16S rRNA gene diversity in cyanobacterial-bacterial mat consortia of King George Island, Maritime Antarctica

Cecilia Callejas1 Emanuel M. de Souza2 and Silvia Batista1

1 Molecular Microbiology Unit, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, C.P. 11600, Uruguay 2 Department of Biochemistry and Molecular Biology, Federal University of Paraná , Curitiba, PR, C.P. 19046 Brazil

INTRODUCTION METHODOLGY

IN THE WET LAB IN THE DRY LAB Antarctic environment is being strongly affected by Global

Climate Change. We use a combination of molecular SAMPLE COLLECTION SEQUENCING methods to describe microbial phylotypes present in different bacterial-cyanobacterial mats from King George Island,

Maritime Antarctica. Our results can be used as a baseline to ENVIRONMENTAL DNA SEQUENCE QUALITY assess future impacts on Antarctic microbial communities. PURIFICATION -PHRED (Ewing and Green, 1998)

PCR 16S rRNA MULTIALIGNMENT EUBACTERIA, CYANOBACTERIA AND ARCHAEA -CLUSTAL W (Larkin et al, 2008) -BIOEDIT (Hall, 1999) OBJECTIVE CLONING AND LIBRARY MULTIBLAST CONSTRUCTION -RDP (Cole et al., 2009) Analyze 16S rRNA gene sequence diversity from cyanobacteria, eubacteria and archaea in four terrestrial microbial mats from Fildes PLASMID PURFICATION AND DIVERSITY INDICES Peninsula (King George/ 25 de Mayo Island). SEQUENCING -DOTUR (Schloss and Handelsman, 2005)

RESULTS

16S rRNA GENE LIBRARIES FROM FOUR MICROBIAL COMMUNITIES IDENTIFICATION USING SEQUENCE MATCH IN RDP

•3 sites, 4 different terrestrial microbial mats . % Eubacteria Cyanobacteria

100% % •3 libraries (cyano, eub and archaea) for each 100% site. 80% 80% OBJECTIVE 60% •Each library contains 150 clones approx. 40% 60%

20% 40%

* Lane, D. J. (1991) in Nucleic Acid 0% M7 M8 M15 M16 Sample Techniques in Bacterial Systematics, 20% eds. Acidimicrobidae Actinobacteridae Anaerolineales Stackebrandt, E. & Goodfellow, M. Bdellovibrionales Burkholderiales Caulobacterales 0% M7 M8 M15 M1 6 (Wiley, New York), pp. 115–175. Clostridiales Family I Family XIII Flavobacteriales Gemmatimonadales Gp3 Sample Myxococcales Family I Family XIII Chlo roplast F amily I F amily IV ** Nubel et al., (1997) PCR primers to Flavobacteriales Rhodobacterales Spartobacteria_genera_incertae_sedis F amily VI Family XIII G p4 amplify 16S rRNA genes from Sphingomonadales unclassified_"Chloroflexi" unclassified_Alphaproteobacteria Planctomycetales Sparto bacteria_genera_incertae_sed is u nclass ified_ Cyan obacte ria unclassified_Betaproteobacteria unclassified_Cyanobacteria unclassified_Deltaproteobacteria cyanobacteria Verrucom icro biales Appl. Environ. Microbiol. 63: 3327- Verrucomicrobiales Xanthomonadales 3332. •Proteobacteria was the most abundant •Cyanobacterial libraries from the four samples RAREFACTION CURVES phylum in eubacterial libraries from M7 and analyzed were dominated by Family IV and a high M8. percentage of unclassified sequences were found. •Some unculturable groups such as Nº OTUs Cyanobacteria Eubacteria •On the other hand, cyanobacteria was the Verrucomicrobia, Plantomyces and Acidobacteria 30 Nº OTUs most abundant group in eubacterial libraries were found in low abundance in all cyanobacteria 60 from M15 and M16. libraries. 25 50 20 40 NUMBER AND DISTRIBUTION OF OTUs 15 30

10 20 Eubacteria Nº OTUs 10 5 m7 m8 m15 m16 35 60 eub 3% 18 18 9 30 16 16 8 0 14 25 14 0 7 12 cia 3% 12 20 6 10 10 5 0 20 40 60 80 100 50 m7 m8 8 m15 m16 15 4 8 6 0 20 40 60 80 100 6 10 3 4 4 2 2 5 2 1 0 0 0 Nº seq 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 M7 M8 M15 M16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 3 5 7 9 11 13 15 17 19 21 23 25 M7 M8 M15 M16 Nº seq 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 30 Cyanobacteria DIVERSITY INDICES 20

m7 m8 m15 m16 10 35 9 18 18 8 16 16 30

7 14 14 25 Cianobaterias Eubacterias 6 12 12 H H 20 5 10 10 Shannon (H) Shannon (H) m7 m8 m15 m16 4 8 8 0 15 3.5 4.5 3 6 6 10 2 4 4

5 4 Sample 1 2 2 3 M7 M8 M15 M16 0 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 3 5 7 9 11 13 15 17 19 21 23 25 3.5 2.5 3

2 2.5

2 1.5 •Samples M7 and M8 had a higher number of eubacterial OTUs compared to M15 and 1.5 1 M16. 1

0.5 0.5 •There were no differences in the number of cyanobacterial OTUs in any of the four 0 0 0 1 2 3 4 5 0 1 2 3 4 5 libraries. Muestra Muestra •Some OTUs were in a much greater abundance in relation to the others in all the

Chao 1 libaries. Cianobacterias Chao 1 Eubacterias

Chao 1 350 Chao 1 300

300 250 REFERNCES 250

200 200 Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. (2009) The Ribosomal Database 150 150 Project: improved alignments and new tools for rRNA analysis Nucleic Acids Res 37(Database issue):D141-5. Schloss PD, Handelsman J. (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ 100 100 Microbiol 71(3):1501-6. Ewing, B. and Green, P.(1998) Basecalling of automated sequencer traces using Phred. II. Error probabilities. Genome Res 8:186-194. 50 50 Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H.*, Valentin F.*, Wallace I.M., Wilm A., Lopez R.*, Thompson J.D., Gibson T.J. and Higgins D.G. (2007) ClustalW and ClustalX version 2. Bioinformatics 23(21): 2947-2948. 0 0 0 1 2 3 4 5 Hall, T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser 41:95-98. 0 1 2 3 4 Muestra5 Muestra

AKNOWLEDGMENT We wish to thank all the Institutions that supported this work. RESEARCH • Diversity indices did not show clear differences between the four COUNCIL OF NORWAY, TINKER FOUNDATION, SCAR, ANII, PEDECIBA, INSTITUTE PASTEUR communities analyzed. MONTEVIDEO , URUGUAYAN ANTARCTIC INSTITUTE.,