Mer Operon Research Articles

Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay

Thirty mercury-resistant (Hg R) Bacillus strains were isolated from mercury-polluted sediment of Minamata Bay, Japan. Mercury resistance phenotypes were classified into broad-spectrum (resistant to inorganic Hg 2+ and organomercurials) and narrow-spectrum (resistant to inorganic Hg 2+ and sensitive to organomercurials) groups. Polymerase chain reaction (PCR) product sizes and the restriction nuclease site maps of mer operon regions from all broad-spectrum Hg R Bacillus were identical to that of Bacillus megaterium MB1. On the other hand, the PCR products of the targeted merP (extracellular mercury-binding protein gene) and merA (intracellular mercury reductase protein gene) regions from the narrow-spectrum Hg R Bacillus were generally smaller than those of the B. megaterium MB1 mer determinant. Diversity of gene structure configurations was also observed by restriction fragment length polymorphism (RFLP) profiles of the merA PCR products from the narrow-spectrum Hg R Bacillus. The genetic diversity of narrow-spectrum mer operons was greater than that of broad-spectrum ones.

Bio-affecting mercury detection using mercury resistance gene module fused with bioluminescence reporter genes

Bioluminescence sensor systems were developed for monitoring environmental mercury contamination. The biological mercury measurement sensor systems were constructed by DNA recombination technique. A bacterial mercury-resistant operon (meroperon) from Pseudomonas sp. K-6y4 and a bacterial bioluminescence operon (lux operon) from an ocean bacterium Vibrio fischeri were fused in avector plasmid. The resulting recombinant plasmids were cloned in Escherichia coli cells. The bioluminescence sensor systems responded to mercury chloride of 0.1 nM to 100 nM. The mercury bioluminescence sensor developed in this study can be used for monitoring of the bio-affecting mercury instead of total mercury that is measured by conventional analytical equipment. The fundamental feature of the bioluminescence sensor system is attractive for use as a monitoring system for bio-affecting environmental mercury contamination.

English

5kg of mercury to the global atmosphere with a residence time of approximately one year. This phenomenon changes the flux of biologically available mercury in natural microbial communities where enzymatic actions, including mercuric reductase and organomercurial lyase activities, underpin the biogeochemical cycling of mercury with repercussions for human exposure to toxic forms of the element. To elucidate the impact of episodic mercury bioavailability on the response of microbial communities, the expression of microbial proteins and nucleic acids in environmental strains of Pseudomonas species were evaluated under various concentrations of mercury ranging from 0 to 500 µM. Routine cultivation of Pseudomonas aeruginosa PU21 containing the 142.5 kb plasmid Rip64 in medium containing 100 µg of Hg ++ /ml (500 µM) exhibited a prolonged lag phase survived by hyper-resistant cells able to grow in medium containing 200 µg of Hg ++ /ml. Nucleic acid analyses showed a distinct mutation in the merA gene encoding for mercuric reductase activity in cells able to grow at elevated mercury concentrations. A similar mutation was detected in the merR locus which serves as the regulator of the mer operon. Mutations were not detected in merC which encodes for a hydrophobic membrane-associated protein implicated in active mercury transport. Protein profiles of cells grown with elevated mercury concentrations were associated with a stable increase in the production of specific polypeptides. In addition, the survival and genetic response of naturally-occurring mercury resistant bacteria inoculated into contaminated environmental samples were monitored in microcosm experiments over a 30 day period. The results suggest that sudden exposure to high concentrations of mercury either decimates the bacterial population or selects for hyper-resistant strains with high level of constitutive expression of active proteins, including mercuric reductase. Methyl mercury was observed to cause a higher level of induction for mercuric reductase than the specific substrate, inorganic mercury. The selection of hyper-resistant strains is potentially useful for biotechnological strategies to control the bioavailability of mercury, and thereby potentially reducing the re-uptake of mercury into vegetation in regions frequently subjected to wildfires.

Tn5041-like transposons: molecular diversity, evolutionary relationships and distribution of distinct variants in environmental bacteria.

