Variability of biochemical compounds in surface sediments along the eastern margin of the Arabian Sea.

Different fractions of organic matter in surface sediments from three transects along the eastern margin of the Arabian Sea (AS) were quantified to determine the sources of organic matter, and also to study its impact on microbial community structure. From the extensive analyses of different biochemical parameters, it was evident that the distribution of total carbohydrate (TCHO), total neutral carbohydrate (TNCHO), proteins, lipids, and uronic acids (URA) concentrations and yield (% TCHO-C/TOC) are affected by organic matter (OM) sources and microbial degradation of sedimentary OM. Monosaccharide compositions from surface sediment was quantified to assess the sources and diagenetic fate of carbohydrates, suggesting that the deoxysugars (rhamnose plus fucose) had significant inverse relationship (r = 0.928, n = 13, p < 0.001) with hexoses (mannose plus galactose plus glucose) and positive relationship (r = 0.828, n = 13, p < 0.001) with pentoses (ribose plus arabinose plus xylose). This shows that marine microorganisms are the source of carbohydrates and there is no influence of terrestrial OM along the eastern margin of AS. During the degradation of algal material, the hexoses seem to be preferentially used by heterotrophic organisms in this region. Arabinose plus galactose (glucose free wt %) values between 28 and 64 wt% indicate that OM was derived from phytoplankton, zooplankton, and non-woody tissues. In the principal component analysis, rhamnose, fucose, and ribose form one cluster of positive loadings while glucose, galactose, and mannose form another cluster of negative loadings which suggest that during OM sinking process, hexoses were removed resulting in increase in bacterial biomass and microbial sugars. Results indicate sediment OM to be derived from marine microbial source along the eastern margin of AS.

Evaluation of the Physiological Bacterial Groups in a Tropical Biosecured, Zero-Exchange System Growing Whiteleg Shrimp, Litopenaeus vannamei.

To elucidate the individual and multiple roles of physiological bacterial groups involved in biogeochemical cycles of carbon, nitrogen, phosphorus and sulfur, the changes in the abundance of aerobic bacteria (heterotrophs, methane oxidizers, ammonia oxidizers, sulfur oxidizers, phosphate solubilizers, phosphate accumulators) and anaerobic bacteria (total anaerobes, nitrate reducers, denitrifiers and sulfate reducers) were investigated in a biosecured, zero-exchange system stocked with whiteleg shrimp, Litopenaeus vannamei for one production cycle. Key water quality parameters during the 96-day production cycle fell within the normal range for L. vannamei culture. Results of Spearman's correlation matrix revealed that different sets of variables correlated at varying levels of significance of the interrelationships between bacterial abundances and water quality parameters. The three nitrogenous species (ammonia, nitrite and nitrate) strongly influenced the physiological bacterial groups' abundance. The strong relationship of bacterial groups with phytoplankton biomass and abundance clearly showed the trophic interconnections in nutrient exchange/recycling. Canonical correspondence analysis performed to assess the total variation revealed that the three dissolved nitrogen species followed by salinity, temperature, phytoplankton biomass and pH collectively accounted for as much as 82% of the total variation. In conclusion, the results of the study revealed that the major drivers that interweaved biogeochemical cycles are the three dissolved nitrogen species, which microbially mediated various aerobic-anaerobic assimilation/dissimilation processes in the pond ecosystem. Considering the pond microbial ecology becoming an important management tool where applied research could improve the economic and environmental sustainability of the aquaculture industry, the findings of the present study are practically relevant.

Bacterial Community Composition Markedly Altered by Coastal Hypoxia.

