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DNA barcoding in Microorganisms

Alexandre Soares RosadoAlexandre Soares Rosado

Institute of MicrobiologyInstitute of Microbiology

UFRJ - BrazilUFRJ - Brazil

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Bergey’s Manual = 4500 SpeciesSome studies DNA reassociation= 10.000 genomes / g soil; 1% - 5% of microorganismos are culturableThe majority = uncuturable

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BRAZILIAN BIODIVERSITYFonte: Lewinsohn & Prado, 2000

TAXON kNOWN ESTIMATED

VIRUS 350 55.000

BACTÉRIA 400 136.000

FUNGI 13.000 205.000

ALGAE 10.000 55.000

PLANTS 47.500 52.000

PROTOZOA 3.500 27.000

ANIMALS 132.000 1.337.000

TOTAL 207.000 1.867.000

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DNA barcoding

DNA barcoding is a methodology for identifying species using a short DNA sequence. It is purported to be a reliable, inexpensive and easily accessible tool for both taxonomic specialists and non-specialists (e.g., government officials, professionals in health and agriculture).

To date, rRNA genes are the most frequently used target for identifying microorganisms, because not only do they occur in all living organisms, but they typically are also present in several copies that are distributed over the genome.

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Background information

Based on the information, many molecular tools (e.g. FISH, T-RFLP, PCR, RAPD, Sequencing etc.) have been developed to discriminate many microorganisms. Of the methods, DNA sequencing generally provides the most accurate means for identifying them. For species identities, investigators generally compare their own DNA sequence to GenBank database by BLAST search. In each case, result quality depends mainly on the reference databank being used. However, each of these databases contains many errors, putting limits on their value for diagnosing differently originated organisms. Further, in cases that DNA sequence matched is not found and hit score queried is low, it is difficult to identify them based on BLAST search.

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Top-to-bottom analysis:Sequencing of differentiating

bandsTaxon-specific primersUse of probes to identify bacterial

isolatescorresponding to differentiating

bands

Application of molecular fingerprinting techniques to study the

composition and dynamics of soil microbial communities

Cultivation-independent analysis of large numbersof samples

Advantages

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Study of Microbial CommunitiesStudy of Microbial Communities

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The rRNA-gene - an ideal molecular marker?

„The molecular clock“

Ubiquitous distribution

Functionally conserved in all forms of life

Regions of different degrees of conservation

Disadvantages

Different number of ribosomal operons

Sequence heterogeneities

A given variable region allows a different resolution for

different taxa

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Figure 1: gene rpoB

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Figure 2: Distance (p) among strains of Paenibacillus 16S and rpoB

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Many equally abundant populations (high evenness): patterns with many bands

Few prominent populations (low evenness): patterns with few bands

Molecular fingerprints of microbial communities

Denaturing gradient gel electrophoresis(DGGE, TGGE)

PCR amplification of 16Sor 18S rDNA fragments

„community“ DNA or RNA

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Soil aggregation and bacterial community structure asaffected by tillage and cover cropping in the

Brazilian CerradosPeixoto et al., 2006

The Cerrados region in central Brazil occupies 22% of the country. It is characterized by high average temperature (22 - 270C), rainfall (800 - 1600 mm) and solar radiation (475- 500 Cal/cm2/day).

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0 -5 cm 5 -1 0 cm

W ith w in ter cover crop s

0 -5 cm 5 -1 0 cm

W ith ou t w in ter cover crop s

N o T illa ge

0 -5 cm 5 -1 0 cm

W ith w in ter cover crop s

0 -5 cm 5 -1 0 cm

W ith ou t w in ter cover crop s

T illa ge

0 -5 cm 5 -1 0 cm

N a tive F ores t

E x per im en ta l S ta tion

No Till X Till

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L TW1 TW2 T1 T2 NTW1 F2 NT1 NT2 F1 NTW2 L

A

B

L TW1 TW2 T1 T2 NTW1 NTW2 NT1 NT2 F1 F2 L

Linkage Distance

F2/5-10 F1/5-10 F2/0-5 F1/0-5

NT2/5-10NT1/5-10

NTw2/5-10NTw1/5-10

NT2/0-5 NT1/0-5

NTw2/0-5NTw1/0-5

T2/5-10 T1/5-10

Tw2/5-10Tw1/5-10

T2/0-5 T1/0-5

Tw2/0-5 Tw1/0-5

0 2 4 6 8 10

16S

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L TW1 TW2 T1 T2 NTW1 NTW2 NT1 NT2 F1 F2 L

