Deep-sea mercury resistant bacteria from the Central Indian Ocean: A potential candidate for mercury bioremediation.

Deep-sea bacteria when grown in normal environmental conditions get morphologically and genetically adapted to resist the provided culture conditions for their survival, making them a possible aspirant in mercury bioremediation. In this study, seawater samples were collected from different depths of the Central Indian Ocean and seven mercury resistant bacteria (resistant to 100 mg L-1 concentration of inorganic Hg as HgCl2) were isolated. Based on 16S rRNA gene sequencing, the identified isolates belong to the genera Pseudomonas, Bacillus and Pseudoalteromonas. The presence of the merA gene in the isolates contributes to the effective volatilization of mercury. The Inductively Coupled Plasma Mass-Spectroscopy analysis revealed that the isolates can reduce up to >80% of inorganic mercury. Moreover, Fourier Transform Infrared spectrum analysis indicates that functional groups play a key role in the mechanism of adaptation towards Hg2+ reduction. Thus, the deep-sea bacteria expressed significant tolerance and reduction potential towards ionic mercury.

Existence in cellulose shelters: industrial and pharmaceutical leads of symbiotic actinobacteria from ascidian Phallusia nigra, Andaman Islands.

The diversity of actinobacteria associated with marine ascidian Phallusia nigra from Andaman Islands was investigated. A total of 10 actinobacteria were isolated and based on the biochemical and molecular characterization, the isolates were assigned to 7 different actinobacterial genera. Eight putatively novel species belonging to genera Rhodococcus, Kineococcus, Kocuria, Janibacter, Salinispora and Arthrobacter were identified based on 16S rDNA sequence similarity with the NCBI database. The organic extracts of ten isolates displayed considerable bioactivity against test pathogens, which were Gram-positive and Gram-negative in nature. PCR-based screening for type I and type II polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) revealed that, 10 actinobacterial isolates encoded at least one type of polyketide synthases biosynthesis gene. Majority of the isolates found to produce industrially important enzymes; amylase, protease, gelatinase, lipase, DNase, cellulase, urease, phosphatase and L-asparaginase. The present study emphasized that, ascidians are a prolific resource for novel bioactive actinobacteria with potential for novel drug discovery. This result expands the scope to functionally characterize the novel ascidian associated marine actinobacteria and their metabolites could be a source for the novel molecules of commercial interest.

Taxonomic Composition and Biological Activity of Bacterial Communities Associated with Marine Ascidians from Andaman Islands, India.

Marine invertebrates, particularly ascidians, constitute an important source of potential active and biofunctional natural products. The microbial diversity associated with ascidians is little recognized, although these microorganisms play a vital role in marine ecosystems. The objective of this study was to investigate bacterial population diversity in four ascidian samples: Phallusia nigra, Phallusia fumigata, Eudistoma viride, and Rhopalaea macrothorax, collected from the North Bay, Andaman and Nicobar Islands. Microbial strains identified up to the species level revealed 236 distinct species/ribotypes out of 298 bacterial strains. Of 298 ascidian-associated bacteria, 72 isolates belong to the class Gammaproteobacteria and the genus Endozoicomonas. The results from this investigation will contribute a broaden knowledge of microbial diversity associated to marine ascidians, and as a promising source for the discovery of new natural products.

Molecular characterization, phylogenetic and in silico sequence analysis data of trehalose biosynthesis genes; otsA and otsB from the deep sea halophilic actinobacteria, Streptomyces qinglanensis NIOT-DSA03.

Trehalose, a non-reducing disaccharide (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside) is a natural compound, which serves as a protective substance in halophilic bacterial cells. Trehalose biosynthesis genes (otsA and otsB) were PCR amplified from the genomic DNA of deep sea actinobacteria, Streptomyces qinglanensis NIOT-DSA03. The amplified genes were cloned and nucleotide sequences were determined. In silico sequence and phylogenetic analysis of nucleotides and amino acids of otsA and otsB sequences of S. qinglanensis were also determined. The experimental data described in this study will be helpful to develop a recombinant expression system to produce trehalose for biotechnological applications.

