Cellular and genetic mechanism of bacterial mercury resistance and their role in biogeochemistry and bioremediation.

Mercury (Hg) is a highly toxic element that occurs at low concentrations in nature. However, various anthropogenic and natural sources contribute around 5000 to 8000 metric tons of Hg per year, rapidly deteriorating the environmental conditions. Mercury-resistant bacteria that possess the mer operon system have the potential for Hg bioremediation through volatilization from the contaminated milieus. Thus, bacterial mer operon plays a crucial role in Hg biogeochemistry and bioremediation by converting both reactive inorganic and organic forms of Hg to relatively inert, volatile, and monoatomic forms. Both the broad-spectrum and narrow-spectrum bacteria harbor many genes of mer operon with their unique definitive functions. The presence of mer genes or proteins can regulate the fate of Hg in the biogeochemical cycle in the environment. The efficiency of Hg transformation depends upon the nature and diversity of mer genes present in mercury-resistant bacteria. Additionally, the bacterial cellular mechanism of Hg resistance involves reduced Hg uptake, extracellular sequestration, and bioaccumulation. The presence of unique physiological properties in a specific group of mercury-resistant bacteria enhances their bioremediation capabilities. Many advanced biotechnological tools also can improve the bioremediation efficiency of mercury-resistant bacteria to achieve Hg bioremediation.

Alternatives to amelogenin markers for sex determination in humans and their forensic relevance.

Forensic DNA typing and subsequent molecular methods of sex determination in humans have been proven to be an imperious tool to criminal justice system. In current practice, most of the short tandem repeat (STR) based commercial kits contain amelogenin as the sexing marker. Amelogenin gene which contributes to the tooth enamel formation is present on both X and Y chromosome with a variation in base pair size. However, huge discrepancies have been observed with amelogenin based sex determination mostly due to X and Y deletion in the population and mutation in primer binding sites. Some ethnicities such as those in Indian population are affected badly with inappropriate sex determination by amelogenin marker due to the presence of high frequency of Y deletion in the population. Presence of PCR inhibitors, degradation in the DNA samples and presence of mixed DNA also contribute to the discrepancy in results obtained by amelogenin analysis. To overcome this problem, many alternative markers/techniques such as STS, SRY, TSPY, DXYS156, SNPs, DYZ1 and Next generation sequencing have been discussed in much detail with their respective pros and cons. In this regard, inclusion of one or more alternative markers along with amelogenin will decrease the anomalies in sex determination observed while using the amelogenin marker alone in forensic sample analysis.

Functional efficiency of MerA protein among diverse mercury resistant bacteria for efficient use in bioremediation of inorganic mercury.

MerA protein of mer operon in mercury resistant bacteria influences transformation of Hg2+ to Hg0. Both in-silico and in-vivo studies have been carried out and MerA sequences, conserved motifs for mercury binding and NADPH (GCVPSK and LSCCA) varied widely in both Gram-positive and Gram-negative bacteria. As MerA-NADPH-FAD complex plays an important role in mercury volatilization, molecular interaction studies between MerA, NADPH, FAD and Hg2+ was carried out to study the efficiency of transformation of Hg2+ to Hg0 in mercury resistant bacteria. After the prediction of suitable models and molecular interaction analysis, the potential energies in the selected bacteria were as follows: Bacillus thuringiensis (NADPH: -5.15 kcal/mol and FAD: -9.63 kcal/mol), Pseudomonas aeruginosa (NADPH: -3.8 kcal/mol and FAD: -8.56 kcal/mol), Exiguobacterium sp. (NADPH: -3.37 kcal/mol and FAD: -8.42 kcal/mol), Vibrio sp. (NADPH: -3.3 kcal/mol and FAD: -6.7 kcal/mol) and Escherichia coli (NADPH: -3.28 kcal/mol and FAD: -5.69 kcal/mol). Additionally, the binding scores between MerA and Hg2+ followed the similar trend and found higher in B. thuringiensis (3.79) followed by P. aeruginosa (3.57), Exiguobacterium sp. (2.37), Vibrio sp. (1.47) and E. coli (1.07). ANOVA (2-way) result showed the significant (P < 0.05) variation among the energy values obtained after interaction studies. In-vivo analysis of expression of merA gene and Hg2+ removal efficiency also followed the same pattern with a highly significant correlation (P < 0.001) between the binding energy, binding score and expression pattern of merA gene as well as Hg2+ volatilization. Thus, the mercury removal efficiency of bacteria is genera specific which is correlated with the binding efficiency between MerA-NADPH complex and Hg2+ in mer operon mediated mercury resistant bacteria.

Evidence of mercury trapping in biofilm-EPS and mer operon-based volatilization of inorganic mercury in a marine bacterium Bacillus cereus BW-201B.

Biofilm-forming mercury-resistant marine bacterium Bacillus cereus BW-201B has been explored to evident that the bacterial biofilm-EPS (exopolymers) trap inorganic mercury but subsequently release EPS-bound mercury for induction of mer operon-mediated volatilization of inorganic mercury. The isolate was able to tolerate 50 ppm of mercury and forms biofilm in presence of mercury. mer operon-mediated volatilization was confirmed, and -SH was found to be the key functional group of bacterial EPS responsible for mercury binding. Biofilm-EPS-bound mercury was found to be internalized to the bacterial system as confirmed by reversible conformational change of -SH group and increased expression level of merA gene in a timescale experiment. Biofilm-EPS trapped Hg after 24 h of incubation, and by 96 h, the volatilization process reaches to its optimum confirming the internalization of EPS-bound mercury to the bacterial cells. Biofilm disintegration at the same time corroborates the results.

