Shotgun metagenome guided exploration of anthropogenically driven resistomic hotspots within Lonar soda lake of India.

Anthropogenic activities mediated antibiotic resistance genes (ARGs) in the pristine aquatic bodies (lakes) is raising concern worldwide. Long read shotgun sequencing was used to assess taxonomic diversity, distribution of ARGs and metal resistance genes (MRGs) and mobile genetic elements (MGEs) in six sites within hypersaline Lonar soda lake (India) prone to various anthropogenic activities. Proteobacteria and Euryarchaeota were dominant phyla under domain Bacteria and Archaea respectively. Higher abundance of Bacteroidetes was pragmatic at sites 18LN5 and 18LN6. Functional analysis indicated 26 broad-spectrum ARGs types, not reported earlier in this ecosystem. Abundant ARG types identified were multidrug efflux, glycopepetide, bacitracin, tetracycline and aminogylcoside resistance. Sites 18LN1 and 18LN5 depicted 167 and 160 different ARGs subtypes respectively and rpoB2, bcrA, tetA(48), mupA, ompR, patA, vanR and multidrug ABC transporter genes were present in all samples. The rpoB2 gene was dominant in 18LN1, whereas bcrA gene in 18LN2-18LN6 sites. Around 24 MRGs types were detected with higher abundance of arsenic in 18LN1 and copper in 18LN2-18LN6, signifying metal contamination linked to MRGs. The bacterial taxa Pseudomonas, Thioalkalivibrio, Burkholderia, Clostridium, Paenibacillus, Bacillus and Streptomyces were significantly associated with ARGs. This study highlights the resistomic hotspots in the lake for deploying policies for conservation efforts.

Genetic basis and importance of metal resistant genes in bacteria for bioremediation of contaminated environments with toxic metal pollutants.

Metal pollution is one of the most persistent and complex environmental issues, causing threat to the ecosystem and human health. On exposure to several toxic metals such as arsenic, cadmium, chromium, copper, lead, and mercury, several bacteria has evolved with many metal-resistant genes as a means of their adaptation. These genes can be further exploited for bioremediation of the metal-contaminated environments. Many operon-clustered metal-resistant genes such as cadB, chrA, copAB, pbrA, merA, and NiCoT have been reported in bacterial systems for cadmium, chromium, copper, lead, mercury, and nickel resistance and detoxification, respectively. The field of environmental bioremediation has been ameliorated by exploiting diverse bacterial detoxification genes. Genetic engineering integrated with bioremediation assists in manipulation of bacterial genome which can enhance toxic metal detoxification that is not usually performed by normal bacteria. These techniques include genetic engineering with single genes or operons, pathway construction, and alternations of the sequences of existing genes. However, numerous facets of bacterial novel metal-resistant genes are yet to be explored for application in microbial bioremediation practices. This review describes the role of bacteria and their adaptive mechanisms for toxic metal detoxification and restoration of contaminated sites.

Marine bacteria: potential candidates for enhanced bioremediation.

Bacteria are widespread in nature as they can adapt to any extreme environmental conditions and perform various physiological activities. Marine environments are one of the most adverse environments owing to their varying nature of temperature, pH, salinity, sea surface temperature, currents, precipitation regimes and wind patterns. Due to the constant variation of environmental conditions, the microorganisms present in that environment are more suitably adapted to the adverse conditions, hence, possessing complex characteristic features of adaptation. Therefore, the bacteria isolated from the marine environments are supposed to be better utilized in bioremediation of heavy metals, hydrocarbon and many other recalcitrant compounds and xenobiotics through biofilm formation and production of extracellular polymeric substances. Many marine bacteria have been reported to have bioremediation potential. The advantage of using marine bacteria for bioremediation in situ is the direct use of organisms in any adverse conditions without any genetic manipulation. This review emphasizes the utilization of marine bacteria in the field of bioremediation and understanding the mechanism behind acquiring the characteristic feature of adaptive responses.

Spatio-temporal resolution of taxonomic and functional microbiome of Lonar soda lake of India reveals metabolic potential for bioremediation.