A multiplex PCR kit for the detection of three major virulent genes in Enterococcus faecalis.

A multiplex PCR kit that detects three major virulence genes, gelE, hyl and asaI, in Enterococcus faecalis was developed. Analyses of the available sequences of three major virulence genes and designed primers allowed us to develop the three-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting three amplicon bands for gelE, hyl and asaI were even and distinct with product sizes of 213, 273 and 713 bp, respectively. The multiplex PCR procedure was validated with a total of 243 E. faecalis strains that included 02 ATCC strains, 109 isolates from marine samples (sediment, water and sea foods), 22 isolates from cattle fodder, 79 isolates fresh water samples and 31 isolates from nosocomial samples. Specificity of the kit was indicated by amplification of only three major virulent genes gelE, hyl and asaI without any nonspecific bands. Tests for the limit of detection revealed that amplified genes from the sample with a minimum of 104 CFU/g or CFU/mL (10 cells/reaction) of E. faecalis and lower cell load samples, after a 3 h enrichment in NIOT-E. faecalis enrichment medium at 37 °C, a sensitivity level of 10 CFU/g or CFU/mL was achieved.

Characteristics and dynamics of Salmonella diversity and prevalence of biomarker genes in Port Blair Bays, South Andaman, India.

Salmonella is a major cause of morbidity and mortality in humans worldwide, and the infection with multidrug-resistant strains can cause severe diseases. Many coastal cities around the world discharge their wastewaters into the marine environment. These wastewaters contain a variety of pathogenic microorganisms that may have a role in the contamination of this ecosystem and have potential risks for public health. Using an environmental approach, the present study investigated the presence of Salmonella in sediment and water samples collected from Port Blair Bays. In this environmental approach, the provided information about the diversity of the Salmonella serovars, antibiotic resistance and the prevalence of virulence factors in Salmonella, especially from the coastal waters of Port Blair Bays. The occurrence of Salmonellae was significantly higher in water column samples (2.9%) than in those taken from the marine sediments (0.7%). Of the 133 positive Salmonella strains, 22 different serovars were identified. Salmonella enterica serovar Senftenberg was the predominant serovar, being represented by 54 isolates (42.5%), followed by serovar Typhimurium (19 isolates [15%]) and serovar Agona (12 isolates [9.4%]). The presence of virulence genes (filC, sitC, hilA, invA, sipC, hilD, hilC, invF, invE, invH, sipF, aadA, pare, gyrA, spaP and parC) and susceptibility studies with 10 selected antibiotics were also performed. The results of this study revealed that all Salmonella isolates were positive for targeted virulence genes and were resistant to at least one antibiotic. Antibiotic susceptibility studies revealed the presence of multidrug resistant Salmonella strains in coastal water, which usually from land base sources end up in the marine environment and may pose a significant risk on public health.

Ectoine biosynthesis genes from the deep sea halophilic eubacteria, Bacillus clausii NIOT-DSB04: Its molecular and biochemical characterization.

Ectoine, the most prominent osmolyte in nature, is a vital compatible solute present in halophilic bacterium. It protects the cellular biomolecules of the halophilic bacteria and retains their intrinsic function from extreme circumstances. In the current research, ectoine biosynthesis gene cluster (ectABC) in Bacillus clausii NIOT-DSB04 was expressed heterologically in E. coli M15 (pREP4). RP-HPLC resolved several fractions of the purified recombinant product, one of which had been confirmed as ectoine. The recombinant ectoine was further characterized by 1H and 13C Nuclear Magnetic Resonance. The purified recombinant ectoine was also authenticated by FT-IR studies with the existence of ester carbonyl and C-H group. In IPTG induced E. coli M15 transgenic cells, the enzymatic activity of the ectA, B and C genes were found to be higher than that of uninduced cells.

Studies on diversity of Vibrio sp. and the prevalence of hapA, tcpI, st, rtxA&C, acfB, hlyA, ctxA, ompU and toxR genes in environmental strains of Vibrio cholerae from Port Blair bays of South Andaman, India.

