Effect of thiol compounds and flavins on mercury and organomercurial degrading enzymes in mercury resistant aquatic bacteria.
01 Feb 1990-Bulletin of Environmental Contamination and Toxicology (Bull Environ Contam Toxicol)-Vol. 44, Iss: 2, pp 216-223
TL;DR: The authors report on enzymatic volatilization of HgCl{sub 2} by fourteen Hg-resistant bacterial strains and studied thiol specificity of HG-reductases and organomercurial lyases isolated from the above bacterial species.
Abstract: Plasmid-determined mercuric and organomercurial resistance in microorganisms has been studied by several workers. Mercury reductase, catalyzing the reduction of mercury depends on sulfhydryl compounds. Organomercurial lyase that catalyzes the splitting of C-Hg linkages also needs thiol compounds for its activity. Until recently, no study has been reported on thiol specificity of these enzymes from various sources. In the present study, the authors report on enzymatic volatilization of HgCl{sub 2} by fourteen Hg-resistant bacterial strains. They have also studied thiol specificity of Hg-reductases and organomercurial lyases isolated from the above bacterial species. Hg-reductase is known to have FAD-moiety which stimulates enzyme activity whereas FMN and riboflavin are ineffective in this regard. The effect of flavins, namely FAD, FMN and riboflavin, on Hg-reductase and organomercurial lyase activity is also reported here.
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TL;DR: The mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.
Abstract: Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistance to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.
333 citations
TL;DR: It is reported that resting cells of mercury-resistant bacteria survive in a buffer system for several hours, synthesize inducible mercury-degrading enzymes and volatilize mercury from a mercury-containing buffer system.
Abstract: The mercuric ion reduction system encoded by the Hg2+ inducible mer operon confers bacterial resistance to mercuric ion. The mer A gene product which is a FAD-containing enzyme catalyzes the reduction of Hg2+ to volatile elemental mercury with the help of intracellular thiols and NADPH as a cofactor (Schottel 1974; Summers and Silver 1978; Fox and Walsh 1982; Misra 1992). Our earlier studies have shown that growing cells of different mercury-resistant bacteria reduce Hg2+ compounds to Hg(O) (Ray et al. 1989; Pahan et al. 1990a; Gachhui et al. 1989). We have also shown the effect of thiol compounds and flavins on mercury-degrading enzyme activities in mercury-resistant bacteria (Pahan et al. 1990b). Here we report that resting cells of mercury-resistant bacteria survive in a buffer system for several hours, synthesize inducible mercury-degrading enzymes and volatilize mercury from a mercury-containing buffer system. We know of no information regarding studies of mercury-degrading enzymes in resting mercury-resistant bacterial cells.
16 citations
TL;DR: The author isolated a broad-spectrum Hg-resistant Bacillus pasteurii strain DR{sub 2} which could volatilize different mercury compounds and utilize various aromatic compounds as sole sources of carbon and preferentially utilized benzene in a medium containing both glucose and benzene.
Abstract: Aquatic ecosystems may receive aromatic compounds through various routes These compounds can cause cancerous diseases in aquatic animals and enhance mutagenicity of the sediments The persistence of aromatic compounds deposited in sediments is affected by microbial degradation Plasmid-determined mercuric and organomercurial resistance in microorganisms has also been studied by several workers Utilization of various aromatic compounds as sole sources of carbon by an Hg-resistant bacterial strain has not been reported The author isolated a broad-spectrum Hg-resistant Bacillus pasteurii strain DR{sub 2} which could volatilize different mercury compounds and utilize various aromatic compounds as sole sources of carbon This strain preferentially utilized benzene in a medium containing both glucose and benzene To their knowledge, until recently there has been no report on preferential utilization of other compounds, particularly an aromatic compound to glucose in a mixture
10 citations
TL;DR: Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials and NADPH and GSH might have a role in suppressing the inhibition of N 2 -fixation in the presence of Hg compounds.
Abstract: Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10-50 μmol l -1 HgCl 2 . Nitrogen-fixing ability of these isolates was slightly inhibited when the mercury-resistant bacterial cells were preincubated with 10 μmol l -1 HgCl 2 . Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol l -1 HgCl 2 . However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 nmol l -1 HgCl 2 , only 42-50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N 2 -fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.