Monsoonal upwelling along south and central west coast of India leads to intense biological productivity. As a consequence of excess organic matter production following upwelling during June-October and low dissolved oxygen in the upwelled waters, denitrification occurs in the near shore waters. Implicitly, these processes ought to bring alterations in microbial communities. Therefore, diversity and community structure of bacteria from subsurface layers of a tropical region experiencing intense seasonal lows of oxygen were analyzed through sequencing of 16S rRNA gene clones. The overall diversity was more during hypoxic period of Fall intermonsoon (FIM, October) compared either to Spring intermonsoon (SIM, March) or summer monsoon (SuM, June) months. As many as 14 lineages of bacterial domains: Gammaproteobacteria (37%), Alphaproteobacteria (21%), Cyanobacteria (20%), Deltaproteobacteria (3%), Firmicutes (3%), Betaproteobacteria (2%), Acidobacteria (2%), Actinobacteria (7%), Marinimicrobia (2%), Bacteroidetes (1%), Verrucomicrobia (1%), Planctomycetes (0.4%), Chloroflexi (0.2%) and Omnitrophica bacterium (0.2%), were recognized from our coastal location. Notably, sequences of Planctomycetes, Chloroflexi and Omnitrophica bacterium were found exclusively during FIM. A generally higher representation of sequences of Betaproteobacteria during SuM and of Actinobacteria and Firmicutes during SIM was discernible. This study is thus useful to recognize that microbial community might undergo strong temporal shifts in the monsoon affected tropical coastal ecosystems.

Full-length cloning and phylogenetic analyses of translationally controlled tumour protein and ferritin genes from the Indian white prawn, Fenneropenaeus indicus (H. Milne Edwards).

Elucidation, through molecular analyses, of bacterial afflictions in commercially important aquaculture-reared shrimps is pivotal for the prevention and/or control of disease outbreaks. In this study, we examined the phylogenetic relatedness and compared the possible immune-related functional roles of both translationally controlled tumour protein (TCTP) and ferritin genes with previous studies. Both TCTP and ferritin genes were substantially upregulated in the Indian white prawn, Fenneropenaeus indicus (H. Milne Edwards), post-larvae following bath challenge with the virulent strain of bacteria, Vibrio harveyi D3. Full-length cloning of these genes by rapid amplification of complementary DNA ends -polymerase chain reaction (RACE-PCR) yielded 727-base pair (bp)-long TCTP and 1212-bp-long ferritin gene sequences. Their open reading frames (ORFs) were 507 and 510 bp, respectively. The TCTP-ORF coded for 168 amino acids with three substitutions at positions 37, 141, 155, and the ferritin ORF coded for 170 amino acids with no species-specific substitutions. Phylogenetic analysis suggested the closest relatedness of both TCTP and ferritin from F. indicus to Chinese white prawn, Fenneropenaeus chinensis (Osbeck). In addition to reporting the full-length sequences of these immune-relevant genes, this study highlighted their conserved natures, which perhaps make them important defence-related proteins in the innate immune system of F. indicus.

Molecular characterization of 2-chlorobiphenyl degrading Stenotrophomonas maltophilia GS-103.

The catabolic potential of transformer oil contaminated soil bacteria in aerobic degradation of polychlorinated biphenyls (PCB) were assessed. Transformer oil contaminated soil sample was subjected to microcosm enrichment experiments (PAS medium/biphenyl as sole carbon source). PCB-degrading activity of the enrichment cultures in PAS medium with the addition of 2-chlorobiphenyl were analysed by GC-MS indicated that, although the isolates differed in PCB-degrading capabilities, all of the enrichment cultures expressed activity toward at least some of the lower chlorinated congeners. Biphenyl-utilizing bacteria isolated from the most active PCB-degrading mixed cultures showed little taxonomic diversity and identified as Stenotrophomonas maltophilia GS-103.

Profiling of a few immune responsive genes expressed in postlarvae of Fenneropenaeus indicus challenged with Vibrio harveyi D3.

We identified 38 genes and eight hypothetical proteins by sequencing of 1200 clones from a Vibrio harveyi challenged Fenneropenaeus indicus subtracted cDNA library. Based on physiological roles and functions these genes were categorized into 10 groups with ∼29% of the sequences having no matches in the databases. Immune related transcripts in the library were carboxypeptidase B, ferritin, translationally controlled tumor proteins (TCTP), hemocyanin, chitinase and serine carboxy peptidase. Remarkably, qPCR results imply 4.15, 3.45 and 1.86-fold rises in expression of ferritin, TCTP and hemocyanin transcripts respectively. Additionally, minor upregulation of other immune relevant genes lectin, penaeidin, crustin, MnSOD was observed in the challenged postlarvae.

Distribution of diatom Pseudo-nitzschia and dinoflagellates of Dinophysis spp along coast off Goa.

