Mercury Resistance Genes Research Articles

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326.

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K(D)) for binding of MerR were: binding site I, 8.5 x 10(-9) M; binding site II, 1.2 x 10(-8) M; and for the complete promoter/operator region 1 x 10(-8) M. The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively. The K(D) value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 x 10(-7) M.

A single procedure to recover DNA from the surface or inside aggregates and in various size fractions of soil suitable for PCR-based assays of bacterial communities

A single DNA procedure to recover bacterial DNA from various soil microenvironments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (outside and surface of aggregates), the inner part or microporosity (inside of aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved sample homogenization and cell disruption by grinding in liquid nitrogen, followed by enzymatic lysis with lysozyme and proteinase K. High yields of high molecular weight DNA (≥ 23 kb) were obtained for all microenvironments. Crude DNA yields for the various soil microenvironments were between 0.7 and 51.4 μg DNA·g −1 soil sample and were positively correlated with bacterial cell abundance ( r = 0.91). Further purification steps allowed to recover at least 60 % of the DNA extracted from the various microenvironments. The suitability of the extracted DNA to undergo enzymatic amplification reactions and the effectiveness of the extraction procedure in recovering DNA from various native bacterial groups was tested using primers for archaebacterial 16S rDNAs, universal and group-specific eubacterial 16S rDNAs primers (β- and γ-proteobacteria, High G+C Gram-positive bacteria, and Bacillus species and relatives). Successful amplification of less ubiquitous genes was also obtained with primers targeting nitrogen fixation ( nifH) and mercury resistance ( merRTΔP) genes.

Analysis of mer Gene Subclasses within Bacterial Communities in Soils and Sediments Resolved by Fluorescent-PCR-Restriction Fragment Length Polymorphism Profiling

Bacterial mer (mercury resistance) gene subclasses in mercury-polluted and pristine natural environments have been profiled by Fluorescent-PCR-restriction fragment length polymorphism (FluRFLP). For FluRFLP, PCR products were amplified from individual mer operons in mercury-resistant bacteria and from DNA isolated directly from bacteria in soil and sediment samples. The primers used to amplify DNA were designed from consensus sequences of the major subclasses of archetypal gram-negative mer operons within Tn501, Tn21, pDU1358, and pKLH2. Two independent PCRs were used to amplify two regions of different lengths (merRT(Delta)P [ca. 1 kb] and merR [ca. 0.4 kb]) starting at the same position in merR. The oligonucleotide primer common to both reactions (FluRX) was labelled at the 5(prm1) end with green (TET) fluorescent dye. Analysis of the mer sequences within databases indicated that the major subclasses could be differentiated on the basis of the length from FluRX to the first FokI restriction endonuclease site. The amplified PCR products were digested with FokI restriction endonuclease, with the restriction digest fragments resolved on an automated DNA sequencing machine which detected only those bands labelled with the fluorescent dye. For each of the individual mer operon sources examined, this single peak (in bases) position was observed in separate digests of either amplified region. These peak positions were as predicted on the basis of DNA sequence. mer PCR products amplified from DNA extracted directly from soil and sediment bacteria were studied in order to determine the profiles of the major mer subclasses present in each natural environment. In addition to peaks of the expected sizes, extra peaks were observed which were not predicted on the basis of DNA sequence. Those appearing in the restriction endonuclease digests of both study regions were presumed to be novel mer types. Genetic heterogeneity within and between mercury-polluted and pristine sites has been studied by this technique. Profiles generated were highly similar for samples taken within the same soil type. The profiles, however, changed markedly on crossing from one soil type to another, with gradients of the different groupings of mer genes identified.

Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site.

Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate. Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates. Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems. Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes. Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants. These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil. In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates.

Mercury resistance as a selective marker for recombinant mycobacteria.

