METAGENOMIC APPROACH TO STUDY MICROBES FROM HIMALAYAN GEOTHERMAL SPRINGS

Abstract

The present study includes a comprehensive analysis of microbial population and their functional properties from two Himalayan geothermal springs through culture independent approach. The first part of the study includes metagenomic DNA isolation and purification from environmental sample of Himalayan thermal springs. Subsequently, total community based bacterial diversity of the thermal springs was assessed. The metagenomic functional studies included cloning and characterization of endoglucanase gene responsible for cellulose hydrolysis. In addition to the above, L-asparaginase-II enzyme from a culturable bacterial isolate was also studied. Real time PCR based transporter gene identification from metagenomic DNA pool of Himalayan hot spring was also carried out. Microbial communities of the sulphur hot spring at Tattapani geothermal area of Himachal Pradesh state and Tapovan geothermal spring located in Chamoli District of Uttarakhand state, India were analyzed. 16S rRNA gene based microbial identification and phylogenetic affiliation of microorganisms from these hot springs was carried out by culture independent approach. 16S rRNA gene based metagenomic libraries were constructed from the hot spring mat DNA. Restriction Fragment Length Polymorphism (RFLP) patterns of cloned 16S rRNA gene from both the springs were analyzed. About 89 clones from both the constructed libraries were sequenced and their non-chimeric validation was done by using pintail version 1.1. The non-redundant nucleotide sequences were deposited in the GenBank database and have accession numbers JN613324, JN896893 to JN896938, JN934657 to JN934666, JN967771 to JN967773, KC608724 to KC608751. Among Tattapani hot spring derived clones, 35% of the clones belonged to the phyla Proteobacteria which consist of Alpha-proteobacteria (4%), Betaproteobacteria (7%), Gamma-proteobacteria (11%), and Delta-proteobacteria (13%) respectively. Other phyla identified were 25% of Acidobacteria, 5% of Planctomycetes, 14% of Verrucomicrobia, 4% each of Bacteroidetes, Chloroflexi, Gemmatimonadetes and 9% of total represented unaffiliated clones. These were classified into 8 distinct phyla. Similarly 3 distinct phyla were identified in Tapovan spring 16S rRNA library. 79% of the clones belonged to the phyla Firmicutes, 14% to Proteobacteria and 7% represented unclassified bacteria found in Tapovan library. The Tapovan clone sequences showed similarity to various species of Anoxybacillus and Aneurinibacillus genera. About 37 clones found collectively in the libraries ii showed <97% sequence similarity with the known database of NCBI. Hence, these organisms were affiliated as new species as per the species concepts of microorganisms. Operational Taxonomic Unit was calculated to about 24 for Tattapani and 8 for Tapovan derived clones at 97% similarity cutoff. Shannon-weiner index was calculated as 3.923088 and 1.6705014 for Tattapani and Tapovan springs respectively. The Maximum possible value for species evenness was found to approach towards one i.e. 0.9834805 and 0.8033413 for Tattapani and Tapovan springs respectively. The Neighbor-Joining based phylogenetic tree deduced from both the hot springs showed that microbial signatures differ significantly. The overall microbial diversity of Tattapani spring was higher than Tapovan spring. This is the first report of comprehensive analysis of microbes and their diversity in these Himalayan thermal springs using metagenomics approach. The aim to conduct functional study of hot spring DNA led to the construction of metagenomic libraries in easily culturable host Escherichia coli. Small insert metagenomic libraries were constructed using plasmid pNYL-rygC. The Tapovan and Tattapani metagenomic DNA was partially digested with Sau3A I and the vector pNYL-rygC was digested with Bam HI. About 2000 clones from each of the libraries were screened for cellulase and xylanase using direct agar plate containing specified substrate. But no visible clone was detected with hydrolysis activity. The predictable cause for this lack of activity may be attributed to difficulty in heterologous gene expression or with the vector system used for the study. To alleviate the limitations imposed by this approach, an alternative method using direct polymerase chain reaction based method was applied. Metagenomic DNA from Tapovan hot spring was used to identify endo beta- glucanase gene (TM-cel5A) using direct