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.     

Mercury-Resistant Bacteria from Permafrost Sediments and Prospects for their Use in Comparative Studies of Mercury Resistance Determinants

Mercury-resistant bacteria were isolated from permafrost sediments of Kolyma lowland and Canada existing over five thousand to two million years. Their content was shown to vary within the range 0.001–2.9% and to depend on the amount of mercury in sampling sites (coefficient of correlation 0.75). A collection of mercury-resistant bacterial strains was created. In this collection, various representatives of both Gram-positive bacteria (Bacillus, Exiguobacterium, Micrococcus, Arthrobacter) and Gram-negative bacteria (Pseudomonas, Acinetobacter, Plesiomonas, Myxobacteriales) were identified. Most resistant bacteria were found to contain determinants homologous to mer-operons of contemporary bacteria. The isolated strains of paleobacteria are proposed to be used for a comparative structural study of contemporary and ancient plasmids and transposons carrying mercury resistance determinants.     

Distribution of transposons Tn<Emphasis Type="Italic">5044</Emphasis> and Tn<Emphasis Type="Italic">5070</Emphasis> with noncanonical <Emphasis Type="Italic">mer</Emphasis> operons in environmental bacterial populations

The distribution of noncanonical 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 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 Tn5044- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1717–1721.Original Russian Text Copyright © 2004 by Gorlenko, Kalyaeva, Bass, Petrova, Mindlin.     

Novel mercury resistance determinants carried by IncJ plasmids pMERPH and R391

Summary HgCl₂ resistance (Hg^r) in a strain of Pseudomonas putrefaciens isolated from the River Mersey was identified as plasmid-borne by its transfer to Escherichia coli in conjugative matings. This plasmid, pMERPH, could not be isolated and was incompatible with the chromosomally integrated IncJ Hg^r plasmid R391. pMERPH and R391 both express inducible, narrow-spectrum mercury resistance and detoxify HgCl₂ by volatilization. The cloned mer determinants from pMERPH (pSP100) and R391 (pSP200) have very similar restriction maps and express identical polypeptide products. However, these features show distinct differences from those of the Tn501 family of mer determinants. pSP100 and pSP200 failed to hybridize at moderate stringency to merRTPA and merC probes from Tn501 and Tn21, respectively. We conclude that the IncJ mer determinants are only distantly related to that from Tn501 and its closely homologous relatives and that it identifies a novel sequence which is relatively rare in bacteria isolated from natural environments.     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

Comparative genomics reveals the acquisition of mobile genetic elements by the plant growth-promoting Pantoea eucrina OB49 in polluted environments

Heavy metal-tolerant plant growth-promoting bacteria (PGPB) have gained popularity in bioremediation in recent years. A genome-assisted study of a heavy metal-tolerant PGPB Pantoea eucrina OB49 isolated from the rhizosphere of wheat grown on a heavy metal-contaminated site is presented. Comparative pan-genome analysis indicated that OB49 acquired heavy metal resistance genes through horizontal gene transfer. On contigs S10 and S12, OB49 has two arsRBCH operons that give arsenic resistance. On the S12 contig, an arsRBCH operon was discovered in conjunction with the merRTPCADE operon, which provides mercury resistance. P. eucrina OB49 may be involved in an ecological alternative for heavy metal remediation and growth promotion of wheat grown in metal-polluted soils. Our results suggested the detection of mobile genetic elements that harbour the ars operon and the fluoride resistance genes adjacent to the mer operon.     

Bacterial <Emphasis Type="Italic">mer</Emphasis> operon-mediated detoxification of mercurial compounds: a short review

Mercury pollution has emerged as a major problem in industrialized zones and presents a serious threat to environment and health of local communities. Effectiveness and wide distribution of mer operon by horizontal and vertical gene transfer in its various forms among large community of microbe reflect importance and compatibility of this mechanism in nature. This review specifically describes mer operon and its generic molecular mechanism with reference to the central role played by merA gene and its related gene products. The combinatorial action of merA and merB together maintains broad spectrum mercury detoxification system for substantial detoxification of mercurial compounds. Feasibility of mer operon to coexist with antibiotic resistance gene (amp ^r , kan ^r , tet ^r ) clusters enables extensive adaptation of bacterial species to adverse environment. Flexibility of the mer genes to exist as intricate part of chromosome, plasmids, transposons, and integrons enables high distribution of these genes in wider microbial gene pool. Unique ability of this system to manipulate oligodynamic property of mercurial compounds for volatilization of mercuric ions (Hg²⁺) makes it possible for a wide range of microbes to tolerate mercury-mediated toxicity.     