As a part of an annual analysis on the phytoplankton distribution and composition, regular monthly sampling carried out during October 2007- September 2008 from salinity gradient zones in the intertidal waters along the coast of Goa. Among the 179 species of phytoplankton observed during this study, as many as 11 of them are recognized, potentially toxic ones. The toxic diatom species, Pseudo-nitzschia pungens was quite preponderant, in particular during the pre-monsoon month of May 2008 off Chapora, a perennially low salinity location. Among the 10 toxic dinoflagellate species detected, the known toxic species, Alexandrium minutum followed by Dinophysis acuminata were found to attain maximum cell numbers in the study area. It is apparent from our results that the toxic species do occur in all salinity zones sampled and during many months of the year in coastal waters off Goa. Though directly governed by the variations in nutrient concentrations, some of these toxic phytoplankton species attain high cell numbers. It is reasonable for us to therefore caution that the toxic species do prevail in these waters.

Identification of upregulated immune-related genes in Vibrio harveyi challenged Penaeus monodon postlarvae.

A subtracted cDNA library was constructed and analyzed to elucidate the response of Penaeus monodon postlarvae challenged with Vibrio harveyi. As many as 960 randomly selected cDNA fragments generated through suppression subtractive hybridization were single pass sequenced. Forty five genes and 20 hypothetical proteins were identified, a few being first reports from shrimps. The most abundant immune relevant genes were ferritin, hemocyanin, and TCTP (translationally controlled tumor protein) indicating their upregulation as also confirmed through qPCR. Post-infection qPCR analyses confirmed 2.04, 2.09, 3.28, 5.49, 6.47, and 11.63 fold rise respectively in ferritin, penaeidin, MnSOD, lysozyme, TCTP, and hemocyanin genes. These genes may be involved in the regulation of the host defense against V. harveyi.

Arsenite tolerance and biotransformation potential in estuarine bacteria.

Bacterial isolates from water and sediment samples from freshwater, estuarine and marine regions were tested for their growth in the presence of different concentrations of arsenic. Despite the generation times being longer in case of all bacterial isolates tested in nutrient broth with 200 ppm Arsenite (As(3+)), many of them were able to attain log phase and substantial growth variously between 36 and 96 h. The isolates tolerating >or=200 ppm arsenic (As) were found to belong to Enterobacteriaceae, Pseudomonas, Corynebaterium, Xanthomonas, Acinetobacter, Flavimonas and Micrococcus. Some of these environmental strains tolerant to 1,000 ppm arsenic were tested to realize their potential to detoxify arsenic. The rate of As biotransformation was faster by many of these strains. The percent of arsenite biotransformed/removed from the growth medium was the highest by a strain of Enterobacteriaceae (as much as 92% of the As in the growth medium by 120 h) followed by that of Corynebaterium and Acinetobacter strains. From these observations it is clear that many environmental strains are capable of quite rapid biotransformation of As. Contamination of drinking water by toxic metalloid arsenic affects thousands of people worldwide. Many environmental isolates of bacteria which detoxify this metalloid would serve beneficial in the depuration processes. We suggest that only such strains capable of high tolerance to toxic arsenite, would biotransform As in polluted estuarine environments and would prove useful in As bioremediation applications.

Dispersion and retrievability of water quality indicators during tidal cycles in coastal Salaya, Gulf of Kachchh (West coast of India).

Bacterial indicators in relation to tidal variations were studied at five locations for over 2 days covering three tidal cycles in the southwestern region of Gulf of Kachchh, India. Tidal flow here is predominantly in the east west direction and can transport particles up to 32 km. Tidal amplitude appears to play a prominent role in abundance, distribution, and dispersal of coliform bacteria examined during this study. Shallow depths, clayey sediments, strong currents, and higher tidal amplitudes appear to rise by an order of magnitude in total bacterial abundance up to 2.4 x 10(4) ml(-1) due to their resuspension from the churned up sediments. Dispersal of allochthonous microflora far into coastal marine regions appears to be governed by the strong tidal amplitude in this region.

Abundance of sewage-pollution indicator and human pathogenic bacteria in a tropical estuarine complex.

