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.     

Phenotypic and genotypic adaptation of aerobic heterotrophic sediment bacterial communities to mercury stress

The effects of mercury contamination of lake sediments on the phenotypic and genotypic mercury resistance of the indigenous heterotrophic aerobic bacterial communities were investigated. Strong positive correlations between mercury sediment concentration and the frequency of the gene coding for mercury volatilization (mer) (r = 0.96) or the phenotypic mercury resistance (r = 0.86) of the studied communities suggested that the inheritance via selection or genetic exchange of the mer gene had promoted bacterial adaptation to mercury. Failure to detect the mer gene in one mercury-contaminated sediment where phenotypic expression was low suggested that other mechanisms of resistance may partially determine the presence of mercury-resistant organisms in mercury-contaminated sediment or that the mercury in this particular sediment was very chemically limited in its availability to the microorganisms.     

Phenotypic and genotypic adaptation of aerobic heterotrophic sediment bacterial communities to mercury stress.

The effects of mercury contamination of lake sediments on the phenotypic and genotypic mercury resistance of the indigenous heterotrophic aerobic bacterial communities were investigated. Strong positive correlations between mercury sediment concentration and the frequency of the gene coding for mercury volatilization (mer) (r = 0.96) or the phenotypic mercury resistance (r = 0.86) of the studied communities suggested that the inheritance via selection or genetic exchange of the mer gene had promoted bacterial adaptation to mercury. Failure to detect the mer gene in one mercury-contaminated sediment where phenotypic expression was low suggested that other mechanisms of resistance may partially determine the presence of mercury-resistant organisms in mercury-contaminated sediment or that the mercury in this particular sediment was very chemically limited in its availability to the microorganisms.     

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.     

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.     

Horizontal transfer of mercury resistance genes in natural 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.     

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.     

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.     

Environmental significance of the potential for mer(Tn21)-mediated reduction of Hg2+ to Hg0 in natural waters.

The role of mer(Tn21) in the adaptation of aquatic microbial communities to Hg2+ was investigated. Elemental mercury was the sole product of Hg2+ volatilization by freshwater and saline water microbial communities. Bacterial activity was responsible for biotransformation because most microeucaryotes did not survive the exposure conditions, and removal of larger microbes (greater than 1 micromole) from adapted communities did not significantly (P greater than 0.01) reduce Hg2+ volatilization rates. DNA sequences homologous to mer(Tn21) were found in 50% of Hg2+-resistant bacterial strains representing two freshwater communities, but in only 12% of strains representing two saline communities (the difference was highly significant; P less than 0.001). Thus, mer(Tn21) played a significant role in Hg2+ resistance among strains isolated from fresh waters, in which microbial activity had a limited role in Hg2+ volatilization. In saline water environments in which microbially mediated volatilization was the major mechanism of Hg2+ loss, other bacterial genes coded for this biotransformation.     

Environmental significance of the potential for mer(Tn21)-mediated reduction of Hg2+ to Hg0 in natural waters

The role of mer(Tn21) in the adaptation of aquatic microbial communities to Hg2+ was investigated. Elemental mercury was the sole product of Hg2+ volatilization by freshwater and saline water microbial communities. Bacterial activity was responsible for biotransformation because most microeucaryotes did not survive the exposure conditions, and removal of larger microbes (greater than 1 micromole) from adapted communities did not significantly (P greater than 0.01) reduce Hg2+ volatilization rates. DNA sequences homologous to mer(Tn21) were found in 50% of Hg2+-resistant bacterial strains representing two freshwater communities, but in only 12% of strains representing two saline communities (the difference was highly significant; P less than 0.001). Thus, mer(Tn21) played a significant role in Hg2+ resistance among strains isolated from fresh waters, in which microbial activity had a limited role in Hg2+ volatilization. In saline water environments in which microbially mediated volatilization was the major mechanism of Hg2+ loss, other bacterial genes coded for this biotransformation.     

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.     

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.     

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments.

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Acclimation of subsurface microbial communities to mercury

We studied the acclimation to mercury of bacterial communities of different depths from contaminated and noncontaminated floodplain soils. The level of mercury tolerance of the bacterial communities from the contaminated site was higher than those of the reference site. Furthermore, the level of mercury tolerance and functional versatility of bacterial communities in contaminated soils initially were higher for surface soil, compared with the deeper soils. However, following new mercury exposure, no differences between bacterial communities were observed, which indicates a high adaptive potential of the subsurface communities, possibly due to differences in the availability of mercury. IncP-1 trfA genes were detected in extracted community DNA from all soil depths of the contaminated site, and this finding was correlated to the isolation of four different mercury-resistance plasmids, all belonging to the IncP-1beta group. The abundance of merA and IncP-1 plasmid carrying populations increased, after new mercury exposure, which could be the result of selection as well as horizontal gene exchange. The data in this study suggest a role for IncP-1 plasmids in the acclimation to mercury of surface as well as subsurface soil microbial communities.     

Intergeneric homology of the speC gene encoding biosynthetic ornithine decarboxylase in Escherichia coli.

A 32P-labeled fragment of DNA containing the speC gene, which encodes the biosynthetic enzyme ornithine decarboxylase of Escherichia coli, was used as a hybridization probe for homologous sequences in the genomes of gram-negative and gram-positive bacteria. The speC probe detected homologous sequences in the DNA of only four members of the Enterobacteriaceae (Citrobacter freundii, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes); no homology was detected with the DNA of other representative members of the Enterobacteriaceae and gram-negative and gram-positive bacteria.     

用反向斑点杂交方法检测猪常见致病菌

目的:建立检测猪常见致病菌的反向斑点杂交方法。方法:将23S rRNA基因芯片用的针对12种细菌的25～30 mer探针加长到30～38 mer,2对通用引物序列不变。用地高辛标记下游引物,以尼龙膜为载体制备膜芯片,检验探针/膜杂交的特异性和敏感性;另外设计1条大肠杆菌K88基因探针、一段带K88探针的报告基因和1对报告基因的反向PCR引物,在PCR体系中增加封口的K88报告基因和反向引物对,被检样品扩增后进行膜杂交。结果:修改的13条探针与参考目标菌株在膜上成特异性杂交,对52个参考菌株和野外分离株的检测准确率为92%;膜杂交的敏感性与玻片芯片接近,最小检出量为100 fg DNA;在尼龙膜上增加K88探针,与3重PCR产物杂交,可以检测到大肠杆菌K88毒力基因。结论:建立的反向斑点杂交方法简便快速,检测成本低,可用于仪器设备不足的实验室,同时可以加入检测如大肠杆菌K88等致病基因,提高基于保守基因的芯片的诊断能力。     

Translation of merD in Tn21.

All four sequenced examples of the mercury resistance (mer) operon of gram-negative bacteria have a promoter-distal reading frame, merD, whose removal has little effect on the resistance phenotype and whose translation has not previously been observed. Using merD-lacZ protein fusions, we show that merD is translated. However, Hg(II)-induced merD expression, as measured by beta-galactosidase activity and immunoblotting, is 10- to 15-fold lower than that of fusions to the gene immediately preceding it, merA.     

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.