Mercury pollution: an emerging problem and potential bacterial remediation strategies

Heavy metal toxicity represents an uncommon but clinically significant medical condition, which if unrecognized or inappropriately treated results in significant morbidity and mortality. Mercury is recognized as a potent and widely distributed toxicant in the global environment having ability to accumulate at various levels of food chain besides possessing ability to cross placental and blood–brain barrier. It has been seen that bacteria growing near mercury polluted sites have evolved various means of resistance based on the expression of different genes of mer operon against different forms of mercury. Microbe based remediation/detoxification of mercury is on forefront due to low cost and less health hazardous compared to physicochemical based strategies, which are cost intensive and hazardous to human health. However, strategies based on the modern aspects of biological technologies employing mer operon genes in different combination are needed to be designed for exploitation in the remediation of mercury completely from mercury contaminated environments.     

Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria

Individual merRTΔP regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one ‘pristine’ (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (= 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria. Forty-five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRTΔP probe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.     

New mobile genetic elements in <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34, their possible roles and occurrence in other bacteria

Cupriavidus metallidurans strain CH34 is a β-Proteobacterium that thrives in low concentrations of heavy metals. The genetic determinants of resistance to heavy metals are located on its two chromosomes, and are particularly abundant in the two megaplasmids, pMOL28 and pMOL30. We explored the involvement of mobile genetic elements in acquiring these and others traits that might be advantageous in this strain using genome comparison of Cupriavidus/Ralstonia strains and related β-Proteobacteria. At least eleven genomic islands were identified on the main replicon, three on pMOL28 and two on pMOL30. Multiple islands contained genes for heavy metal resistance or other genetic determinants putatively responding to harsh environmental conditions. However, cryptic elements also were noted. New mobile genetic elements (or variations of known ones) were identified through synteny analysis, allowing the detection of mobile genetic elements outside the bias of a selectable marker. Tn4371-like conjugative transposons involved in chemolithotrophy and degradation of aromatic compounds were identified in strain CH34, while similar elements involved in heavy metal resistance were found in Delftia acidovorans SPH-1 and Bordetella petrii DSM12804. We defined new transposons, viz., Tn6048 putatively involved in the response to heavy metals and Tn6050 carrying accessory genes not classically associated with transposons. Syntenic analysis also revealed new transposons carrying metal response genes in Burkholderia xenovorans LB400, and other bacteria. Finally, other putative mobile elements, which were previously unnoticed but apparently common in several bacteria, were also revealed. This was the case for triads of tyrosine-based site-specific recombinases and for an int gene paired with a putative repressor and associated with chromate resistance.     

<Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>: evolution of a metal-resistant bacterium

Cupriavidus metallidurans CH34 has gained increasing interest as a model organism for heavy metal detoxification and for biotechnological purposes. Resistance of this bacterium to transition metal cations is predominantly based on metal resistance determinants that contain genes for RND (resistance, nodulation, and cell division protein family) proteins. These are part of transenvelope protein complexes, which seem to detoxify the periplasm by export of toxic metal cations from the periplasm to the outside. Strain CH34 contains 12 predicted RND proteins belonging to a protein family of heavy metal exporters. Together with many efflux systems that detoxify the cytoplasm, regulators and possible metal-binding proteins, RND proteins mediate an efficient defense against transition metal cations. To shed some light into the origin of genes encoding these proteins, the genomes of C. metallidurans CH34 and six related proteobacteria were investigated for occurrence of orthologous and paralogous proteins involved in metal resistance. Strain CH34 was not much different from the other six bacteria when the total content of transport proteins was compared but CH34 had significantly more putative transition metal transport systems than the other bacteria. The genes for these systems are located on its chromosome 2 but especially on plasmids pMOL28 and pMOL30. Cobalt–nickel and chromate resistance determinants located on plasmid pMOL28 evolved by gene duplication and horizontal gene transfer events, leading to a better adaptation of strain CH34 to serpentine-like soils. The czc cobalt–zinc–cadmium resistance determinant, located on plasmid pMOL30 in addition copper, lead and mercury resistance determinants, arose by duplication of a czcICAB core determinant on chromosome 2, plus addition of the czcN gene upstream and the genes czcD, czcRS, czcE downstream of czcICBA. C. metallidurans apparently evolved metal resistance by horizontal acquisition and by duplication of genes for transition metal efflux, mostly on the two plasmids, and decreased the number of uptake systems for those metals. This paper is dedicated to Dr. Max Mergeay for a long time of cooperation, constructive competition and friendship.     