Studies on abundance and types of various pollution indicator bacterial populations from tropical estuaries are rare. This study was aimed to estimate current levels of pollution indicator as well as many groups of human pathogenic bacteria and their seasonal variations in different locations in Mandovi and Zuari Rivers in the central west coast of India. The sampling covered the estuarine and upstream regions of these rivers representing premonsoon (May 2005), monsoon (September 2006) and post-monsoon (November 2005). Both the abundance and types of autochthonous and allochthonous microbial populations in the near shore environments are affected by land drainages, domestic sewage outfalls and other discharges. The overall ranges (and their mean abundance; no. ml(-1)) of the monitored groups of bacteria were: total coliforms: 0-29,047 (3,134 ml(-1)); total streptococci: 3-14,597 (798); total vibrios: 13-42,275 (2,530); Escherichia coli: 0-1,333 (123); Vibrio cholerae: 0-3,012 (207); Salmonella spp: 0-1,646 (90); Streptococcus faecalis: 0-613 (88) and Aeromonas spp: 0-2,760 (205). In general, abundance of sewage pollution indicator bacteria such as total coliforms and total streptococci was lower than that reported from many other locations worldwide.

Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury.

Pollution in industrial areas is a serious environmental concern, and interest in bacterial resistance to heavy metals is of practical significance. Mercury (Hg), Cadmium (Cd), and lead (Pb) are known to cause damage to living organisms, including human beings. Several marine bacteria highly resistant to mercury (BHRM) capable of growing at 25 ppm (mg L(-1)) or higher concentrations of mercury were tested during this study to evaluate their potential to detoxify Cd and Pb. Results indicate their potential of detoxification not only of Hg, but also Cd and Pb. Through biochemical and 16S rRNA gene sequence analyses, these bacteria were identified to belong to Alcaligenes faecalis (seven isolates), Bacillus pumilus (three isolates), Bacillus sp. (one isolate), Pseudomonas aeruginosa (one isolate), and Brevibacterium iodinium (one isolate). The mechanisms of heavy metal detoxification were through volatilization (for Hg), putative entrapment in the extracellular polymeric substance (for Hg, Cd and Pb) as revealed by the scanning electron microscopy and energy dispersive x-ray spectroscopy, and/or precipitation as sulfide (for Pb). These bacteria removed more than 70% of Cd and 98% of Pb within 72 and 96 h, respectively, from growth medium that had initial metal concentrations of 100 ppm. Their detoxification efficiency for Hg, Cd and Pb indicates good potential for application in bioremediation of toxic heavy metals.

Nutrient regimes and their effect on distribution of phytoplankton in the Bay of Bengal.

The seasonal dynamics of nutrient ratios and abundance of phytoplankton cells from the central (CB) and western (WB) Bay of Bengal (BOB) were studied during the fall intermonsoon (FIM; September-October 2002) and spring intermonsoon (SpIM; April-May 2003). The nutrient molar ratios of macronutrients such as nitrate to phosphate (N:P), nitrate to silicate (N:Si) and silicate to phosphate (Si:P) in the top 120 m were calculated for both FIM and SpIM. During both the seasons, the N:P ratios along the CB and WB were lower than 16, indicating nitrate deficiency. Whereas, along both transects the N:Si ratio was <1 and Si:P>3 in the top 20 and 40 m during FIM and SpIM, respectively, indicating Si enrichment. Relatively greater nutrient concentrations along the WB than the CB appear to contribute to higher phytoplankton abundance. The preponderance of diatoms in the Bay could be attributed to rapid utilization of available nutrients in particular during FIM thus resulting in low N:Si ratios in the water column. Among diatoms, pennales were predominantly controlled by nutrients and their ratios. While, apart from nutrients, physical stratification, light and eddies also seem to influence the distribution and abundance of centrales.

Spatial variations of DMS, DMSP and phytoplankton in the Bay of Bengal during the summer monsoon 2001.

Data on the distribution of dimethylsulphide (DMS) and dimethylsulphoniopropionate (DMSP) in relation to phytoplankton abundance in different oceanic environments is important to understand the biogeochemistry of DMS, which plays an important role in the radiation balance of the earth. During the summer monsoon of 2001 measurements were made for DMS and DMSPt (total DMSP) together with related biological parameters in the Bay of Bengal. Both DMS and DMSPt were restricted to the upper 40 m of the water column. Diatoms accounted for more than 95% of the phytoplankton and were the major contributors to the DMS and DMSPt pool. The mean concentration of DMS in the upper 40 m was observed to be around 1.8+/-1.9 nM in the study area, while DMSPt concentrations varied between 0.7 nM and 40.2 nM with a mean of 10.4+/-8.2 nM. The observed lower DMSPt in the northern Bay in spite of higher mean primary productivity, chlorophyll a and phytoplankton cell counts seemed to result from grazing. Though salinity divides the Bay into different biogeochemical provinces there is no relation between salinity and DMS or DMSPt. On the other hand DMS was linearly related to chlorophyll a:phaeopigments ratio. The results suggest the need for deeper insight into the role of diatoms in the biogeochemical cycling of DMS.