A detailed study on the geographic distribution, molecular diversity and evolutionary relationships of 24 closely related variants of the Tn5041 transposon found among 182 mercury resistant environmental Gram-negative strains from the IMG-Hg Reference Collection is reported here. RFLP analysis, followed by the determination of partial DNA sequences, identified 14 distinct types of these transposons, which differed from each other by 1-7 single-event DNA polymorphisms. No polymorphisms were detected at the right arm of the transposons except an insertion of a new mobile DNA element carrying a mer operon (named the mer2 cassette) within the Tn5041 mer operon. According to the model presented here, the insertion occurred via homologous recombination with a circular form of the mer2 cassette. A total of 8 point mutations, 1 internal deletion, 2 end-involving deletions, 3 mosaic regions and 2 insertions were detected at the left arm of the transposons. The insertions were a transposon closely related to Tn21 but lacking the integron and a new group II intron (named INT5041C). Inspection of the geographic distribution of the Tn5041 variants suggested that at least three long-distance waves of dissemination of these variants had occurred, accompanied by homologous recombination between different Tn5041 lineages. Movements of circular DNAs by homologous recombination as a source of mosaic genes and new mer genes, and formation of unusual mosaics ending or beginning at the Tn5041 att site are discussed.

Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon Tn MERI1.

Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.

Relationship between the persistence of mer operon sequences in Escherichia coli and their resistance to mercury.

Studies related to geographic distribution of E. coli carrying mer operon sequences were carried out on the Indian subcontinent. Out of the 80 E. coli isolates, collected from five geographically distinct regions of India, 68 were found to be resistant to one or the other heavy metal used in the study. Among these isolates, 36 were found to be resistant to the inorganic form (HgCl2) and only 5 to resist both the inorganic and organic forms of mercury. Colony hybridization studies revealed 35 isolates out of 68 to hybridize with the probe. Interestingly, some of the mercury-sensitive isolates (Hgs), especially from the Dal Lake, were found positive in hybridization studies. These findings, supported by mercury volatilization studies, indicate the presence of nonfunctional/vestigial mer sequences in the isolates collected from different environments. On the other hand, few of the mercury-resistant isolates (Hgr) from the Yamuna River did not show any sign of hybridization. Further, volatilization studies also indicated an alternate mode of resistance mechanism operating in them. The studies demonstrate that the mer operon sequences share very high homology among the E. coli isolates collected from different geographical locations, and this metal resistance may be a genetic character that arose from a common ancestral background.

Polyphosphate produced in recombinant Escherichia coli confers mercury resistance

An Escherichia coli strain was generated by fusion of a merA-deleted broad-spectrum mer operon from Pseudomonas K-62 with a bacterial polyphosphate kinase gene ( ppk) from Klebsiella aerogenes in vector pUC119. A large amount of the ppk-specified polyphosphate was identified in the mercury-induced bacterium with the fusion plasmid designated pMKB18 but not in the cells without mercury induction. These results suggest that the synthesis of polyphosphate as well as the expression of the mer genes is mercury-inducible and regulated by merR. The E. coli strain with pMKB18 was more resistant to both Hg 2+ and C 6H 5Hg + than its isogenic strain with cloning vector pUC119. The recombinant strain accumulated more mercury from Hg 2+- and C 6H 5Hg +-contaminated medium. Hg 2+ transported into the cytoplasm appeared to be bound by chelation with the polyphosphate produced by the recombinant cells. The transported phenylmercury was degraded to Hg 2+ before the chelation since polyphosphate did not directly chelate with C 6H 5Hg +. These results indicate that polyphosphate is capable of reducing the cytotoxicity of the transported Hg 2+ probably via chelation between polyphosphate and Hg 2+.

Horizontal Transfer of Mercury Resistance Genes in Environmental Bacterial Populations

The results of studying the horizontal transfer of mercury resistance determinants in environmental bacterial populations are reviewed. Identical or highly homologous mercury resistance (mer) operons and transposons were found in bacteria of different taxonomic groups from geographically distant regions. Recombinant mer operons and transposons were revealed. The data suggest high frequencies of horizontal transfer and of recombination for mercury resistance determinants. The mechanisms of horizontal gene transfer were elucidated in Gram-negative and Gram-positive bacteria. New transposons were found and analyzed.

Tn5044-conferred mercury resistance depends on temperature: the complexity of the character of thermosensitivity.

