Comparative analysis of microbiome inhabiting oxygenated and deoxygenated habitats using V3 and V6 metabarcoding of 16S rRNA gene.

We examine how oxygen levels and the choice of 16S ribosomal RNA (rRNA) tags impact marine bacterial communities using Next-Generation amplicon sequencing. Analyzing V3 and V6 regions, we assess microbial composition in both Oxygen minimum zones (OMZ) and non-OMZ (NOMZ) areas in the Arabian Sea (AS) and the Central Indian Ocean basin (CIOB) respectively. Operational taxonomic units (OTUs) at 97% similarity showed slightly higher richness and diversity with V6 compared to V3. Vertical diversity patterns were consistent across both regions. NOMZ showed greater richness and diversity than OMZ. AS and CIOB exhibited significant differences in bacterial community, diversity, and relative abundance at the order and family levels. Alteromonadaceae dominated the OMZ, while Pelagibacteraceae dominated the NOMZ. Synechococcaceae were found exclusively at 250 m in OMZ. Bacteria putatively involved in nitrification, denitrification, and sulfurylation were detected at both sites. Dissolved oxygen significantly influenced microbial diversity at both sites, while seasonal environmental parameters affected diversity consistently, with no observed temporal variation.

Diversity and Quantitative Detection of Clade I Type nosZ Denitrifiers in the Arabian Sea Oxygen Minimum Zone.

A significant amount of nitrous oxide (N2O) is effluxed into the atmosphere as a result of marine denitrification in the Arabian Sea (AS) oxygen minimum zone (OMZ). An assessment of temporal variations in the diversity and abundance of nosZ denitrifiers was performed to establish the relative importance of these bacteria in denitrification. Sampling was conducted at the Arabian Sea Time Series (ASTS) location and a quantitative PCR (qPCR) ana-lysis was performed. We detected a high abundance of the nosZ gene at core OMZ depths (250| |m and 500 m), indicating the occurrence of denitrification in the AS-OMZ. The maximum abundance of the nosZ gene was observed during the Spring Intermonsoon (SIM) at 250| |m (1.32×106 copies L-1) and 500| |m (1.50×106 copies L-1). Sequencing ana-lysis showed that nosZ denitrifiers belonged to the classes Alpha-, Beta-, and Gammaproteobacteria. Taxonomic ana-lysis revealed that most OTUs were affiliated with Pseudomonas, Rhodopseudomonas, and Bradyrhizobium. Diversity indices and richness estimators confirmed a higher diversity of nosZ denitrifiers at 250| |m than at 500| |m during all three seasons. The present results also indicated that dissolved oxygen (DO) and total organic carbon (TOC) are critical factors influencing the diversity and abundance of the nosZ-denitrifying bacterial community.

Chromate detoxification potential of Staphylococcus sp. isolates from an estuary.

Chromium (Cr) pollution is an emerging environmental problem. The present study was carried out to isolate Cr-resistant bacteria and characterize their Cr detoxification and resistance ability. Bacteria screened by exposure to chromate (Cr6+) were isolated from Mandovi estuary Goa, India. Two isolates expressed high resistance to Cr6+ (MIC ≥ 300 µg mL-1), Cr3+ (MIC ≥ 900 µg mL-1), other toxic heavy metals and displayed a pattern of resistance to cephalosporins and ß-lactams. Biochemical and 16 S rRNA gene sequence analysis indicated that both isolates tested belonged to the Staphylococcus genus and were closely related to S. saprophyticus and S. arlettae. Designated as strains NIOER176 and NIOER324, batch experiments demonstrated that both removed 100% of 20 and 50 µg mL-1 Cr6+ within 4 and 10 days, respectively. The rate of reduction in both peaked at 0.260 µg mL-1 h-1. ATP-binding cassette (ABC) transporter gene involved in transport of a variety of substrates including efflux of toxicants was present in strain NIOER176. Through SDS-PAGE analysis, whole-cell proteins extracted from both strains indicated chromium-induced specific induction and up-regulation of 24 and 40 kDa proteins. Since bacterial ability to ameliorate Cr6+ is of practical significance, these findings demonstrate strong potential of some estuarine bacteria to detoxify Cr6+ even when its concentrations far exceed the concentrations reported from many hazardous effluents and chromium contaminated natural habitats. Such potential of salt tolerant bacteria would help in Cr6+ bioremediation efforts.