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.

Diversity, community structure, and bioremediation potential of mercury-resistant marine bacteria of estuarine and coastal environments of Odisha, India.

Both point and non-point sources increase the pollution status of mercury and increase the population of mercury-resistant marine bacteria (MRMB). They can be targeted as the indicator organism to access marine mercury pollution, besides utilization in bioremediation. Thus, sediment and water samples were collected for 2 years (2010-2012) along Odisha coast of Bay of Bengal, India. Mercury content of the study sites varied from 0.47 to 0.99 ppb irrespective of the seasons of sampling. A strong positive correlation was observed between mercury content and MRMB population (P < 0.05) suggesting the utilization of these bacteria to assess the level of mercury pollution in the marine environment. Seventy-eight percent of the MRMB isolates were under the phylum Firmicutes, and 36 and 31% of them could resist mercury by mer operon-mediated volatilization and mercury biosorption, respectively. In addition, most of the isolates could resist a number of antibiotics and toxic metals. All the MRMB isolates possess the potential of growth and survival at cardinal pH (4-8), temperature (25-37 °C), and salinity (5-35 psu). Enterobacteria repetitive intergenic consensus (ERIC) and repetitive element palindromic PCR (REP-PCR) produced fingerprints corroborating the results of 16S rRNA gene sequencing. Fourier transform infrared (FTIR) spectral analysis also revealed strain-level speciation and phylogenetic relationships.

Production, optimization and characterization of polyhydroxybutyrate, a biodegradable plastic by Bacillus spp.

Poly-ß-hydroxybutyrate (PHB) is the intracellular lipid reserve accumulated by many bacteria. The most potent terrestrial bacterium Bacillus cereus SE-1 showed more PHB accumulating cells (22.1 and 40% after 48 and 72 h) than that of the marine Bacillus sp. CS-605 (5 and 33% after 48 and 72 h). Both the isolates harbored phbB gene and the characteristics C=O peak was observed in the extracted PHB by Fourier transformed infrared spectroscopy analysis. Maltose was found to be the most suitable carbon source for the accumulation of PHB in B. cereus SE-1. The extracted PHB sample from B. cereus SE-1 was blended with a thermoplastic starch (TS) and an increased thermoplasticity and decreased crystallinity were observed after blending in comparison to the standard PHB. The melting temperature (Tm), melting enthalpy (∆Hf), and crystallinity (Xc) of the blended PHB sample were found to be 109.4 °C, 64.58 J/g, and 44.23%, respectively.

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.

Toll-like receptors (TLRs) in aquatic animals: signaling pathways, expressions and immune responses.

The innate system's recognition of non-self and danger signals is mediated by a limited number of germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are single, non-catalytic, membrane-spanning PRRs present in invertebrates and vertebrates. They act by specifically recognizing PAMPs of a variety of microbes and activate signaling cascades to induce innate immunity. A large number of TLRs have been identified in various aquatic animals of phyla Cnidaria, Annelida, Mollusca, Arthropoda, Echinodermata and Chordata. TLRs of aquatic and warm-blooded higher animals exhibit some distinctive features due to their diverse evolutionary lineages. However, majority of them share conserve signaling pathways in pathogen recognition and innate immunity. Functional analysis of novel TLRs in aquatic animals is very important in understanding the comparative immunology between warm-blooded and aquatic animals. In additions to innate immunity, recent reports have highlighted the additional roles of TLRs in adaptive immunity. Therefore, vaccines against many critical diseases of aquatic animals may be made more effective by supplementing TLR activators which will stimulate dendritic cells. This article describes updated information of TLRs in aquatic animals and their structural and functional relationship with warm-blooded animals.

Characterization and potential application in mercury bioremediation of highly mercury-resistant marine bacterium Bacillus thuringiensis PW-05.

Bacillus thuringiensis PW-05 was isolated from the Odisha coast and was found to resist 50 ppm of Hg as HgCl2 as well as higher concentrations of CdCl2, ZnSO4, PbNO3 and Na2HAsO4. Resistance towards several antibiotics, viz amoxycillin, ampicillin, methicillin, azithromycin and cephradine (CV) was also observed. The mer operon possessed by most of the mercury-resistant bacteria was also found in this isolate. Atomic absorption spectroscopy revealed that the isolate can volatilize >90 % of inorganic mercury. It showed biofilm formation in the presence of 50 ppm HgCl2 and can produce exopolysaccharide under same conditions. The isolate was found to volatilize mercury efficiently under a wide range of environmental parameters, i.e. pH (7 to 8), temperature (25 °C to 40 °C) and salinity (5 to 25 ppt). merA gene expression has been confirmed by real-time reverse transcriptase PCR study. Fourier transform infrared study revealed that -SH and -COOH groups play a major role in the process of adaptation to Hg. Hence, this isolate B. thuringiensis PW-05 shows an interesting potential for bioremediation of mercury.

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.