The use of antibiotic-resistance markers for the selection of recombinant mycobacteria is widespread but questionable considering the development of live recombinant BCG vaccines. In contrast, vector-encoded resistance to heavy metals such as mercury may represent an interesting alternative for the development of live vaccines compatible with use in humans and in animals. The mercury resistance genes (mer) from Pseudomonas aeruginosa and from Serratia marcescens were cloned into the Escherichia coli-Mycobacterium shuttle vector pRR3. The resulting vectors, designated pMR001 and pVN2, were introduced by electroporation into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. The recombinant mycobacteria were stable in vitro and in vivo, and had high-level mercury resistance, thus indicating that the mer genes can be useful as selective markers in mycobacteria.

Sequence Conservation between Regulatory Mercury Resistance Genes in Bacteria from Mercury Polluted and Pristine Environments

Summary The regulatory gene merR , and the adjacent operator/promoter region was amplified from the mercury resistance (Hg R ) determinants from 10 Gram-negative bacterial isolates from mercury polluted and pristine environments using the polymerase chain reaction. These mer regions showed polymorphism in size of PCR amplification products with those from isolates SE3, SE11, SE12, SE31, SO1 and T217 being of 557 base pairs in size, whilst those from isolates SE20, T238, SB3, SB4 and the positive control (Tn 501 ) were 536 base pairs in size. From the sequence analysis of these mer regions and comparison with previously sequenced Hg R determinants an evolutionary tree was constructed which showed there to be a significant difference between Gram- negative merR genes and those found in Gram-positive organisms. With the exception of the Thiobacillus Hg R determinants, merR genes from Gram negative bacteria were strongly conserved and could be grouped closely around the previously sequenced determinants of Tn 501 , Tn 21 , Tn 5053 (pMER327/419) and pKLH2. Only the merR genes of pDU1358 and T238 showed significant variation from these subgroups. The regions of greatest variation were the carboxyl terminal coding region of the merR gene and the operator/promoter region. It is suggested that, due to the global nature of inducible mercury resistance and its strong sequence conservation across large geographical distances, bacterial resistance to mercury is an ancient genetic character.

Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil

Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.

The very large amplifiable element AUD2 from Streptomyces lividans 66 has insertion sequence-like repeats at its ends

In a spontaneous, chloramphenicol-sensitive (Cms), arginine-auxotrophic (Arg-) mutant of Streptomyces lividans 1326, two amplified DNA sequences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second one was a 92-kb sequence called ADS2. ADS2 encoding the previously isolated mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking the corresponding amplifiable element called AUD2 in the wild-type strain were isolated by using another genomic library prepared from S. lividans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of an 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. This insertion sequence-like structure was confirmed by the DNA sequence determined from the amplified copy of the direct repeats which demonstrated a high degree of similarity of 65% identity in nucleic acid sequence to IS112 from Streptomyces albus. The recombination event leading to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was correlated with a deletion on one side of the flanking chromosomal region beginning very near or in the amplified DNA. Strains of S. lividans like TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides.

Survival and impact of genetically engineered Pseudomonas putida harboring mercury resistance gene in soil microcosms.

The survival of genetically engineered and wild-type Pseudomonas putida PpY101, that contained a recombinant plasmid pSR134 conferring mercury resistance, were monitored in andosol and sand microcosms. The survival of genetically engineered and wild-type P. putida was not significantly different in andosol. The population change of the two strains was dissimilar in andosol and sand. The survival of genetically engineered and wild-type P. putida strains was affected by the water content of andosol, and increased with the increment of the water content. The impact of the addition of genetically engineered and wild-type P. putida strains on indigenous bacteria and fungi was examined. Inoculation of both strains had no apparent effect on the density of indigenous microorganisms.

Plasmids pJP4 and r68.45 Can Be Transferred between Populations of Bradyrhizobia in Nonsterile Soil

IncP plasmid r68.45, which carries several antibiotic resistance genes, and IncP plasmid pJP4, which contains genes for mercury resistance and 2,4-dichlorophenoxyacetic acid degradation, were evaluated for their ability to transfer to soil populations of rhizobia. Transfer of r68.45 was detected in nonsterile soil by using Bradyrhizobium japonicum USDA 123 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid transfer frequencies ranged up to 9.1 x 10 in soil amended with 0.1% soybean meal and were highest after 7 days with strain 3G4b4-RS as the recipient. Transconjugants were detected in 7 of 500 soybean nodules tested, but the absence of both parental strains in these nodules suggests that plasmid transfer had occurred in the soil, in the rhizosphere, or on the root surface. Transfer of degradative plasmid pJP4 was also evaluated in nonsterile soil by using B. japonicum USDA 438 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid pJP4 was transferred only when strains USDA 110-ARS and 3G4b4-RS were the recipients. The plasmid transfer frequency was highest for strain 3G4b4-RS (up to 7.4 x 10). Mercury additions to soil, ranging from 10 to 50 mug/g of soil, did not affect population levels of parental strains or the plasmid transfer frequency.