Determinants of site-specific recombination in the lambdoid coliphage HK022. An evolutionary change in specificity.

The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.

Tolerance to various toxicants by marine bacteria highly resistant to mercury.

Bacteria highly resistant to mercury isolated from seawater and sediment samples were tested for growth in the presence of different heavy metals, pesticides, phenol, formaldehyde, formic acid, and trichloroethane to investigate their potential for growth in the presence of a variety of toxic xenobiotics. We hypothesized that bacteria resistant to high concentrations of mercury would have potential capacities to tolerate or possibly degrade a variety of toxic materials and thus would be important in environmental pollution bioremediation. The mercury-resistant bacteria were found to belong to Pseudomonas, Proteus, Xanthomonas, Alteromonas, Aeromonas, and Enterobacteriaceae. All these environmental bacterial strains tolerant to mercury used in this study were capable of growth at a far higher concentration (50 ppm) of mercury than previously reported. Likewise, their ability to grow in the presence of toxic xenobiotics, either singly or in combination, was superior to that of bacteria incapable of growth in media containing 5 ppm mercury. Plasmid-curing assays done in this study ascertained that resistance to mercury antibiotics, and toxic xenobiotics is mediated by chromosomally borne genes and/or transposable elements rather than by plasmids.

Spectrophotometric titration of bismuth with EDTA.

A new spectrophotometnc method for the estimation of bismuth with EDTA, using iron-salicylate complex as the indicator, has been developed. The determinations were carried out by measuring the absorbance at 520 mmu. of solutions containing bismuth, iron(III), salicylic acid and various quantities of EDTA, at pH 0.5. It has been shown from the stability constants of the complexes present that before the end-point iron(III) will not react appreciably with the Bi-EDTA complex. The interference from iron(III) in the estimation of bismuth, which is a serious drawback in many other methods, is eliminated in the present method, as iron(III) acts as the indicator.

Marine environmental pollution stress detection through direct viable counts of bacteria.

Direct viable counts (DVC) of bacteria were quantified from polluted and relatively less/non-polluted coastal locations during different seasons to assess whether they can be routinely monitored for an understanding of environmental stress(es) that may impede the full functioning of bacterial communities in situ. Most notably, DVC were quite low during pre-monsoon (March-May) in pollution-affected locations when compared to relatively less/non-polluted ones. In contrast, their abundance was significantly higher (up to or > 10%) suggesting a substantially higher microbial activity (thus, a larger turnover of organic matter) during monsoon (June-September) and post-monsoon (October-February) even in pollution-affected locations. The ease of reliably measuring DVC was useful in realising decreased metabolic functioning of bacteria during pre-monsoon, a season where dispersion of land discharges/effluents is much lower. From laboratory and field analyses of this study it is ascertained that DVC are direct indices of potential bacterial metabolic activity, reliable for sensing metabolic stress experienced by bacterial communities in situ and can be useful for evaluating risks in marine environment through human (industrial) activities.

Disruption of microbial biofilms by an extracellular protein isolated from epibiotic tropical marine strain of Bacillus licheniformis.

BACKGROUND: Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. METHODOLOGY/PRINCIPAL FINDINGS: B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. CONCLUSION/SIGNIFICANCE: We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.

Bioremediation of toxic substances by mercury resistant marine bacteria.

Bioremediation of toxic substances includes microbe-mediated enzymatic transformation of toxicants to non-toxic, often assimilable, forms. Mercury-resistant marine bacteria are found to be very promising in dealing with mercury, and a host of other highly toxic heavy metals and xenobiotics. In the present studies we have shown that the Pseudomonas aeruginosa CH07 (NRRL B-30604) has been able to degrade a variety of PCB congeners including a complete degradation of CB-126 and CB-181. The culture was able to remove over 70% Cd from growth medium when supplemented with 100 ppm Cd. The same bacterium rapidly biotransformed/removed toxic mercury from wastewater in a bioreactor system.