DNA barcoding in Microorganisms Alexandre Soares Rosado Institute of Microbiology UFRJ - Brazil

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

BRAZILIAN BIODIVERSITY Fonte: Lewinsohn & Prado, 2000 TAXON kNOWN ESTIMATED VIRUS 350 55.000 BACTÉRIA 400 136.000 FUNGI 13.000 205.000 ALGAE 10.000 PLANTS 47.500 52.000 PROTOZOA 3.500 27.000 ANIMALS 132.000 1.337.000 TOTAL 207.000 1.867.000

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.

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.

Application of molecular fingerprinting techniques to study the composition and dynamics of soil microbial communities Advantages Cultivation-independent analysis of large numbers of samples Top-to-bottom analysis: Sequencing of differentiating bands Taxon-specific primers Use of probes to identify bacterial isolates corresponding to differentiating bands

Study of Microbial Communities

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

Figure 1: gene rpoB

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

Denaturing gradient gel electrophoresis Molecular fingerprints of microbial communities „community“ DNA or RNA PCR amplification of 16S or 18S rDNA fragments Denaturing gradient gel electrophoresis (DGGE, TGGE) Few prominent populations (low evenness): patterns with few bands Many equally abundant populations (high evenness): patterns with many bands

Soil aggregation and bacterial community structure as affected by tillage and cover cropping in the Brazilian Cerrados Peixoto 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).

No Till X Till

16S A B L TW1 TW2 T1 T2 NTW1 F2 NT1 NT2 F1 NTW2 L L TW1 TW2 T1 T2 NTW1 NTW2 NT1 NT2 F1 F2 L 16S

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

Biodiversity DGGE- Fingerprints 16S rDNA Diversity of bacterial comunities Identification/ 16S rDNA-Sequencing 1b Bacillus megaterium 2b Arthrobacter sp. 1s Sphinghomonas 2s Streptomyces galbus 3s Streptomyces sp. 4s Nocardia 5s Pseudomonas sp. 6s Pseudomonas 1p Bacillus megaterium 2p unknown Bacterium 3p unknown Bacterium 1r Pseudomonas sp.

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 III IIIb a IV V IIIb III I 2 1 3 5 4 6 7 IIIb IV Cluster Cluster IV Cluster V b V VI Cluster Cluster IV c

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 phyla Further, 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.

Biodiversity and Systematics Important issues Biodiversity and Systematics Our 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. 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.

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.

SBM Acknowledgments: Laboratory of Molecular Microbial Ecology –LMME Petrobras CNPq SBM Sociedade Brasileira de Microbiologia