Construction and characterization of a mercury-independent MerR activator (MerRAC): transcriptional activation in the absence of Hg(II) is accompanied by DNA distortion.

The MeR regulatory protein of transposon Tn501 controls the expression of the mercury resistance (mer) genes in response to the concentration of mercuric ions. MerR is unique among prokaryotic regulatory proteins so far described in that it acts as a repressor [-Hg(II)] and an activator [+Hg(II)] of transcription of the mer genes, but binds to a single site on the DNA in both cases. This transcriptional activation process has been postulated to involve a protein-induced conformational change in the DNA that allows RNA polymerase more readily to form an open complex at the promoter. It has been shown [Frantz and O'Halloran (1990) Biochemistry, 29, 4747-4751] that activation of transcription by MerR in the presence of mercury is accompanied by hypersensitivity of the operator to chemical nucleases that are sensitive to local distortion in DNA structure. Here we describe specific mutations in MerR that allow the protein to stimulate transcription in the absence of the allosteric activator Hg(II). We demonstrate that the degree of activation caused by these mutants directly correlates with the degree of DNA distortion as measured by the hypersensitivity of MerR-DNA complexes to the nuclease Cu-5-phenyl-o-phenanthroline. These results support the model described above.

Survival and impact of genetically engineered Pseudomonas putida harboring mercury resistance gene in aquatic microcosms.

The survival of wild-type and genetically engineered Pseudomonas putida PpY101 that contained a recombinant plasmid pSR134 conferring mercury resistance were monitored in aquatic microcosms. We used lake, river, and spring water samples. The density of genetically engineered and wild-type P. putida decreased rapidly within 5 days (population change rate k -0.87 approximately -1.00 day-1), then moderately after 5 to 28 days (-0.10 approximately -0.14 day-1). The population change rates of genetically engineered and wild-type P. putida were not significantly different. We studied the important factors affecting the survival of genetically engineered and wild-type P. putida introduced in aquatic microcosms. Visible light exerted an adverse effect on the survival of the two strains. The densities of genetically engineered and wild-type P. putida were almost constant until 7 days after inoculation in natural water filtered with a 0.45-micron membrane filter, or treated with cycloheximide to inhibit the growth of protozoa. These results suggested that protozoan predation was one of the most important factors for the survival of two strains. We examined the impact of the addition of genetically engineered and wild-type P. putida on indigenous bacteria and protozoa. Inoculation of genetically engineered or wild-type P. putida had no apparent effect on the density of indigenous bacteria. The density of protozoa increased in microcosms inoculated with genetically engineered or wild-type P. putida at 3 days after inoculation, but after 5 to 21 days, the density of protozoa decreased to the same level as the control microcosms.

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans.

A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cms), arginine auxotrophic (Arg-) mutant of Streptomyces lividans 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cms Arg- mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.

Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction.

Specific DNA sequences from native bacterial populations present in soil, sediment, and sand samples were amplified by using the polymerase chain reaction with primers for either "universal" eubacterial 16S rRNA genes or mercury resistance (mer) genes. With standard amplification conditions, 1.5-kb rDNA fragments from all 12 samples examined and from as little as 5 micrograms of soil were reproducibly amplified. A 1-kb mer fragment from one soil sample was also amplified. The identity of these amplified fragments was confirmed by DNA-DNA hybridization.

Bacterial resistances to inorganic mercury salts and organomercurials

Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the CHg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes ( merR and merD) were identified. The ( merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The −10 and −35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli σ 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).

