Indian Journal of Biotechnology Vol 15, April 2016, pp 201-209 Bacterial diversity in sediments of river Mahananda (Siliguri) as determined by 16S rRNA gene analysis Shriparna Mukherjee1,2, Dhananjay Kumar1,3 and Ranadhir Chakraborty1* 1Omics Laboratory, Department of Biotechnology, University of North Bengal (NBU), PO NBU, Siliguri 734 013, India 2Department of Botany, P D Women’s College, Jalpaiguri 735 101, India 3Department of Biotechnology, Birla Institute of Technology, Mesra, Raanchi 835 215 India Received 4 November 2014; revised 16 April 2015; accepted 10 May 2015 Analyses of 16S ribosomal (r) DNA clone library have enabled to assess the bacterial diversity in the sediment of an anthropogenically stressed river Mahananda at Siliguri, India. A total of 178 clones were examined by amplified rDNA restriction analysis (ARDRA) using HaeIII (four base specific restriction enzyme). According to the restriction patterns generated from ARDRA, 71 clones were selected as operational taxonomic units (OTUs) and sequenced. No clones were identical to any of the known 16S rRNA sequences or to each other. Sequencing analysis revealed eight distinct major lineages of bacteria [α-, β-, γ-, ε-Proteobacteria, Bacteroidetes, Firmicutes, Chloroflexi & Planctomycetes]. About 69.66% of the sequenced clones were grouped into Proteobacteria [comprising α (12.90%), β (45.96%), γ (2.41%) & ε (38.70%)] and 29.21% were included amongst the divisions other than Proteobacteria represented by Bacteroidetes (34.61%), Firmicutes (57.69%), Chloroflexi (3.84%) and Planctomycetes (3.84%). 16S rRNA gene phylotypes only distantly related to any of the previously identified sequences (non-affiliated rRNA genes) represent a significant fraction of the total cloned sequences. This is the first report describing microbial diversity of Mahananda river sediment by using metagenomic approach. The results suggest that the sediment of river Mahananda is the reservoir of unexplored microbial resources. Keywords: ARDRA, 16S rDNA, microbial diversity, sediment Introduction In the past, studies have been carried out to explore A special dominion in aquatic ecosystems is the microbial diversity in freshwater sediments of sediment. Compared to the corresponding water lakes, estuary and marine environments8-10. Despite bodies, the biomass and taxon richness of microbes such studies, data on the bacterial composition of are much higher in sediment part1. Due to their high the surface sediments of organically polluted/ abundance relative to the water column, these bacteria anthropogenically stressed river is very limited11. In play a key role in the decomposition, nutrient cycling one of our earlier reports, bacterial diversity of and carbon flux2. A complete knowledge regarding surface water of the river Mahananda of Siliguri, the resident microbial populations will, therefore, India was studied by analysis of 16S rRNA genes be required to understand how the chemical derived from environmental DNA12. In the present environment of sediment is generated and stabilized3. study, diversity of the benthic bacterial assemblages For centuries, rivers have been used as the dumping from sediments of the anthropogenically stressed river grounds for urban effluents, agricultural and industrial Mahananda was determined using 16S rRNA gene wastes that contain substances varying from simple clone library method. nutrients to highly toxic chemicals including heavy metals4-6. Several studies have been focused on the Materials and Methods adverse effects of different pollutants, fecal pathogens, organic and inorganic fertilizers on the Sediment Sampling, Analyses and Site Climate 7 The river Mahananda flows through the middle of river ecosystem . the Siliguri city. The maximum river gorge below the ——————— main Mahananda bridge is about 50 m wide (varies *Author for correspondence: from 10-50 m in different seasons). Sediment samples Tel: +91+353_2776354; Fax: +91-353-2699001 Mobile: +91-9434872273 were collected from both the banks and mid river. [email protected] An ethanol flamed spatula was used to collect soil 202 INDIAN J BIOTECHNOL, APRIL 2016 samples from the 2 to 10 cm of soil from various sequencing was performed with the ABI PRISMTM points. Collected soil samples were brought to the Dye Terminator Cycle Sequencing Ready Reaction laboratory in properly labeled, autoclaved and sealed kit and the reactions were analyzed on an ABI PRISM polyethylene bags on ice. Prior to the total DNA 377 DNA sequencer. The recombinant plasmids isolation, all the soil samples collected from different were sequenced with T7 and SP6 primers from both spots were mixed to homogeneity to make a single ends and the middle portion was sequenced with composite sample. Textural composition of the 530F primer. Only