Escherichia coli K12 containing the transposon Tn5044 mer operon (merR, T, P, C, and A genes) is resistant to mercuric chloride at 30 degrees C but sensitive to this compound at 37-41.5 degrees C. We have studied the mechanism underlying the temperature-sensitive nature of this mercury resistance phenotype, and found that the expression of the Tn5044 merA gene coding for mercuric reductase (MerA) is severely inhibited at non-permissive temperatures. Additionally, MerA showed a considerably reduced functional activity in vivo at non-permissive temperatures. However, the temperature-sensitive character of the functioning of this enzyme in cell extracts, where it interacted with one of the low-molecular weight SH compounds rather than with the transport protein MerT (as is the case in vivo), was not apparent. These data suggest that the temperature-sensitive mercury resistance phenotype should stay under control at two stages: when the merA gene is expressed and when its product interacts with MerT to accept the mercuric ion.

Mercury resistance transposons of Gram-negative environmental bacteria and their classification

A total of 29 mercury resistance transposons were isolated from mercury-resistant environmental strains of proteobacteria collected in different parts of Eurasia and the USA and tested for hybridization with probes specific for transposase genes of known mercury resistance transposons. 9 were related to Tn 21 in this test, 12 were related to Tn 5053, 4 to Tn 5041 and 1 to Tn 5044; three transposons were negative in this test. Restriction mapping and DNA sequencing revealed that 12 transposons were identical or nearly identical to their corresponding relatives while the rest showed varying divergence from their closest relatives. Most of these previously unknown transposons apparently arose as a result of homologous or site-specific recombination. One of these, Tn 5046, was completely sequenced, and shown to be a chimera with the mer operon and the transposition module derived from the transposons related to Tn 5041 and to Tn 5044, respectively. Transposon Tn 5070, showing no hybridization with the specific probes used in this study, was also completely sequenced. The transposition module of Tn 5070 was most closely related to that of Tn 3 while the mer operon was most closely related to that of plasmid pMERPH. The merR of Tn 5070 is transcribed in the same direction as the mer structural genes, which is typical for mer operons of Gram-positive bacteria. Our data suggest that environmental bacteria may harbor many not yet recognized mercury resistance transposons and warrant their further inventory.

Detection of organomercurials with sensor bacteria.

Mercury and its organic compounds, especially methylmercury, are hazardous compounds that concentrate in biota via biomagnification and cause severe neurological disorders in animals. In this paper, a recombinant whole-cell bacterial sensor for the detection of the organic compounds of mercury was constructed. The sensor carries firefly luciferase gene as a reporter under the control of the mercury-inducible regulatory part of broad spectrum mer operon from pDU1358. In addition, a gene-encoding organomercurial lyase (an enzyme necessary for cleavage of the mercury-carbon bond) was coexpressed in the sensor strain. The sensitivity of the sensor was evaluated on some environmentally important organomercurial compounds. The lowest detectable concentrations were 0.2 nM (50 ng/L), 1 nM (0.34 microg/L), and 10 microM (2.3 mg/L) for methylmercury chloride, phenylmercury acetate, and dimethylmercury, respectively. The sensor responded also to inorganic mercury and, therefore, using the sensor described here together with sensor bacteria responding only to inorganic mercury, it should be possible to characterize the mercury contamination, for example, in environmental samples.

The meroperon of a mercury-resistant Pseudoalteromonas haloplanktis strain isolated from Minamata Bay, Japan.

A mer operon of mercury-resistant Pseudoalteromonas haloplanktis strain M1, isolated from sea water of Minamata Bay, was cloned and analyzed. The mer genes were located in the chromosome and organized as merR-merT-merP-merC-merA-merD, the same order as that in Tn21. However, the orientation of the merR gene is the same as that of other mer genes (opposite direction to Tn21), and merR was cotranscribed with other mer genes, a pattern that has not been previously seen with mer determinants from other Gram-negative bacteria. Furthermore, the amino acid similarities of the corresponding mer gene products between those from strain M1 and Tn21 were unusually low.