Response to chromate challenge by marine Staphylococcus sp. NIOMR8 evaluated by differential protein expression.

Liquid Chromatography-Mass Spectrometry-Quadrupole Time of Flight (LC/MS QToF) protein profiling of marine-derived Staphyloccous gallinarum NIOMR8 was carried out to evaluate proteins conferring chromate (Cr6+) resistance and possible metabolic pathways that were altered as a result. Expressional (up or down-regulation) responses to varying Cr6+ (0, 50, 100, 150, and 200 µg mL- 1) concentrations varied, with as many as 346 proteins identified. Most number of proteins-their numbers in parentheses-were up-regulated when grown in medium with 50 µg mL- 1 (162) and, down-regulated in medium with 100 (281) or 200 µg mL- 1 Cr6+ (280). Among these, eight proteins were commonly up-regulated, while 58 were commonly down-regulated across all conditions of Cr6+. Expression of protein moieties in metabolic pathways like translation (38), transcription (14), replication (18) and repair (4), metabolism of carbohydrates (26), amino acids (27), nucleotides (17), and membrane transport (21) was evidenced. Up-regulation patterns suggest that reduction of molecular oxygen (5), DNA repair (4) and peptide misfolding (7) were the potential protective mechanisms employed to counter Cr6+ stress. Additionally, proteins associated with biofilm and cell wall biogenesis highlight their hypothetical involvement in toxicity tolerance. Results also indicate that at higher concentrations of Cr6+, down-regulation of functional proteins impedes normal cellular functions.

Efficient bioremediation of tannery wastewater by monostrains and consortium of marine Chlorella sp. and Phormidium sp.

This study evaluated the bioremediation potential of two marine microalgae Chlorella sp. and Phormidium sp., both individually and in consortium, to reduce various pollutants in tannery wastewater (TW). The microalgae were grown in hazardous 100% TW for 20 days, and the reductions in biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN), total phosphorous (TP), chromium (Cr) and total dissolved solids (TDS) of the wastewater monitored periodically. Both marine isolates reduced the BOD and COD by ≥90% in the consortium and by over 80% individually. Concentrations of TN and TP were reduced by 91.16% and 88%, respectively, by the consortium. Removal/biosorption efficiencies for chromium ranged from 90.17-94.45%. Notably, the TDS, the most difficult to deal with, were reduced by >50% within 20 days by the consortium. The novel consortium developed in this study reduced most of the ecologically harmful components in the TW to within the permissible limits of discharge in about 5 to 15 days of treatment. Thus, both the tested marine strains of Chlorella and Phormidium sp. are promising for bioremediating/detoxifying TW and adequately improve the water quality for safe discharge into open water bodies, in particular when used as a consortium.

Improved Sprouting and Growth of Mung Plants in Chromate Contaminated Soils Treated with Marine Strains of Staphylococcus Species.

Marine bacteria possess a wide variety of bioremediation potential which is beneficial environmentally and economically. In this study, bacterial isolates from marine waters were screened for tolerance and growth in high concentrations of chromate (Cr6+). Two isolates, capable of tolerating Cr6+ concentrations 300 µg mL-1 or higher, and found to completely reduce 20 µg mL-1 Cr6+ were grown in Cr6+ (50 and 100 mg kg-1) spiked garden soil. Notably, both facilitated normal germination and growth of mung (Vigna radiata) seeds, which could hardly germinate in Cr6+ spiked garden soil without either of these bacteria. In fact, large percent of mung seeds failed to sprout in the Cr6+ spiked garden soil and could not grow any further. Apparently, chromate detoxification by marine bacterial isolates and the ability of mung plants to deal with the reduced form appear to work complementarily. This study provides an insight into marine bacterial abilities with respect to chromium and potential applications in promoting growth of leguminous plants-similar to mung in particular-in Cr6+ contaminated soil.