Vibrio species are widely distributed in the estuarine and coastal waters that possess the greatest threat to human health worldwide. In this study it is aimed to isolate and observe the abundance of Vibrio sp. and prevalence of biomarker genes and antibiotic resistance profile of V. cholerae isolated from the Port Blair bays of South Andaman. A total of 56 water samples were collected from the seven sampling stations of Port Blair bays in which maximum number of Vibrio sp. population density (1.78 × 104) was recorded in Phoenix Bay. Among the 786 isolates 57.38% of the isolates were confirmed as Vibrio sp., Vibrio cholerae and Vibrio parahaemolyticus. PCR results revealed that the prevalence of biomarker genes was recorded maximum in the isolates from Phoenix Bay and Junglighat Bay samples. Upon further analysis, it was observed that the prevalence of hlyA gene (215 bp), was found to be the most widespread biomarker determinant in 84.17% of isolates. Major virulence determinants; ctxA, ompU and toxR genes were not detected in V. cholerae isolates from Port Blair bays. Maximum antibiotic resistance pattern was observed in Phoenix Bay isolates and maximum number of V. cholerae isolates was resistance to tetracycline (60.76%). Cluster and Principal Component Analysis were employed to understand the diversity and distribution of Vibrio isolates and its biomarker genes. Upon PCA analysis seasonal influence was not much perceived in Vibrio species diversity in Port Blair bays and the lack of significant difference in the detection of species diversity in this study is due to resemblance in geographical conditions and sources of pollution.

Biodiversity and antibacterial potential of cultivable halophilic actinobacteria from the deep sea sediments of active volcanic Barren Island.

Deep Sea sediment cores were collected from the surrounding of active volcanic Barren Island, Andaman & Nicobar Islands. A total of 123 cultivable marine actinobacteria were isolated and identified based on their biochemical and 16S rDNA sequences. The isolates were categorized under 10 genera, of which Streptomyces sp., Dietzia sp. and Brevibacterium sp. are the dominant genera. Of 123 isolates, 73 isolates exhibited appreciable inhibitory activity against both Gram positive and Gram negative bacteria. High frequencies of positive PCR amplification were obtained for PKS-I (39.13%), PKS-II (56.52%) and NRPS (69.57%). The highest levels of biosynthetic systems were observed for NRPS and PKS-II. Majority of the actinobacterial isolates revealed excellent potential for bioprospecting of novel byproducts with industrial and pharmaceutical importance.

Functional characterization of a major compatible solute in Deep Sea halophilic eubacteria of active volcanic Barren Island, Andaman and Nicobar Islands, India.

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a compatible solute, serves as a protective compound in many halophilic eubacterial cells under stress. In this study, the ectoine biosynthesis genes (ectA, B and C) from the genomic DNA of a deep sea eubacteria, Bacillus clausii NIOT-DSB04 was PCR amplified, cloned into the expression vector pQE30 with a 6 × histidine tag and expressed in M15 cells. The lysates of induced cells with diaminobutyric acid aminotransferase and ectoine synthase disclosed two clear expressed bands with molecular masses of 46 kDa and 15 kDa as estimated by SDS-PAGE. The recombinant ectoine synthase activity of the expressed cells was at higher level than that of uninduced cells. In silico sequence and phylogenetic analysis of nucleotides and amino acids revealed that the ectA, B and C sequences of Bacillus clausii NIOT-DSB04 were conserved in many eubacteria.

Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy.

l-Asparaginase is an antineoplastic agent that selectively reduces the level of l-asparagine in blood and diminishes the proliferation of cancerous cells. Studies were carried out on the cloning and heterologous expression of l-asparaginase biosynthesis gene (ansA) from Nocardiopsis alba NIOT-VKMA08 to achieve the stable inducible system that overproduces the glutaminase-free recombinant l-asparaginase. Overexpression of recombinant l-asparaginase was achieved with an optimized final concentration of 1.5mM of isopropyl-ß-d-thiogalactoside (IPTG) and the enzyme was expressed as a soluble protein. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and the purified enzyme disclosed an elevated level of asparaginase activity (158.1IU/mL). Optimum pH and temperature of the purified l-asparaginase for the hydrolysis of l-asparagine were 8.0 and 37°C and it was very specific for its natural substrate, l-asparagine. Detailed studies were carried out on the kinetics of enzyme reaction, catalytic activity, temperature and ionic strength and the thermostability of the l-asparaginase enzyme. The functional characterisation of the recombinant l-asparaginase was studied through Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), in silico sequence analysis and protein structural modelling. Glutaminase activity was not detected in the recombinant l-asparaginase, which could reduce the probable side effects during leukaemia therapy.

L-Asparaginase from Streptomyces griseus NIOT-VKMA29: optimization of process variables using factorial designs and molecular characterization of L-asparaginase gene.

Marine actinobacteria are known to be a rich source for novel metabolites with diverse biological activities. In this study, a potential extracellular L-asparaginase was characterised from the Streptomyces griseus NIOT-VKMA29. Box-Behnken based optimization was used to determine the culture medium components to enhance the L-asparaginase production. pH, starch, yeast extract and L-asparagine has a direct correlation for enzyme production with a maximum yield of 56.78 IU mL(-1). A verification experiment was performed to validate the experiment and more than 99% validity was established. L-Asparaginase biosynthesis gene (ansA) from Streptomyces griseus NIOT-VKMA29 was heterologously expressed in Escherichia coli M15 and the enzyme production was increased threefold (123 IU mL(-1)) over the native strain. The ansA gene sequences reported in this study encloses several base substitutions with that of reported sequences in GenBank, resulting in altered amino acid sequences of the translated protein.

Enterococcus species diversity and molecular characterization of biomarker genes in Enterococcus faecalis in Port Blair Bay, Andaman and Nicobar Islands, India.

This study was performed to evaluate the abundance and diversity of Enterococcus sp. and the distribution of biomarker genes in Enterococcus faecalis in Port Blair Bay, Andaman and Nicobar Islands. The Enterococcus sp. densities at the seven sampling stations were highly influenced by tidal fluctuations and season. The distributions and diversities of species varied in the inner and outer regions of Port Blair Bay. Among the 1816 total isolates, the occurrence of fecal Enterococcus was high (1.78×10(4) CFU/100 mL) in Phoenix Bay. Moreover, 67.76% of the isolates were identified as Enterococcus, and the most frequently identified species were E. hirae, E. avium and E. faecalis. Assessments of antibiotic resistance and biomarker genes revealed the maximum occurrence in the Aberdeen Bay isolates. The most prevalent biomarker genes observed in the E. faecalis isolates were gelE and asa1, whereas cyl was not found among the isolates. In silico sequence analysis of biomarker genes of E. faecalis also revealed that they are evolutionarily well conserved with those of earlier reports. Further, multivariate analysis distinguished the JB, PB and OS stations from the other stations according to distinctive microbial densities and compositions. In addition, the Shannon-Wiener diversity indices and box-whisker plots further facilitated and supported the multivariate results.

Heterologous expression, purification, and phylogenetic analysis of oil-degrading biosurfactant biosynthesis genes from the marine sponge-associated Bacillus licheniformis NIOT-06.

Surfactin is a lipopeptide, composed of one ß-hydroxy fatty acid, a long fatty acid moiety, and seven amino acids. In this study, the biosurfactant biosynthesis genes; 4'-pantetheinyl transferase (sfp), phosphopantetheinyl transferase (sfpO), and surfactin synthetase (srfA) have been characterized from the marine sponge-associated Bacillus licheniformis NIOT-06 from the Andaman and Nicobar Islands. The purified recombinant biosurfactant revealed excellent emulsification activity with crude oil and kerosene. Reverse-phase high-performance liquid chromatography resolved the purified recombinant biosurfactant into several fractions and one of which had significant surface tension reducing property. Fourier transform infrared spectroscopy spectrum also revealed the presence of C-N-N, alkenes, and N-H as the functional groups, and a similar overlapping pattern was observed with that of standard lipopeptide surfactin. The diversity and phylogeny of sfp, sfpO, and srfA gene sequences were compared with other eubacteria. The sfp, sfpO, and srfA gene sequences obtained from Bacillus licheniformis NIOT-06 were diverse and appeared to be partially conserved when compared with the GenBank reported sequences of several eubacteria.