SENSITIVE DETERMINATION OF URINARY MERCURY(II) BY A BIOLUMINESCENT TRANSGENIC BACTERIA-BASED BIOSENSOR

We used a luminescence-based biosensor to determine mercury in urine samples of subjects with or without dental amalgam restorations. The system utilizes Escherichia coli as a host organism and firefly luciferase luc gene as a reporter under the control of the mercury-inducible promoter from the mer operon from transposon Tn21. The presence of mercury induces the specific gene sequence with consequent synthesis of the luciferase enzyme, and luciferin/luciferase-mediated light output occurs. The method showed a linear relationship between the light signal intensity and the Hg2+ concentration in the range of 1.67 × 10−13–1.67 × 10−7 M with a detection limit of 1.67 × 10−13 M, corresponding to 4.0 × 10−18 mol/tube. The system proved precise, accurate, and highly selective for mercury and methylmercury, with no light emission induced by other heavy metals; the matrix effect caused by urine components was minimal. The analysis of urine specimens showed that urinary mercury levels in subjects with amalgam fillings were slightly significantly higher than those in subjects without amalgam fillings (p < 0.1) and the statistically significant difference improved when creatinine-normalized values were considered (p < 0.05). In summary, from the analytical point of view this luminescent microbial biosensor represents an easy, rapid, and sensitive tool to analyse Hg2+ in biological and environmental samples. Along with the possibility of using 384-well microtitre plates requiring very low reagent volumes, these characterisitcs make the system suitable for the high-throughput screening of Hg2+ on large numbers of specimens. This method could help better clarify the relationship between dental amalgam and urinary mercury excretion by permitting large-scale analysis of many selected groups of subjects, with strict control of all the relevant parameters affecting Hg2+ concentration in urine, such as diet and environmental and occupational exposure.

新たに発見された有機水銀分解遺伝子ｍｅｒＢ３とその発現調節系を利用した有機水銀の検出方法に関する研究

Mercurials have been released into the environment by geological or anthropogenic activities. Organomercurial compounds such as methylmercury are known as toxic compounds. Therefore, organomercurials should be monitored to keep clean environments. However, chemical species of organomercurials are not easily detected separately or individually.In this study, we tried to detect organomercurial compounds by using a bacterial gene-expression system. A series of mer operon-luciferase (mer-lux) transcriptional fusion plasmids (pHYΔB3Lux and pHYB3Lux) was constructed to evaluate the gene expression system with a new organomercury lyase gene merB3 from Bacillus megaterium MB1. A plasmid (pGR1A) encoding mer operon genes from O/PmerR1 to merA isolated from B. megaterium MB1 was used as a transacting gene expression vector with the mer-lux transcriptional fusion plasmids into a same host bacterial cell. The transformants that carry a set of two plasmids (pHYΔB3Lux+pGR1A or pHYB3Lux+pGR1A), respectively) were used for detection of organomercurials. The experimental results showed that the transformant with pHYΔB3Lux+pGR1A responded to only mercury chloride (MC). On the other hand, the transformant with pHYB3Lux+pGR1A responded and detected MC and the all organomercurials tested in the study. Therefore, the bacterial strain which possess a gene expression system of mer-lux transcriptional fusion was useful to detect organomercurials with distinction from inorganic mercury.

The quality of merC, a module of the mer mosaic.

We examined a region of high variability in the mosaic mercury resistance (mer) operon of natural bacterial isolates from the primate intestinal microbiota. The region between the merP and merA genes of nine mer loci was sequenced and either the merC, the merF, or no gene was present. Two novel merC genes were identified. Overall nucleotide diversity, pi (per 100 sites), of the merC gene was greater (49.63) than adjacent merP (35.82) and merA (32.58) genes. However, the consequences of this variability for the predicted structure of the MerC protein are limited and putative functional elements (metal-binding ligands and transmembrane domains) are strongly conserved. Comparison of codon usage of the merTP, merC, and merA genes suggests that several merC genes are not coeval with their flanking sequences. Although evidence of homologous recombination within the very variable merC genes is not apparent, the flanking regions have higher homologies than merC, and recombination appears to be driving their overall sequence identities higher. The synonymous codon usage bias (EN(C)) values suggest greater variability in expression of the merC gene than in flanking genes in six different bacterial hosts. We propose a model for the evolution of MerC as a host-dependent, adventitious module of the mer operon.

Development and Validation of a Detection System for Wild-type Vibrio cholerae in Genetically Modified Cholera Vaccine

Orochol®, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intactctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol®.

Identification and characterization of anaerobic mercury-resistant bacteria isolated from mercury-polluted sediment

To develop biotechnology for biological treatment of mercury-contaminated wastes or for bioremediation of mercury-polluted sites, mercury-resistant microorganisms have been isolated and characterized. However, understanding of the mercury resistance mechanism by anaerobic bacteria is lacking. In this study, we tried to isolate anaerobic mercury-resistant bacteria from mercury-polluted sediment in Minamata Bay, Japan. One strain of the bacterial isolates, designated Mersaru, was used for the identification and for the growth capability of mercurials. We also analyzed genetic characteristics of mercury resistance genes (merA and merB gene) from the strain Mersaru. The strain Mersaru, which was isolated from Minamata Bay sediment, was identified as Clostridium butyricum and showed resistance to both inorganic mercury and organomercurials. Furthermore, nucleotide sequence analysis showed that merA and merB genes of the strain Mersaru were identical to those of the B. cereus RC607 an aerobic mercury-resistant bacterium in the nucleotide sequence level. From the pulsed field gel electrophoresis analysis, it is suggest that the mer operon of the strain Mersaru is located on chromosomal DNA.

