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Keywords:

  • Bacterial mercury resistance;
  • mer Operon;
  • Evolution;
  • Horizontal gene transfer

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistances to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.


1Introduction and overview

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

1.1Metal requirement and toxicity

Metals are required in many aspects of the growth, metabolism and differentiation of organisms [1]. Some are considered essential for normal function e.g. potassium, sodium, calcium and copper [2], whilst others have no known essential biological function and are toxic to the cell e.g. mercury, cadmium and arsenic [1]. In addition some metals, e.g. copper, are essential at low concentrations but toxic at high concentrations [2]. Three mechanisms have been proposed for the toxic action of metals on biological systems [3]: (i) the blocking of functional groups of important molecules such as enzymes and transport systems; (ii) displacement and/or substitution of essential ions; and (iii) modification of the active conformation of biomolecules. In response to this toxic assault, bacteria have developed an astonishing array of resistance mechanisms.

1.2Bacterial heavy metal resistance mechanisms

A number of mechanisms which impart resistance to heavy metals have been identified [4]. These are: (i) blocking, in which the toxic ion is prevented from entering the cell, e.g. Cu2+[5]; (ii) active efflux of the metal ion from the cell by highly specific systems encoded by resistance genes, e.g. Cd2+[6], AsO21− and AsO43−[7]; (iii) intracellular physical sequestration of the metal by binding proteins, e.g. Cd2+ and Zn2+[8]; (iv) extracellular sequestration, often by extracellular polysaccharides on the cell wall, e.g. Pb2+[9]and Cu2+[10]; and v) enzymatic conversion of the metal to a less toxic form, e.g. CH3Hg and Hg2+[11]. Many of these heavy metal resistance mechanisms are encoded by genetic systems which have been extensively studied and are well understood, and have been reviewed elsewhere: arsenic and antimony [7]; cadmium, cobalt, zinc and nickel [6]; copper [12]; chromate [13]; germanium and silver [14]and tellurium [15]. In this review we focus on the most extensively studied metal resistance, that of resistance to mercury (HgR) and its compounds.

1.3Environmental cycling and toxicity of mercury and its compounds

A global mercury cycle exists whereby mercury and its compounds are released from both geological and industrial processes into water and the air. Microbial activity plays an important role in this cycle, resulting in the conversion of mercury compounds from one form to another. Mercury has a ubiquitous distribution occurring in all classes of igneous rock, and it is likely that huge amounts of mercury were released into the early Earth's atmosphere from ancient volcanic activity [16]. Today an average soil will contain between 20 and 150 ppb mercury in a number of chemical species, although much of this will be biologically unavailable due to binding to organic compounds. Large deposits of mercury have been found in Spain, China, and the former Soviet Union occurring as mercury(II) oxide and mercury (II) sulphide (cinnabar). In addition to the mining of mercury ores, anthropogenic activity has resulted in a widescale release of large quantities of biologically available mercury from activities such as the burning of fossil fuels and the production of chloroalkali [16]. Of more recent concern is the use of mercury in extraction of metallic gold from alluvial washings in the Brazilian gold mining fields, where 650000 people are believed to be exposed to levels of mercury in the air of nearly 300 times those usually found [17]. In the general population, the worldwide use of dental amalgam represents the major source of exposure to mercury [18]. The toxic effects of mercury and its compounds are diverse. Inorganic mercury damages both proteins and DNA and its effects include pharyngitis and hepatitis, whilst the more toxic methylmercury, which became infamous after the Minimata Bay poisonings, leads to a series of neurological disorders including encephalopathy and in some cases death. A more detailed coverage of mercury toxicity has been presented by von Burg and Greenwood [16]. Mercuric compounds have also been extensively implicated in a number of DNA mutations which are the subject of a comprehensive review by De Flora et al. [19].