Synthetic oligonucleotide probes for detection of mercury-resistance genes in environmental freshwater microbial communities in response to pollutants

Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the(32)P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria.

Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR

Positive control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase. Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions. Activator-induced DNA bending is typically assigned a role in binding-site recognition, alterations in DNA loop structures or optimal positioning of the activator for interaction with polymerase. Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA. The allosterically modulated transcription factor MerR is a repressor and an Hg(II)-responsive activator of bacterial mercury-resistance genes. Escherichia coli RNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref. 6). Chemical nuclease studies show that the activator form, but not the repressor, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site. Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33 degrees relative to the MerR-operator complex. The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex.

Distribution of DNA Sequences Encoding Narrow- and Broad-Spectrum Mercury Resistance

The distribution of DNA sequences homologous with three mer genes was determined in unselected and mercury-resistant water and sediment isolates. The maximum proportions of unselected bacterial isolates containing DNA hybridizing with the 358merA, 358merB, and 501merR probes, derived from gram-negative organisms, were 93.8, 21, and 100%, respectively. Up to 53.3% of mercury chloride-resistant isolates and 54% of methylmercury hydroxide-resistant isolates did not contain DNA homologous with 358merA or 358merB, respectively. Hybridizations performed at high and low stringencies demonstrated that divergence of the merA gene accounted for many of the mercury-resistant but probe-negative isolates. Sixteen mercury-resistant Bacillus spp. isolated from the least contaminated site all contained DNA homologous with 258merA, originally from a gram-positive organism, but only four hybridized weakly with 358merA. The results demonstrate the wide distribution of mercury resistance genes but, because of the diversity of genetic determinants, highlight the importance of using multiple detection techniques and gene probes derived from a variety of origins for such studies.

The Bradyrhizobium japonicum nolA gene and its involvement in the genotype-specific nodulation of soybeans.

Several soybean genotypes have been identified which specifically exclude nodulation by members of Bradyrhizobium japonicum serocluster 123. We have identified and sequenced a DNA region from B. japonicum strain USDA 110 which is involved in genotype-specific nodulation of soybeans. This 2.3-kilobase region, cloned in pMJS12, allows B. japonicum serocluster 123 isolates to form nodules on plants of serogroup 123-restricting genotypes. The nodules, however, were ineffective for symbiotic nitrogen fixation. The nodulation-complementing region is located approximately 590 base pairs transcriptionally downstream from nodD2. The 5' end of pMJS12 contains a putative open reading frame (ORF) of 710 base pairs, termed nolA. Transposon Tn3-HoHo mutations only within the ORF abolished nodulation complementation. The N terminus of the predicted nolA gene product has strong similarity with the N terminus of MerR, the regulator of mercury resistance genes. Translational lacZ fusion experiments indicated that nolA was moderately induced by soybean seed extract and the isoflavone genistein. Restriction fragments that hybridize to pMJS12 were detected in genomic DNAs from both nodulation-restricted and -unrestricted strains.

Effects of Hg 2+ , CH 3 -Hg + , and Temperature on the Expression of Mercury Resistance Genes in Environmental Bacteria

Twenty different bacterial isolates obtained from a mercury-contaminated site in Oak Ridge, Tenn., were grown on plate count agar amended with 25 mug of Hg or 3 mug of CH(3)-Hg (R-Hg) per ml. The total cellular RNA was extracted from each isolate by an acid-guanidine-thiocyanate-phenol-chloroform method. The transcripts of merA and merB were detected and quantitated by Northern (RNA) hybridization. A qualitative assay of mercuric reductase was used to confirm the enzyme activity. Low temperature (4 degrees C) with the presence of Hg (25 mug/ml) significantly increased the net merA transcripts of mid-log-phase cells of six environmental isolates. The net merA transcript production by 18 of the isolates increased when they were grown on 50% plate count broth with 15 mug of Hg per ml, but only 8 isolates showed increased production of merB transcripts. The MICs of Hg and R-Hg for 10 methyl mercury-resistant isolates ranged from 45 to 110 mug of Hg and 0.6 to 4.5 mug of R-Hg per ml. R-Hg was able to induce the expression of merB in 70% of methyl mercury-resistant strains.