Lonar Lake, India; a hypersaline and hyperalkaline extremophilic ecosystem having a unique microbial population has been rarely explored for bioremediation aspects. MinION-based shotgun sequencing was used to comprehensively compare the microbial diversity and functional potential of xenobiotic degradation pathways with seasonal changes. Proteobacteria and Firmicutes were prevalent bacterial phyla in the pre-monsoon and post-monsoon samples. Functional analysis from SEED-subsystem and KEGG database revealed 28 subsystems and 18 metabolic pathways for the metabolism of aromatic compounds and xenobiotic biodegradation respectively. Occurrence of N-phenyl alkanoic, benzoate, biphenyl, chloroaromatic, naphthalene, and phenol degradation genes depicted varied abundance in the pre-monsoon and post-monsoon samples. Further, KEGG analysis indicated nitrotoluene degradation pathway (ko00633) abundant in post-monsoon samples, and the benzoate degradation pathway (ko00362) predominant in 19LN4S (pre-monsoon) than 18LN7S (post-monsoon) samples. The abundant genes for benzoate degradation were pcaI: 3-oxoadipate CoA-transferase, alpha subunit, pcaH: protocatechuate 3,4-dioxygenase, beta subunit, and pcaB: 3-carboxy-cis, cis-muconate cycloisomerase, and 4-oxalocrotonate tautomerase. This metagenomic study provides a unique blueprint of hitherto unexplored xenobiotic biodegradation genes/pathways in terms of seasonal variations in the Lonar Lake, and warrants active exploitation of microbes for bioremediation purposes.

Characterization of the metabolic pathway and catabolic gene expression in biphenyl degrading marine bacterium Pseudomonas aeruginosa JP-11.

Metabolic pathway of biphenyl assimilation and the catabolic gene expression in a marine bacterium Pseudomonas aeruginosa JP-11, isolated from the coastal sediments of Odisha, India have been studied. This strain utilized 98.86% ± 2.29% of biphenyl within 72 h when supplied as the sole source of carbon, however, preferential utilization of glucose was observed over catechol and biphenyl when grown in a complex medium. Combination of chromatographic and spectrophotometric techniques confirmed the catechol pathway and identified 2-Hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate as the intermediate metabolic product. Assimilation of biphenyl was initiated by its dioxygenation, forming cis-2, 3-dihydro-2, 3-dihydroxybiphenyl subsequently transformed to 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate. In the lower pathway, cis-1, 6-dihydroxy-2, 4-cyclohexadiene-1-carboxylic acid was detected which formed catechol before entering into the Krebs cycle. Detection of key enzyme catechol-1, 2-dioxygenase in the cell-free extract of P. aeruginosa JP-11 supported the proposed degradation pathway. The primary enzyme for biphenyl assimilation, biphenyl dioxygenase encoded by bphA gene was found in the genome of the isolate. On increasing biphenyl stress (50, 100, 150 and 200 mg L(-1)), bphA gene showed a significant (P < 0.01) up-regulation upto 43.5 folds. Production of biosurfactant was confirmed and the rhamnolipid synthesizing gene rhlAB was amplified. This gene also showed a significant (P < 0.01) up-regulation upto 258 folds on increasing biphenyl stress.

Molecular perspectives and recent advances in microbial remediation of persistent organic pollutants.

Nutrition and pollution stress stimulate genetic adaptation in microorganisms and assist in evolution of diverse metabolic pathways for their survival on several complex organic compounds. Persistent organic pollutants (POPs) are highly lipophilic in nature and cause adverse effects to the environment and human health by biomagnification through the food chain. Diverse microorganisms, harboring numerous plasmids and catabolic genes, acclimatize to these environmentally unfavorable conditions by gene duplication, mutational drift, hypermutation, and recombination. Genetic aspects of some major POP catabolic genes such as biphenyl dioxygenase (bph), DDT 2,3-dioxygenase, and angular dioxygenase assist in degradation of biphenyl, organochlorine pesticides, and dioxins/furans, respectively. Microbial metagenome constitutes the largest genetic reservoir with miscellaneous enzymatic activities implicated in degradation. To tap the metabolic potential of microorganisms, recent techniques like sequence and function-based screening and substrate-induced gene expression are proficient in tracing out novel catabolic genes from the entire metagenome for utilization in enhanced biodegradation. The major endeavor of today's scientific world is to characterize the exact genetic mechanisms of microbes for bioremediation of these toxic compounds by excavating into the uncultured plethora. This review entails the effect of POPs on the environment and involvement of microbial catabolic genes for their removal with the advanced techniques of bioremediation.