Tn 5044, a novel Tn 3 family transposon coding for temperature-sensitive mercury resistance

We report the discovery and characterization of the mercury resistance transposon, Tn 5044, from a Xanthomonas strain from the Kamchatka peninsula. In addition to the standard set of merRTPCAD genes, the mer operon of Tn 5044 contains a gene named sigY that encodes the RNA polymerase sigma factor-like protein. Mercury resistance determined by Tn 5044 is expressed at low (30 °C) but not at elevated temperatures (37 °C). None of the mer operon genes downstream of merA is responsible for the temperature-sensitive mercury resistance. The transposition module of Tn 5044 is closely related to those of Tn 1412 isolated from medical sources and to Tn 5563 and IS Xc5 from environmental sources. However, Tn 5044 differs from these transposons in that it has unusually long terminal inverted repeats. Sequence analysis of the transposase ( tnpA) genes places Tn 5044 and its close relatives into the Tn 3 subgroup of the Tn 3 family. However, the orientation of their resolvase and transposase genes is unusual for the Tn 3 family: tnpR is proximal to the end of the transposon, while divergently transcribed tnpA is oriented inwardly. The region between tnpA and tnpR genes is unusually large and contains two short conserved open reading frames. In addition to the complete set of sequence motifs common to true resolvases, the resolvase of Tn 5044 and its close relatives possesses a C-terminal extension showing no homology to known proteins. Despite this peculiarity, Tn 5044 resolvase can resolve cointegrates formed during Tn 5044 transposition controlled by tnpA. Genetic data suggest that the extension is essential for TnpR functioning.

Involvement of merB in the Expression of the pMR26 mer Operon Induced by Organomercurials

The inducibility by organomercury of the broad-spectrum mer operon on pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 was assessed in the absence of a functional merB gene. The mer polypeptides encoded by the mer genes on pMRA17 were almost identified in maxicell induced not only by Hg2+ but also by C6H5Hg+ or CH3Hg+. Maxicell with pMRD103, a merB-deletion plasmid constructed from pMRA17, also produced the corresponding mer polypeptides when maxicell was induced by Hg2+, whereas no mer polypeptides were detected in the maxicell induced by C6H5Hg+ or CH3Hg+. These results suggest that merB is needed for induction of the pMRA17 mer operon expression by organomercurials. Next, to test the inducibility of pMRA17 mer operon expression from its own promoter, a promoterless lacZ was fused with the mer operon, where merB was deleted in plasmid pB43merlacZ. Only Hg2+, but not C6H5Hg+ or CH3Hg+, can activate β-galactosidase expression in bacteria with pB43merlacZ. These results not only imply that the pMRA17 MerR is a narrow-spectrum regulator that did not recognize organomercury as a direct inducer, but also confirms that merB is required for induction of the pMRA17 mer operon expression by organomercurials.

Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1.

The complete structure of a broad-spectrum mercury resistance module was shown by sequencing the Gram-positive bacterial transposon TnMERI1 of Bacillus megaterium MB1. The regions encoding organomercury resistance were identified. Upstream of a previously identified organomercurial lyase merB (merB1) region of TnMERI1, a second merR (merR2) and a second merB gene (merB2) were found. These genes constitute a second operon (mer operon 2) following a promoter/operator (P(merR2)) region. A third organomercurial lyase gene (merB3) was found immediately upstream of the mer operon (mer operon 1) followed by a promoter/operator (P(merB3)) region homologous to that of the mer operon 1 (P(merR1)-merR1-merE-like-merT-merP-merA). The complete genetic structure of the mercury resistance module is organized as P(merB3)-merB3-P(merR1)-merR1-merE-like-merT+ ++ -merP-merA-P(merR2)-merR2 -merB2-merB1. The subcloning analysis of these three merB genes showed distinct substrate specificity as different organomercury lyase genes.