the sequences with overlapping sediment samples (determination of sand, silt & clay) reads were included in further analysis. was not determined. Phylogeny and Statistical Analysis Isolation of Total Soil DNA A CHIMERA_CHECK program of the Ribosomal Total soil (sediment) DNA was isolated using a Database Project II (RDP II) was used to detect the modified CTAB-SDS based DNA extraction 14 13 presence of possible chimeric sequences . In order to technique . RNase (10 mg/mL) treatment, followed find closely related sequence(s) and to obtain a by phenol extraction was carried out prior to PCR preliminary phylogenetic affiliation of the clones, amplification of 16S rRNA gene sequences. submissions of unaligned sequences were made to PCR Amplification and 16S rRNA gene Library Construction the sequence match program of the RDP and to the PCR was performed in 50 µL reaction mixtures advanced BLAST search program of NCBI. The most (40 µM deoxynucleoside triphosphates, 12 pMol of similar reference sequences were retrieved and each primer, 1 U of Taq DNA polymerase [Promega], aligned with clone sequences using Clustal_X15. 1× PCR buffer [Promega] for each DNA sample, Phylogenetic trees were constructed using MEGA using universal bacterial primer 27f (5′- version 4.0 by the neighbor-joining algorithm and the AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′- Jukes-Cantor distance estimation method with TACGGTTACCTTGTTACGACTT-3′). Maintenance bootstrap analyses for 1000 replicates16. of PCR condition and construction of 16S rRNA gene clone library were made according to the methods Nucleotide Sequence Accession Numbers described earlier12. The accession numbers for sequences determined from sediment clone library are as follows: FR690816 ARDRA Method to FR690827, FR691000 to FR691009, FR691051, About 178 clones from sediment clone library were FR691485 to FR691531 and FR729922. chosen for ARDRA analysis. The plasmid inserts were PCR amplified by using vector-specific primers Results SP6 (5′-GATTTAGGTGACACTATAG-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′). All the DNA Extraction, Library Construction and Sequencing PCR assays were carried out in a total volume of analysis 50 µL mixtures ( (40 µM deoxynucleoside triphosphates, The total DNA was extracted from the sediment of 12 pMol of each primer, 1 U of Taq DNA polymerase river Mahananda using modified CTAB-SDS based [Promega], 1× PCR buffer [Promega] for each DNA DNA isolation technique. One clone library was sample). One cycle of PCR was run at 94°C for 5 min, established. The recombinant clones were selected followed by 25 cycles each comprised of 94°C for based on α-complementation (blue-white screening) 30 sec, 47°C for 30 sec, 72°C for 2 min. A final technique and also confirmed by the re-PCR analysis extension was performed at 72°C for 7 min. PCR and restriction enzyme digestion. Sequencing analyses products were digested with HaeIII in accordance included 71 clones. One nonbacterial chloroplastic with the manufacturer’s instructions, electrophoresed DNA was found amongst the recombinant clones. In on a 2% (w/v, solution of agarose in TAE buffer) this study, no primer pair was chosen to amplify the agarose gel, and stained with ethidium bromide. archaeal 16S rRNA gene sequences and, therefore, The band sizes were determined by using 100 bp did not get any archaeal sequences. Our phylogenetic DNA ladder as size standard. analysis revealed that 70 clones from the sediment Sequencing and clone library fell into 5 major lineages of the The clones corresponding to the unique banding domain Bacteria: Proteobacteria, Bacteroidetes, pattern were used for sequencing. Nucleotide Firmicutes, Chloroflexi and Planctomycetes. MUKHERJEE et al: BACTERIAL DIVERSITY IN MAHANANDA RIVER SEDIMENT 203 Proteobacteria α-Proteobacteria Phylum Proteobacteria was represented by 69.66% The subclass ∝- Proteobacteria was represented by of the total sequences. Proteobacterial sequences fell 9 OTUs (16 clones). The cloned sequences belong to within 4 different classes: α-Proteobacteria (12.90%), orders Sphingomonadales, Rhizobiales, Rhodobacterales β-Proteobacteria (45.96%), γ-Proteobacteria (2.41%) and Caulobacterales. The majority of the sequences and ε-Proteobacteria (38.70%). belonged to Sphingomonadales (62.5%). The sequences β-Proteobacteria belonging to Rhodobacterales were 18.75%, while It represented the most dominant subclass of Rhizobiales and Caulobacterales accounted for Proteobacteria comprising of 20 OTUs (consisting of 12.5 and 6.25%, respectively. OTU MS004 was 57 clones) forming four clusters in the phylogenetic related (98%) to uncultured bacterial clone tree. The clusters included four orders, Burkholderiales (DQ337059) isolated from the subsurface water (represented by