Extracellular polymeric substances of a marine bacterium mediated synthesis of CdS nanoparticles for removal of cadmium from aqueous solution.

HYPOTHESIS: Microbial extracellular polymeric substances (EPS) are natural metal adsorbent and effective bio-reductant. In the biosynthesis of CdS NPs, functional groups of EPS act as capping and stabilizing agents. The NPs enriched EPS have enhanced adsorption capacity of cadmium ions, which may be due to increased adsorptive sites. EXPERIMENT: The current study demonstrates, an efficient biosynthesis method to prepare CdS NPs using EPS extracted from a marine bacterium Pseudomonas aeruginosa JP-11 and its comparison with chemical method using D-glucose. The synthesized NPs were characterised by Ultraviolet-visible spectroscopy, ATR-FTIR spectrometry, X-ray diffraction, Field emission scanning electron microscopy and Transmission electron microscopy. Atomic absorption spectroscope (AAS) was used to study adsorption capacity of pristine EPS, functionalized EPS and NPs incorporated functionalized EPS. FINDINGS: Spherical CdS NPs of 20-40nm diameter were synthesized with high crystallinity confirmed by XRD, FESEM and TEM analysis. The ATR-FTIR peaks within 2300-2600cm(-1) range showed a prominent shift in sulphydryl group (SH). The cadmium removal efficiency by CdS NPs incorporated functionalized EPS (88.66%) was higher than functionalized EPS (80.81%) and pristine EPS (61.88%) after 48h of incubation. The experimental data of adsorption thermodynamics and kinetics of Cd by NPs incorporated functionalized EPS was fitted in Langmuir isotherm model.

Characterization and cadmium-resistant gene expression of biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11.

Biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11 was isolated from coastal marine sediment of Paradeep Port, Odisha, East Coast, India, which resisted up to 1,000 ppm of cadmium (Cd) as cadmium chloride in aerobic conditions with a minimal inhibitory concentration of 1,250 ppm. Biomass and extracellular polymeric substances (EPS) secreted by the cells effectively removed 58.760 ± 10.62 and 29.544 ± 8.02 % of Cd, respectively. The integrated density of the biofilm-EPS observed under fluorescence microscope changed significantly (P ≤ 0.05) in the presence of 50, 250, 450, 650 and 850 ppm Cd. ATR-FTIR spectroscopy showed a peak at 2,365.09/cm in the presence of 50, 250, 450 and 650 ppm Cd which depicts the presence of sulphydryl group (-SH) within the EPS, whereas, a peak shift to 2,314.837/cm in the presence of 850 ppm Cd suggested the major role of this functional group in the binding with cadmium. On exposure to Cd at 100, 500 and 1,000 ppm, the expression profiles of cadmium resistance gene (czcABC) in the isolate showed an up-regulation of 3.52-, 17- and 24-fold, respectively. On the other hand, down-regulation was observed with variation in the optimum pH (6) and salinity (20 g l(-1)) level. Thus, the cadmium resistance gene expression increases on Cd stress up to the tolerance level, but an optimum pH and salinity are the crucial factors for proper functioning of cadmium resistance gene.

Understanding molecular identification and polyphasic taxonomic approaches for genetic relatedness and phylogenetic relationships of microorganisms.

The major proportion of earth's biological diversity is inhabited by microorganisms and they play a useful role in diversified environments. However, taxonomy of microorganisms is progressing at a snail's pace, thus less than 1% of the microbial population has been identified so far. The major problem associated with this is due to a lack of uniform, reliable, advanced, and common to all practices for microbial identification and systematic studies. However, recent advances have developed many useful techniques taking into account the house-keeping genes as well as targeting other gene catalogues (16S rRNA, rpoA, rpoB, gyrA, gyrB etc. in case of bacteria and 26S, 28S, ß-tubulin gene in case of fungi). Some uncultivable approaches using much advanced techniques like flow cytometry and gel based techniques have also been used to decipher microbial diversity. However, all these techniques have their corresponding pros and cons. In this regard, a polyphasic taxonomic approach is advantageous because it exploits simultaneously both conventional as well as molecular identification techniques. In this review, certain aspects of the merits and limitations of different methods for molecular identification and systematics of microorganisms have been discussed. The major advantages of the polyphasic approach have also been described taking into account certain groups of bacteria as case studies to arrive at a consensus approach to microbial identification.