Plant growth promoting potential and phylogenetic characteristics of a lichenized nitrogen fixing bacterium, Enterobacter cloacae.

Lichens are complex symbiotic association of mycobionts, photobionts, and bacteriobionts, including chemolithotropic bacteria. In the present study, 46 lichenized bacteria were isolated by conventional and enrichment culture methods on nitrogen-free bromothymol blue (NFb) medium. Only 11 of the 46 isolates fixed nitrogen on NFb and had reduced acetylene. All these 11 isolates had also produced siderophore and 10 of them the IAA. Further, ammonia production was recorded from nine of these nitrogen fixers (NF). On molecular characterization, 16 S rRNA sequencing recorded that, nine NF belonged to Proteobacteria, within Gammaproteobacteria, and were closely related to Enterobacter sp. with a maximum similarity to Enterobacter cloacae. Each one of our NF isolates was aligned closely to Enterobacter pulveris strain E443, Cronobacter sakazakii strain PNP8 and Providencia rettgeri strain ALK058. Notably, a few strains we examined found to possess plant growth promoting properties. This is the first report of Enterobacter sp. from lichens which may be inhabit lichen thalli extrinsically or intrinsically.

PCR detection of ansA from marine bacteria and its sequence characteristics from Bacillus tequilensis NIOS4.

As many as 71 marine bacterial DNA extracts were PCR screened for L-asparaginase (ansA), a key gene in anti-cancer molecular-searches. Over 62% (44) of them were positive for ansA gene. The positive cultures were from genera Bacillus and Staphylococcus. The ansA gene cloned from isolate NIOS4 belonging to recently described Bacillus tequilensis is 1099 bp in length with a 990 bp ORF coding for 329 amino acids. BLASTx analysis revealed this sequence to be 98% similar to earlier reported ansA sequence from B. subtilis (Accession no. NP390239.1). By comparing its deduced amino acid sequence with other bacterial asparaginase sequences six substitutions at positions 305(Thr), 313(Lys), 314(Leu), 315(Asp), 318(Arg), and 320(Gln) are observed. Key residues like Thr(12), Thr(85), Asp(86), Lys(156), and Phe(165) taking part in active-site formation and imparting catalytic properties are conserved. The phylogenetic tree based of the ansA amino acid sequences revealed close relatedness of the NIOS4 ansA sequence with B. subtilis (Accession no. NP 390239.1). It's very close genetic resemblance to B. subtilis and conservation of certain key amino acid residues suggest it as a prospective candidate for evaluation and, production of L-asparaginases.

Microbial community response during the iron fertilization experiment LOHAFEX.

Iron fertilization experiments in high-nutrient, low-chlorophyll areas are known to induce phytoplankton blooms. However, little is known about the response of the microbial community upon iron fertilization. As part of the LOHAFEX experiment in the southern Atlantic Ocean, Bacteria and Archaea were monitored within and outside an induced bloom, dominated by Phaeocystis-like nanoplankton, during the 38 days of the experiment. The microbial production increased 1.6-fold (thymidine uptake) and 2.1-fold (leucine uptake), while total cell numbers increased only slightly over the course of the experiment. 454 tag pyrosequencing of partial 16S rRNA genes and catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) showed that the composition and abundance of the bacterial and archaeal community in the iron-fertilized water body were remarkably constant without development of typical bloom-related succession patterns. Members of groups usually found in phytoplankton blooms, such as Roseobacter and Gammaproteobacteria, showed no response or only a minor response to the bloom. However, sequence numbers and total cell numbers of the SAR11 and SAR86 clades increased slightly but significantly toward the end of the experiment. It seems that although microbial productivity was enhanced within the fertilized area, a succession-like response of the microbial community upon the algal bloom was averted by highly effective grazing. Only small-celled members like the SAR11 and SAR86 clades could possibly escape the grazing pressure, explaining a net increase of those clades in numbers.

