Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.     

Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities.

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.     

Operon mer: bacterial resistance to mercury and potential for bioremediation of contaminated environments

Mercury is present in the environment as a result of natural processes and from anthropogenic sources. The amount of mercury mobilized and released into the biosphere has increased since the beginning of the industrial age. Generally, mercury accumulates upwards through aquatic food chains, so that organisms at higher trophic levels have higher mercury concentrations. Some bacteria are able to resist heavy metal contamination through chemical transformation by reduction, oxidation, methylation and demethylation. One of the best understood biological systems for detoxifying organometallic or inorganic compounds involves the mer operon. The mer determinants, RTPCDAB, in these bacteria are often located in plasmids or transposons and can also be found in chromosomes. There are two classes of mercury resistance: narrow-spectrum specifies resistance to inorganic mercury, while broad-spectrum includes resistance to organomercurials, encoded by the gene merB. The regulatory gene merR is transcribed from a promoter that is divergently oriented from the promoter for the other mer genes. MerR regulates the expression of the structural genes of the operon in both a positive and a negative fashion. Resistance is due to Hg2+ being taken up into the cell and delivered to the NADPH-dependent flavoenzyme mercuric reductase, which catalyzes the two-electron reduction of Hg2+ to volatile, low-toxicity Hg0. The potential for bioremediation applications of the microbial mer operon has been long recognized; consequently, Escherichia coli and other wild and genetically engineered organisms for the bioremediation of Hg2+-contaminated environments have been assayed by several laboratories.     

Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan.

A unique transposon was found in the chromosome of Bacillus megaterium MB1, a Gram-positive bacterium isolated from mercury-polluted sediments of Minamata Bay, Japan. The transposon region of a 14.5kb DNA fragment was amplified by PCR using a single PCR primer designed from the nucleotide sequence of an inverted repeat of class II transposons. The molecular analysis revealed that the PCR-amplified DNA fragment encodes a transposition module similar to that of Tn21. The transposon also encodes a broad-spectrum mercury resistance region having a restriction endonuclease map identical to that of Bacillus cereus RC607, a strain isolated from Boston Harbor, USA. The result of a phylogenetic analysis of the amino acid sequence of putative resolvase of the transposon showed that the transposon is phylogenetically closer to the transposons of Gram-positive bacteria than those of Gram-negative bacteria. Besides the transposition module and mer operon, the transposon encodes a mobile genetic element of bacterial group II introns between the resolvase gene and mer operon. The intron, however, does not intervene in any exon gene. The discovery of this newly found combination of the complex mobile elements may offer a clue to understanding the horizontal dissemination of broad-spectrum mercury resistance among microbes.     

Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad-spectrum mercury resistance.

A 13.5-kilobase HindIII fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal DNA of broad-spectrum mercury-resistant Bacillus sp. strain RC607 by using as a probe a clone containing the mercury reductase (merA) gene. The new clone, pYW33, expressed broad-spectrum mercury resistance both in Escherichia coli and in Bacillus subtilis, but only in B. subtilis was the mercuric reductase activity inducible. Sequencing of a 1.8-kilobase mercury hypersensitivity-producing fragment revealed four open reading frames (ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 were associated with cellular transport function and the hypersensitivity phenotype. DNA fragments encompassing the merA and the merB genes were sequenced. The predicted Bacillus sp. strain RC607 MerA (mercuric reductase) and MerB (organomercurial lyase) were similar to those predicted from Staphylococcus aureus plasmid pI258 (67 and 73% amino acid identities, respectively); however, only 40% of the amino acid residues of RC607 MerA were identical to those of the mercuric reductase from gram-negative bacteria. A 69-kilodalton polypeptide was isolated and identified as the merA gene product by examination of its amino-terminal sequence.     

Mercuric reductase in environmental Gram-positive bacteria sensitive to mercury

According to existing data, mercury resistance operons (mer operons) are in general thought to be rare in bacteria, other than those from mercury-contaminated sites. We have found that a high proportion of strains in environmental isolates of Gram-positive bacteria express mercuric reductase (MerA protein): the majority of these strains are apparently sensitive to mercury. The expression of MerA was also inducible in all cases. These results imply the presence of phenotypically cryptic mer resistance operons, with both the merA (mercuric reductase) and merR (regulatory) genes still present, but the possible absence of the transport function required to complete the resistance mechanism. This indicates that mer operons or parts thereof are more widely spread in nature than is suggested by the frequency of mercury-resistant bacteria.     

Mercury resistance among clinical isolates of Escherichia coli

The use of organomercurials in liquid detergents and disinfectants promoted resistance to mercury among bacteria. Dental amalgam and industries using mercury are the main source of human exposure to mercury vapor. Release of mercury from dental amalgam contributes to the enrichment of the intestinal flora with mercury resistance plasmids which may be associated with antibiotic resistance. The aim of our study was to evaluate the frequency of E. coli strains resistant to mercury and other antimicrobial agents currently used in therapy. The bacterial mercury and ampicillin, cephalexin, cefotaxime, gentamicin, tetracycline and chloramphenicol resistance was tested against 363 E. coli strains obtained from faeces and urine between 1999-2000. According to the guidelines suggested by NCCLS (1998), minimum inhibitory concentrations (MICs) were determined on Mueller-Hinton agar, using the dilution technique with an inoculum of about 10(5) CFU. The MICs were read after 18 h incubation at 37 degrees C as the lowest concentration that inhibited the development of visible growth. Plasmids in enterobacteria may carry genes encoding resistance to both mercury and antibiotics. Among the tested E. coli strains, mercury resistance rose to 29.2%. Mercury resistance in E. coli is significantly linked to multiresistance to antimicrobial agents. Between 91.5-23.6 of mercury chloride resistant isolates were also resistant to the tested antibiotics. The increased use of non antibiotic antimicrobial agents is a possible selection factor for antibiotic-resistant strains in clinical and domestic environments.     