Linkage Distance

F2/5-10 F1/5-10 F2/0-5 F1/0-5

NT1/5-10NTw2/5-10

NT2/5-10 NT2/0-5 NT1/0-5

NTw1/5-10NTw2/0-5NTW1/0-5

Tw2/0-5 T2/5-10 T1/5-10

Tw2/5-10Tw1/5-10

T2/0-5 T1/0-5

Tw1/0-5

0 2 4 6 8 10

rpoB

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DGGE- Fingerprints 16S

rDNA

Diversity of bacterial comunities

Identification/ 16S rDNA-Sequencing

1b Bacillus megaterium2b Arthrobacter sp.1s Sphinghomonas2s Streptomyces galbus3s Streptomyces sp.4s Nocardia5s Pseudomonas sp.6s Pseudomonas 1p Bacillus megaterium2p unknown Bacterium3p unknown Bacterium1r Pseudomonas sp.

BiodiversityBiodiversity

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Firmicutes

Bacteroidetes

Cyanobacteria

Chlamydiae

Deltaproteobacteria

Gammaproteobacteria

Betaproteobacteria

Alphaproteobacteria

Actinobacteria

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Comparison between Denaturing Gradient Gel Electrophoresis (DGGE) and Phylogenetic Analysis for

characterization of A/H3N2 Influenza Samples detected during 1999-2004 epidemics in Brazil.

Comparison between Denaturing Gradient Gel Electrophoresis (DGGE) and Phylogenetic Analysis for

characterization of A/H3N2 Influenza Samples detected during 1999-2004 epidemics in Brazil.

Cluster I Cluster II Cluster IIIIIIb

a

IIIb IIIb

IVCluster

Cluster IV Cluster V

b

V VICluster V

Cluster

Cluster IVV

c

IV

IV

V

IIIbIII

I

2

1

3

5

4

6

7

RioGdeSul/417/04

RioGdeSul/411/04

Parana/291/04

Parana/298/04

RiodeJaneiro/26/04

Parana/313/04

MinasGerais/163/04

MinasGerais/160/04

RioGdeSul/406/04

StaCatarina/380/04

RiodeJaneiro/17/04

MinasGerais/156/04

StaCatarina/379/04

MinasGerais/154/04

RiodeJaneiro/99/03

RiodeJaneiro/98/03

Parana/308/04

Parana/312/04

Parana/306/04

RioGdeSul/212/04

RioGdeSul/214/04

RioGdeSul/211/04

RioGdeSul/213/04

Fujian/411/02

RiodeJaneiro/346/03

StaCatarina/327/02

RiodeJaneiro/205/02

StaCatarina/311/02

StaCatarina/339/02

RiodeJaneiro/478/01

RiodeJaneiro/565/01

RiodeJaneiro/533/01

EspiritoSanto/3/99

EspiritoSanto/33/99

RiodeJaneiro/57/99

RioGdeSul/25/99

RioGdeSul/21/99

Panama/2007/99

EspiritoSanto/454/01

RiodeJaneiro/28/00

RiodeJaneiro/470/01

RiodeJaneiro/465/01

RiodeJaneiro/471/01

Sydney/5/97

Udorn/307/72

Aichi/2/68100

7594

82

90

98

78

91

88

82

99

87

98

0.02

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Mangroves

Our knowledge of mangrove associated bacteria has been limited by a pronounced sampling, culturing and experimental bias. Prokaryotic organisms recognized so far are only few bacterial phylaFurther, most isolates/ or strains studied hitherto have yet been correctly identified to species-level.

Thus, the aim of this project is to determine accurate, precise 16S DNA sequences of more than 1,300 bp from sediments and rizosphere of mangrove plants tighter with traditional methods, and then to evaluate them to advance phylogentic relationships and DNA barcoding.

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Biodiversity and SystematicsOur knowledge of biodiversity needs to be greatly expanded by doubling the rate of taxonomic inventories and species discovery and description by 2015.

This will require a commensurate increase in taxonomic expertise and infrastructure.

The rapidly developing field of informatics and communications technology must be harnessed both to facilitate scientific work and to disseminate taxonomic products to all users, including the general public.

Important issues

There are several issues and problems, however, that need to be addressed before barcoding can be instituted, especially for single-celled microorganisms . Linking barcodes to accurately identified species represents a large hurdle that must be overcome.

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When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs.

Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.

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Acknowledgments:

SBMSociedadeBrasileira deMicrobiologia

Laboratory of Molecular Microbial Ecology –LMME

Petrobras

CNPq