Denaturing gradient gel electrophoresis profiling of bacterial communities composition in Arabian Sea.

Denaturing gradient gel electrophoresis (DGGE) was used to elucidate spatial and temporal variations in bacterial community composition (BCC) from four locations along the central west coast of India. DNA extracts from 36 water samples collected from surface, mid-depth (-10 m) and dose to bottom (-20 m) during premonsoon, postmonsoon, monsoon were analyzed by PCRfor amplifying variable region of 16S rRNAgene and subsequently through DGGE. Prominent bands were excised, cloned and sequenced indicated the preponderance of gammaproteobacteria, bacteroidetes and cyanobacteria. Non-metric dimensional scaling of the DGGE gels indicated that the spatial variations in BCC were prominent among the sampling locations. Temporal variations in the BCC appear to be influenced by monsoonal processes. The canonical correspondence analyses suggest that the concentration of chlorophyll a and nitrate are two important environmental factors for both spatial and temporal variations in BCC. Chlorophyll a seems to be impart a top-down control of BCC while nitrate, the bottom-up control. Our results also suggest that BCC can vary over a small geographic distance in highly dynamic, seasonally predisposed tropical coastal waters.

Phylogenetic diversity of archaeal 16S rRNA and ammonia monooxygenase genes from tropical estuarine sediments on the central west coast of India.

Phylogenetic diversity analyses of archaeal 16S rRNA and ammonia monooxygenase subunit A (AamoA) genes were carried out on sediment samples from the Mandovi and Zuari estuaries on the central west coast of India. The 16S rRNA gene libraries revealed quite high diversity of archaea in these sediments compared to previous reports from tropical and temperate estuarine sediments. Uncultured members of Crenarchaeota accounted for approximately 78% of 433 archaeal 16S rRNA gene clones from both of the estuaries. We detected archaeal 16S and amoA gene-related organisms capable of ammonia oxidation. Among Crenarchaeota, marine group I (MG I) was the most predominant. Clones matching the uncultured methanobacteria were predominant among the ribogroups of Euryarchaeota. Our results indicate that archaeal diversity in tropical estuarine sediments is influenced by the mangrove vegetation bordering the lower stretches of both estuaries. Higher diversity may be related to elevated land drainage during the monsoon, particularly in the Mandovi estuary sediments. Also, the diversity of AamoA sequences was higher in Mandovi sediments than those from Zuari and other tropical and/or temperate estuaries studied previously.

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay.

A 745-bp luxA fragment was amplified from Vibrio harveyi (UM 1503), radiolabeled, and used as a probe to detect and quantify luxA genotypes in culturable bacterial populations from the Chesapeake Bay. DNA samples from 53 reference strains were also examined for this gene. The luxA-positive bacteria comprised from 0-6% of the culturable heterotrophic bacterial community in samples from the Bay. Only those reference strains known to be luminescent contained the luxA gene, as indicated by PCR. Results in all cases were confirmed by PCR of DNA extracts and Southern hybridization analyses, using an internal probe for confirmation of luxA amplification products. Sequence analysis of luxA genes from three nonluminescent bacteria isolated from the Chesapeake Bay indicated little or no differences when compared with luxA sequences from known marine luminescent bacterial species. These three Chesapeake Bay strains and other luxA-positive strains were tested with a luminometer and confirmed to be nonluminescent. All of over 7800 bacterial colonies enumerated during this study from Chesapeake Bay samples were non-visibly luminescent. Our results indicate that luxA-positive bacteria isolated from the Chesapeake Bay are not generally luminescent on phenotypic examination, implying that gene probe techniques are required for examining luxA gene distribution in microbial populations present in environmental samples.