PCR. Degenerate primers were constructed for endo betaglucanase gene and were used to amplify the gene from Tapovan hot spring DNA. The purified PCR product was cloned in pET28(a) expression vector. The codon plus host system was used to minimize codon biasness. The cellulose hydrolysis activity of the functional gene products derived from clones was analyzed on carboxy methyl cellulose (CMC) agar plate and stained with Congo red. Clones with variable hydrolysis pattern were observed on the screening plate. The clone with largest hydrolysis zone was selected. The over expression of gene product in the selected clone was studied using IPTG at variable concentrations and analyzed on PAGE. TMiii Cel5A was found to be stable at 60 ⁰C for 1hour and its activity decreased to to 12% after 2 hours of incubation. TM-Cel5A showed an optimal activity at pH 8.0. TM-Cel5A showed activity in a wide range of pH i.e. 4 to 9. TM-Cel5A also showed the ability to use a wide range of substrates. It has the ability to hydrolyze natural form of storage glucan (Beta- D- glucan). Sequence analysis showed that TM-Cel5A was identical to endoglucanase of Bacillus sp. and with Bacillus licheniformis. Thus, to the best of our knowledge this is the first report of cloning and characterization of beta-endoglucanase thermostable enzyme from a Himalayan thermal spring. The community level microbial analysis based on Biolog EcoPlate analysis showed that microbes in both the hot springs were able to utilize Pyruvic Acid Methyl Ester, Tween 40, Tween 80, D- Xylose, Mallic acid, L-Asparagine, L-Threonine and Glycyl-L-glutamic acid. LArginine is the amino acid which was exclusively utilized by Tapovan microbes. A bacterial isolate was also identified from Tapovan spring water sample producing L-asparaginase enzyme. The L-asparaginase producer was found to be a closest relative of Ralstonia sp. Its Lasparaginase production was confirmed by growth on Modified Czapek Dox medium containing L-asparagine as sole carbon source and by nessler’s reagent based biochemical assays. The gene namely L-asparaginase-II responsible for asparaginase activity was cloned and expressed in Escherichia coli BL21-CodonPlus cells. L-Asparaginase is the enzyme which is well known for its application in food processing and treatment of acute lymphoblastic leukaemia (ALL). In addition to the above mentioned functional characterization of microbes in Himalayan springs, a real time PCR based detection of genes responsible for transporters and antibiotic resistance markers was done. Inductively coupled plasma mass spectroscopy (ICPMS) and ionic chromatography (IC) analysis of Tapovan water sample showed presence of high loads of sulphur and heavy metals. Hence, bacterial efflux systems were studied that can respond to such stressful environmental conditions. These transporter/efflux systems play dual role in efflux of heavy metals as well as modern antibiotics leading to multidrug resistance in microorganisms. In order to detect genes responsible for efflux systems in Tapovan metagenomic DNA, real time PCR were carried out using degenerate primers. The qPCR results showed detectable amounts of transporter genes cusA, acrB and acrD. CusA is an integral part of CusCFBA system and is responsible for efflux of copper/silver. AcrB, AcrD are part of Resistance –Nodulation- Cell iv division (RND) family of transporters, involved in efflux of amphiphilic substances. No Human mitochondrial DNA (mtDNA) and E. coli plasmid pET23a-GFP DNA were found in the metagenomic DNA. This indicates that the sample was free from anthropogenic and modern routine laboratory contaminations. Analysis and quantification of genes was done using threshold (CT) values. Low CT value of Acriflavin resistance protein B (AcrB) was found. The acrB gene was cloned in pTZ57R/T vector and the insert was sequenced. The sequence analysis showed highest identity with acrB gene of Escherichia coli APEC O78 and Acriflavin resistance protein of Escherichia coli P12b. The presence of AcrB, AcrD is also supported by the factor that they are the well known members of hydrophobe/amphiphilic efflux-1 (HAE-1) family of transporters. Their overexpression during nutrient deficient condition and involvement in efflux of currently used antibiotics, disinfectants, dyes, detergents further confirms their presence. The presence of AcrB, AcrD and CusA in Himalayan springs can be attributed to the presence of heavy metals like Al, As, Cu, Fe, B, Se and nutrient deprived conditions.