Mercury-resistant plasmids in bacteria from a mercury and antimony deposit area

Summary Most bacterial cells (Pseudomonas, Acinetobacter) obtained from the soil at the Khaidarkan mercury and antimony mine (Kirghiz USSR) contain R plasmids with mercury (HgCl₂) resistance determinants. The plasmids have a large molecular mass (about 100 MD, though smaller ones also occur), and at least some of them are transmissive. Many of the Hg^r bacteria also display an elevated antimony (SbCl₃) resistance, though this trait was not shown to be plasmid-dependent. There are practically no Hg^r plasmids in bacteria taken from the soil at different distances from the mine: the saturation of bacteria with Hg^r plasmids is maintained by selective pressure only in the area with a high enough toxin concentration.In the same mercury and antimony deposit area Hg^r plasmids were also found in Escherichia coli isolates from the gut of Mus musculus mice and Bufo viridis toads. At least some of the bacterial plasmids obtained from animals also carry antibiotic-resistance determinants (Tc^r, Cm^r, Sm^r). These plasmids are also transmissive. They display internal instability and lose their resistance determinants after a conjugation transfer to other E. coli trains.     

致病性葡萄球菌cfr基因介导的多重耐药机制研究进展

葡萄球菌是临床常见致病菌及食源性致病菌,可在食品原料加工、包装及运输过程中污染食品,引起人体多种严重感染,其耐药性的不断增强对公共卫生安全产生了重大的威胁。葡萄球菌中cfr (chloramphenicol-florfenicol resistance)基因编码的甲基转移酶,可引起细菌核糖体RNA的甲基化,从而阻碍或减弱多种化学结构不同的抗生素与肽基转移酶活性中心(peptidyl transferase center,PTC)的结合,导致葡萄球菌多重耐药表型的出现。噁唑烷酮类药物−利奈唑胺是继万古霉素后治疗耐药革兰氏阳性菌所致感染的最后一道防线,cfr基因的出现大大加速了利奈唑胺耐药性的传播。cfr基因广泛分布于多种致病性葡萄球菌中,cfr基因与各类型可转移元件(质粒、转座子和整合相关元件等)密切关联的遗传环境是其广泛传播的结构基础。在cfr基因水平传播的过程中,食源性致病葡萄球菌作为中间者扮演着重要的角色。本文就近年来国内外对致病性葡萄球菌中cfr基因的分布状况、耐药机制、遗传环境、传播机制等进行综述,以期为防控致病性葡萄球菌的传播提供参考,以遏制多重耐药菌的进一步传播。     

Evolution of mercuric reductase (<Emphasis Type="Italic">merA</Emphasis>) gene: A case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA gene is key gene in mer operon and has been used for this swtudy. Phylogenetic analysis of aligned merA gene sequences shows broad similarities to the established 16S rRNA gene phylogeny. But there is no separation of bacterial merA gene from archael merA gene which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA gene and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.     

细菌中介导多重耐药的Tn7转座子研究进展

转座子是介导细菌耐药性传播的重要可移动遗传元件。Tn7转座子与细菌耐药密切相关,其携带转座模块和Ⅱ类整合子系统。Tn7编码转座相关蛋白TnsABCDE进行“剪切-粘贴”机制转座,转座核心TnsABC也可与三链DNA或Cas-RNA复合物结合实现转座。近年来新发现了多种介导多重耐药的Tn7转座子,其在介导细菌抗生素、消毒剂和重金属抗性基因的获得、传播扩散等方面发挥了重要作用。本文综述了细菌中Tn7转座子的遗传结构、转座机制、流行以及新发现的介导多重耐药的Tn7转座子,以期为细菌中Tn7转座子的深入研究提供参考。     

Intercontinental spread of promiscuous mercury-resistance transposons in environmental bacteria

We demonstrate that horizontal spread of mer operons similar to worldwide spread of antibiotic-resistance genes in medically important bacteria occurred in bacteria found in ores, soils and waters. The spread was mediated by different transposons and plasmids. Some of the spreading transposons were damaged in different ways but this did not prevent their further spread. Certain transposons are mosaics composed of segments belonging to distinct sequence types. These mosaics arose as a result of homologous and site-specific recombination. Our data suggest that the mercury-resistance operons of Gram-negative environmental bacteria can be considered as a worldwide population composed of a relatively small number of distinct recombining clones shared, at least partially, by environmental and clinical bacteria.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Molecular Characterization of an Aberrant Mercury Resistance Transposable Element from an Environmental Acinetobacter Strain

We present the complete nucleotide sequence of a mer operon located on a 60-kb conjugative plasmid pKLH2 from an environmental bacterium, Acinetobacter calcoaceticus , isolated from a mercury mine. The pKLH2 mer operon has essentially the same gene organization as that of Tn21 and Tn501 from clinical bacteria. The pKLH2 mer operon nucleotide sequence shows 85.5% identity with the Tn501 and 80.9% identity with the Tn21 sequences. Vestigial sequences have been found at the ends of the pKLH2 mer operon, indicating that the pKLH2 mer operon was once a part of a Tn21-like transposon, which had committed suicide by an aberrant resolution event.     

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.