Novel glutaminase free L-asparaginase from Nocardiopsis alba NIOT-VKMA08: production, optimization, functional and molecular characterization.

Studies were carried out for the optimization and production of novel extracellular glutaminase-free L-asparaginase from Nocardiopsis alba NIOT-VKMA08. Among the tested carbon and nitrogen sources, maximum L-asparaginase production was observed with a combination of L-asparagine and maltose (1.5%) and twofold increase in yield (18.47 IU mL(-1)) was observed with newly optimized NIOT-asparaginase medium. Activity of the purified enzyme was moderately inhibited by various divalent cations and thiol group blocking reagents, with K(m) and V(max) of 0.127 mM and 5.50 U µg(-1). Optimum pH and temperature of purified L-asparaginase for the hydrolysis of L-asparagine was 8.0 and 37 °C, respectively. The enzyme inhibited polyacrylamide formation in 10% solution and it was very specific for its natural substrate L-asparagine. Partial glutaminase activity was not detected, which could reduce the possibility of side effects during cancer therapy. L-Asparaginase biosynthesis gene (ansA) was cloned and transformed in E. coli JM109. The ansA gene sequence reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in altered amino acid sequences of the translated protein.

Novel marine actinobacteria from emerald Andaman & Nicobar Islands: a prospective source for industrial and pharmaceutical byproducts.

BACKGROUND: Andaman and Nicobar Islands situated in the eastern part of Bay of Bengal are one of the distinguished biodiversity hotspot. Even though number of studies carried out on the marine flora and fauna, the studies on actinobacteria from Andaman and Nicobar Islands are meager. The aim of the present study was to screen the actinobacteria for their characterization and identify the potential sources for industrial and pharmaceutical byproducts. RESULTS: A total of 26 actinobacterial strains were isolated from the marine sediments collected from various sites of Port Blair Bay where no collection has been characterized previously. Isolates were categorized under the genera: Saccharopolyspora, Streptomyces, Nocardiopsis, Streptoverticillium, Microtetraspora, Actinopolyspora, Actinokineospora and Dactylosporangium. Majority of the isolates were found to produce industrially important enzymes such as amylase, protease, gelatinase, lipase, DNase, cellulase, urease and phosphatase, and also exhibited substantial antibacterial activity against human pathogens. 77% of isolates exhibited significant hemolytic activity. Among 26 isolates, three strains (NIOT-VKKMA02, NIOT-VKKMA22 and NIOT-VKKMA26) were found to generate appreciable extent of surfactant, amylase, cellulase and protease enzyme. NIOT-VKKMA02 produced surfactant using kerosene as carbon source and emulsified upto E(24)-63.6%. Moreover, NIOT-VKKMA02, NIOT-VKKMA22 and NIOT-VKKMA26 synthesized 13.27 U/ml, 9.85 U/ml and 8.03 U/ml amylase; 7.75 U/ml, 5.01 U/ml and 2.08 U/ml of cellulase and 11.34 U/ml, 6.89 U/ml and 3.51 U/ml of protease enzyme, respectively. CONCLUSIONS: High diversity of marine actinobacteria was isolated and characterized in this work including undescribed species and species not previously reported from emerald Andaman and Nicobar Islands, including Streptomyces griseus, Streptomyces venezuelae and Saccharopolyspora salina. The enhanced salt, pH and temperature tolerance of the actinobacterial isolates along with their capacity to secrete commercially valuable primary and secondary metabolites emerges as an attractive feature of these organisms. These results are reported for the first time from these emerald Islands and expand the scope to functionally characterize novel marine actinobacteria and their metabolites for the potential novel molecules of commercial interest.