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

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

3. BRAZILIAN BIODIVERSITY Fonte: Lewinsohn & Prado, 2000
TAXON
kNOWN
ESTIMATED
VIRUS
350
55.000
BACTÉRIA
400
FUNGI
13.000
ALGAE
10.000
PLANTS
47.500
52.000
PROTOZOA
3.500
27.000
ANIMALS
TOTAL

4. 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.

5. 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.

6. 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

7. Study of Microbial Communities

8. 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

9. Figure 1: gene rpoB

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

11. 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

12. 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 ( C), rainfall ( mm) and solar radiation ( Cal/cm2/day).

13. No Till X Till

14. 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 F L
16S

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

16. 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.

18. Comparison between Denaturing Gradient Gel Electrophoresis (DGGE) and Phylogenetic Analysis for characterization of A/H3N2 Influenza Samples detected during 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

20. 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.

21. 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.

22. 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.

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