7 citations
TL;DR: Inhibition of the glucose oxidation rate by these chemicals in lysozyme-treated cells indicated that these compounds facilitated the transport of glucose across the cell wall and thereby stimulated growth.
Abstract: A broad spectrumbroad spectrum mercury-resistant organism, Bacillus pasteurii DR 2 , was isolated from the effluents of Durgapur Steel Plant, India. In the presence of phenylmercuric acetate or benzene the rate of glucose uptake and the level of some metabolic enzymes increased significantly. Inhibition of the glucose oxidation rate by these chemicals in lysozyme-treated cells indicated that these compounds facilitated the transport of glucose across the cell wall and thereby stimulated growth
7 citations
References
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TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: The mercury cycle in the biosphere and biological methylation of mercury and microbial resistance to mercury and organomercurials are studied.
Abstract: BIOTRANSFORMA nONS OF TOXIC MET AL CAnONS . Mercury . The mercury cycle in the biosphere .. Biological methylation of mercury . Microbial resistance to mercury and organomercurials .
413 citations
"Effect of thiol compounds and flavi..." refers background in this paper
...A requirement of large molar excess of -SH compounds indicates that the true substrate for Hg- -reductase is not Hg +z but its thioi or dithiol adducts ( Summers and Silver 1978 )....
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TL;DR: The nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 is determined and the structure of the gene product, mercuric reductase, is predicted and it is shown that codons with C or G at the third position are preferentially utilized.
Abstract: We have determined the nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 and have predicted the structure of the gene product, mercuric reductase. The DNA sequence predicts a polypeptide of Mr 58 660, the primary structure of which shows strong homologies to glutathione reductase and lipoamide dehydrogenase, but mercuric reductase contains as additional N-terminal region that may form a separate domain. The implications of these comparisons for the tertiary structure and mechanism of mercuric reductase are discussed. The DNA sequence presented here has an overall G+C content of 65.1 mol%, typical of the bulk DNA of Pseudomonas aeruginosa from which Tn501 was originally isolated. Analysis of the codon usage in the merA gene shows that codons with C or G at the third position are preferentially utilized.
233 citations
TL;DR: Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831), suggesting that the native enzyme is composed of three identical subunits.
Abstract: Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831). The mercuric reductase that reduces Hg2+ to volatile Hg0 was purified about 240-fold from the 160,000 X g supernatant of French press disrupted cells. This enzyme contains bound FAD, requires NADPH as an electron donor, and requires the presence of a sulfhydryl compound for activity. The reductase has a Km of 13 micron HgCl2, a pH optimum of 7.5 in 50 mM sodium phosphate buffer, an isoelectric point of 5.3, a Stokes radius of 50 A, and a molecular weight of about 180,000. The subunit molecular weight, determined by gel electrophoresis in the presence of sodium dodecyl sulfate, is about 63,000 +/- 2,000. These results suggest that the native enzyme is composed of three identical subunits. The organomercurial hydrolase, which breaks the mercury-carbon bond in compounds such as methylmercuric chloride, phenylmercuric acetate, and ethylmercuric chloride, was purified about 38-fold over the starting material. This enzyme has a Km of 0.56 micron for ethylmercuric chloride, a Km of 7.7 micron for methylmercuric chloride, and two Km values of 0.24 micron and over 200 micron for phenylmercuric acetate. The hydrolase has an isoelectric point of 5.5, requires the presence of EDTA and a sulfhydryl compound for activity, has a Stokes radius of 24 A, and has a molecular weight of about 43,000 +/- 4,000.
195 citations
TL;DR: A strain of Escherichia coli carrying genes determining mercury resistance on a naturally occurring resistance transfer factor (RTF) converts 95% of 10(-5)m Hg(2+) (chloride) to metallic mercury at a rate of 4 to 5 nmoles of Hg (2+) per min per 10(8) cells.
Abstract: A strain of Escherichia coli carrying genes determining mercury resistance on a naturally occurring resistance transfer factor (RTF) converts 95% of 10−5m Hg2+ (chloride) to metallic mercury at a rate of 4 to 5 nmoles of Hg2+ per min per 108 cells. The metallic mercury is rapidly eliminated from the culture medium as mercury vapor. The volatilizing activity has a temperature dependence and heat sensitivity characteristic of enzymatic catalysis and is inducible by mercuric chloride. Ag+ and Au3+ are markedly inhibitory of mercury volatilization.
195 citations