Functioning of the mercury resistance operon at extremely high Hg(II) loads in a chemostat: a proteome analysis

The transformation of extremely high concentrations of ionic mercury (up to 500 mg L(-1)) was investigated in a chemostat for two mercury-resistant Pseudomonas putida strains, the sediment isolate Spi3 carrying a regulated mercury resistance (mer) operon, and the genetically engineered strain KT2442Colon, two colonsmer73 expressing the mer operon constitutively. Both strains reduced Hg(II) with an efficiency of 99.9% even at the maximum load, but the concentration of particle bound mercury in the chemostat increased strongly. A proteome analysis using two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) showed constant expression of the MerA and MerB proteins in KT2442Colon, two colonsmer73 as expected, while in Spi3 expression of both proteins was strongly dependent on the Hg(II) concentration. The total cellular proteome of the two strains showed very little changes at high Hg(II) load. However, certain cellular responses of the two strains were identified, especially in membrane-related transport proteins. In Spi3, an up to 45-fold strong induction of a cation efflux transporter was observed, accompanied by a drastic downregulation (106-fold) of an outer membrane porin. In such a way, the cell complemented the highly specific mercury resistance mechanism with a general detoxification response. No indication of a higher demand on energy metabolism could be found for both strains.     

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.     

The distribution and divergence of DNA sequences related to the Tn21 and Tn501 mer operons

The mercury resistance (mer) operons of the Gram-negative bacterial transposons, Tn21 and Tn501, are phenotypically indistinguishable and have extensive DNA identity. However, Tn21 mer has an additional coding region (merC) in the middle of the operon which is lacking in Tn501 and there is also a discrete region of the mercuric ion reductase gene (merA) which differs markedly between the two operons. DNA fragment probes were used to determine the distribution of specific mer coding regions in two distinct collections of mercury-resistant (Hgr) Gram-negative bacteria. Colony blot hybridization analysis showed that merC-positive operons occur almost exclusively in Escherichia, although merC-negative operons can also be found in this genus. The merC-negative operons were found in Citrobacter, Klebsiella, and Enterobacter and in some Pseudomonas. Most of the Pseudomonas did not hybridize detectably with either of the two operons studied, indicating that they harbor an unrelated or more distantly related class of mercury resistance locus. Southern hybridization patterns demonstrated that the merC-positive mer operon is well conserved at the DNA level, whereas the merC-negative operons are much less conserved. The presence of merC also correlated with conservation of a specific variant region of the merA gene and with an antibiotic resistance pattern similar to that of Tn21. Tn501 appears to be an atypical example of the merC-negative subgroup of Hgr loci.     

Distribution of transposons Tn5044 and Tn5070 with unusual mer operons in environmental bacterial populations

The distribution of unusual mercury resistance transposons, Tn5044 and Tn5070, was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase a subunit. Structural organization of mercury operon Tn5070, containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5040- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.     

Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant

Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid.     

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 intact ctxA 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.     

Streptomycin resistance in chloramphenicol-producing strains of Streptomyces species 3022a.

Resistance to low (5 mug/ml) concentrations of streptomycin in agar media was not inherited by all of the surviving population. Outgrowth of cultures in liquid media supplemented with the antibiotic depended upon inoculum size. Antibiotic titers in the supplemented cultures decreased during incubation, and an inactive radioactive product was detected when [14C] streptomycin was used. This low-level resistance is, therefore, attributed to enzymic inactivation of the antibiotic. Growth 10 mug/ml or higher concentrations of streptomycin on agar media was due to selection of resistant variants present in the parent strain. A range of such variants existed, decreasing in frequency as their degree of resistance increased. Examination of one that was resistant to moderate concentrations of streptomycin, (25 mug/ml) and a second that was resistant to high (100 mug/ml) concentrations of streptomycin suggested that both possed ribosomes which had lower affinity for the antibiotic than those of the parent strain, and that tolerance to high levels of streptomycin was due to a resistant ribosomal system for protein biosynthesis.     

Association of mercury resistance with antibiotic resistance in the gram-negative fecal bacteria of primates.

Gram-negative fecal bacterial from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hgr) and mercury-susceptible (Hgs) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hgr strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hgr strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hgr strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hgr strains lacking antibiotic resistance showed no evidence of integron components.     

Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2. The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidance E-15, and Thiobacillus 3.2, respectively. No inducibility of the Tn5037 mer operon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR). However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup. Sequence analysis of the Tn5037 res site suggested its recombinant nature.     

Transposon Tn21, flagship of the floating genome.

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Transposon Tn21, Flagship of the Floating Genome

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Spatial patterns in antibiotic resistance among stream bacteria: effects of industrial pollution

The spatial distribution of antibiotic resistance to streptomycin and kanamycin was examined in natural bacterial communities of two streams. The proportion of resistant bacteria was substantially higher (P < 0.05) in the midreaches of an industrially perturbed stream, but no such pattern was apparent in an undisturbed reference stream. The highest relative frequency of resistance was found at the confluence of a tributary draining a nuclear reactor and industrial complex. Antibiotic resistance increased with distance upstream from the confluence and was positively correlated (r(2) = 0. 54, P = 0.023) with mercury concentrations in the sediments. When the data for two years were compared, this pattern was stable for streptomycin resistance (paired t test, P < 0.05) but not for kanamycin resistance (P > 0.05). Our results imply that heavy metal pollution may contribute to increased antibiotic resistance through indirect selection.