon
insertions into cloned X. oryzae pv. oryzae DNA using a mini Tn5
transposon harboring the E. coli gus gene (Wilson et a/. 1995). The
conditions for marker exchange of these Tn5 insertions into the X
oryzae pv. oryzae chromosome were also standardized. This mini Tn5
transposon functions as a promoter-probe transposon where f\
glucuronidase activity is observed only if the gus gene is driven by
endogenous bacterial promoters. Since both X. oryzae pv. oryzae and
r.ice possess minimal endogenous f\-glucuronidase activity, the use
of the mini:-TnSgus transposon would facilitate studies on X. oryzae
pv. oryzae gene expression in planta.
Materials and Methods
Bacterial strains, media etc.
All bacterial strains used are listed in Table 2.1. BX01 is the
laboratory wild type strain that belongs to pathotype I, the dominant
pathotype of X. oryzae pv. oryzae in India (Reddy and Reddy, 1989;
Yashitola et a/. 1997). All X. oryzae pv. oryzae strains used here
were grown on Peptone-Sucrose (PS) medium (Tsuchiya et a/. 1982)