Who lives at Utsteinen?: a look at heterotrophic bacterial diversity through cultivation

In 2007-2008, the Princess Elisabeth Station was built near the Utsteinen nunatak a few kilometres north of the Sør Rondane Mountains. To monitor the environmental impact, base line data on the biodiversity present before construction are needed. For this purpose, samples were collected during an exploratory expedition to the site in January 2007. Samples (BB50 and BB115) were investigated using a cultivation approach. Dilution series were plated on four different media (MA, R2A, ten-times diluted R2A and PYGV) that were incubated at 20°C, 15°C and 4°C and under aerobic and anaerobic conditions. After a growth period of approximately 14 days, 464 (BB50) and 332 (BB115) isolates were taken from the different plates based on morphological differences of colonies. Using a whole-genome fingerprinting technique (rep-PCR), an initial grouping of similar isolates was made. In total 95 (BB50) and 53 (BB115) clusters were delineated and 82 (BB50) and 46 (BB115) isolates formed single branches. Representatives of each cluster and the separate isolates were used in partial 16S rDNA sequencing to obtain a preliminary identification. After cluster analysis of these sequences, phylotypes were delineated at 99.0% 16S rRNA gene sequence similarity. For each phylotype, the 16S rRNA gene sequence of one representative was completed and an approximate identification found by comparison with the EMBL database using the FASTA-algoritm. To obtain a more precise identification, a phylogenetic analysis was performed using the sequences of type strains from all the species mentioned in the FASTA results completed with the related taxa. Using cultivation, rather high numbers of isolates were obtained. More than three quarters of the isolates belonged to a rep-cluster. From the 259 rep-types defined, only two rep-clusters contained isolates from both samples whereas seven phylotypes were in common. Rarefaction curves were obtained for both rep-types and phylotypes. The curves representing the number of phylotypes approximate a plateau indicating an almost sufficient number of isolates to cover the diversity present. In contrast, the curves for the rep-types do not reach a plateau reflecting that rep-PCR fingerprinting has a higher taxonomic resolution than 16S rRNA gene sequencing. This is confirmed by the fact that most phylotypes consist of several rep-clusters. Results show a large diversity, distributed over the major phylogenetic groups (Proteobacteria, Bacteroidetes, Deinococci, Actinobacteria and Firmicutes). For both samples, more than half of the heterotrophic isolates belong to the class of the Actinobacteria, with Arthrobacter as most common genus. The groups Bacteroidetes, Deinococcus-Thermus, Alphaproteobacteria and Betaproteobacteria were recovered in significant numbers. Only a few isolates belong to the Firmicutes and Gammaproteobacteria. Despite the large diversity and the fact that the samples originate from the same general area, only little overlap between the two samples was observed. Some of the isolated phylotypes show low similarity values with neighbouring sequences in the EMBL-database and may represent new species or even new genera. Some species and genera found in this study have never before been reported from Antarctica whereas some others show only high similarity with sequences from clones and isolates from Antarctica or generally cold environments.