|1205-1||Analysis of the microbial Structure and identification of proteolytic activity from Colombian Andean Glacial ice|
|Autores:||Cesar Augusto Osorio Forero (GEBIX - Centro Colombiano de Genómica Bioinformática de Ambientes) ; Jose Ricardo Bustos (GEBIX - Centro Colombiano de Genómica Bioinformática de Ambientes / GEBIX - Centro Colombiano de Genómica Bioinformática de Ambientes) ; Maria Mercedes Zambrano (GEBIX - Centro Colombiano de Genómica Bioinformática de Ambientes / GEBIX - Centro Colombiano de Genómica Bioinformática de Ambientes / GEBIX - Centro Colombiano de Genómica Bioinformática de Ambientes) |
The composition and structure of microbial communities in diverse and extreme ecosystems in the Colombian Andes is being explored using metagenomic approaches. This study was carried out to analyze the communities present in Andean mountain glacial samples located within the Parque Nacional los Nevados in Colombia.
Material and Methods
Ice samples were collected at two sites (NH2 and NH4) and metagenomic DNA was isolated by filtering thawed ice (5 L per sample) through a 0.22 µm filter. Total DNA was used to PCR amplify and analyze the V5-V6 region of the 16S rRNA gene by 454 pyrosequencing. Whole genome amplification of sample NH2 was done using Multiple Displacement Amplification (MDA) and DNA was used to construct a fosmid library that was used for functional screening of proteases on LB with skim milk. Supernatants of positive clones were then re-assayed for activity using casein as substrate and measuring tyrosine at 280nm.
Results and Discussion
The pyosequencing datasets were filtered, leaving 37,198 and 39,719 sequences for NH2 and NH4 that represented 1,824 and 5,296 OTUs, respectively. Both samples were dominated by the Gamma and Betaproteobacteria at the Class level. Dominant OTUs corresponded to the genera Stenotrophomonas, represented by 1427 sequences from NH4, and Jantinobacterium represented by 2287 sequences from NH2. The metagenomic library from NH2, which contained about 12, 000 clones, was screened for proteases and two clones were identified (NH2f-1 and NH2f-2) with activity of 0.140 and 0.137µmoles/ml/minute at 37oC. DNA from clone NH2f-2 was subcloned in pBluescript II SK, and a single clone with protease activity on LB-skim milk was identified and will be further characterized both enzymatically and by sequence analysis.
Both NH2 and NH4 samples had similar microbial composition and were dominated by Gama and Betaproteobacteria. However, the relative abundances of the different taxa varied. We identified clones with protease activity from sample NH2 that probably come from the dominant Jantinobacterium genus and are currently being analyzed in greater detail. This study shows the presence of microbial communities in ancient Andean mountain surface ice layers.
Palavras-chave: Microbial diversity, Metagenomic, Functional Screening, Glacial Ice