1.4Mechanisms of resistance to mercury compounds

Five types of resistance or detoxification mechanisms to mercury and its compounds have been reported:

(i) Reduced uptake of mercuric ions. This has been reported in a strain of Enterobacter aerogenes where resistance is believed to be due to the expression of two plasmid encoded proteins which cause a reduction in the cellular permeability to Hg2+ ions [20]

(ii) Demethylation of methylmercury followed by conversion to mercuric sulphide compounds. In Clostridium cochlearium T-2P two plasmid encoded genetic factors are believed to be responsible for the demethylation of organomercurial compounds which are subsequently inactivated by reaction with hydrogen sulphide to form insoluble mercuric sulphide [21].

(iii) Sequestration of methylmercury. In Desulfovibrio desulfuricans API, methylmercury is maintained at subtoxic levels by the continuous production of hydrogen sulphide, from the dissimilative reduction of sulphate, which reacts with methylmercury to form insoluble dimethylmercury sulphide [22].

(iv) Mercury methylation. Although methylmercury is generally considered to be more toxic than Hg2+, in some bacteria methylmercury may be the less toxic form, possibly due to subsequent sequestration or volatilisation from the cell. Methylation has been identified in bacteria from sediment, water, soil and the gastrointestinal tract, and is both plasmid and chromosomally encoded [23]. In Desulfovibrio desulfuricans LS the methylation of mercury occurs as a two step process which involves the transfer of a methyl group from methyltetrahydrofolate to methylcobalamin to Hg2+[24].

(v) Enzymatic reduction of Hg2+ to Hg0. Reduction occurs in both Gram-negative and Gram-positive aerobic bacteria from a variety of natural and clinical environments across the globe, and as such has become the best studied of the mercury resistance mechanisms. The distribution, diversity and evolution of the genes and operons conferring this resistance mechanism will be the focus of this review. Mercury resistance is often located on conjugative plasmids and/or transposons [25–28]and in particular is often borne on class II transposable elements, typified by that carried by Tn21[29]. Furthermore, such HgR plasmids or transposons often carry resistances to other heavy metals and/or antibiotics.

The genetics and biochemistry of this resistance mechanism has been extensively reviewed elsewhere [11, 30–33], based on studies predominantly with the mercury resistance (mer) operons carried on the transposons Tn501 and Tn21 and this has led to the proposal of a model for Tn501[30, 33]. These operons consist of a cluster of linked genes which encode polypeptides with regulatory, transport and enzymatic functions. From the similarities between Tn501 and other closely related HgR determinants from Gram-negative bacteria (e.g. Tn21, pKLH2, pMER419 and pDU1358) it is likely that they also employ a similar mechanism. The mechanism is illustrated in Fig. 1 and involves the initial sequestration of mercuric (Hg2+) ions by a pair of cysteine residues on the MerP protein in the periplasm, which is then transferred via a redox exchange mechanism to a pair of cysteine residues on MerT. It is proposed that all subsequent transfers occur by this mechanism. The Hg2+ ion is transferred to the cysteine pair on the cytoplasmic face of MerT, prior to being transferred to a cysteine pair in the amino terminal domain of the dimeric MerA (mercuric reductase), during a proposed transient association between MerT and the MerA [30]. The mercuric ion is transferred to the carboxyl terminal cysteine pair where in association with the cysteines of the active site it is reduced to Hg0 in a NADPH dependent reaction. The non-toxic Hg0 is then released into the cytoplasm and volatilises from the cell.

image

Figure 1. Proposed mechanism of narrow- and broad-spectrum bacterial mercury resistance. Modified from [30]. See text for description.

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Resistance restricted to inorganic mercury is termed narrow spectrum resistance. In some bacteria, however, resistance is also conferred to organomercurial compounds. This is a consequence of the presence of the enzyme organomercurial lyase (MerB) [34–37]. In these broad spectrum resistance determinants the organomercurial compound is cleaved during a protonolytic reaction to leave an organic moiety and a mercuric ion [38], which is subsequently reduced by MerA as before. It has recently been demonstrated in the HgR determinant of Pseudomonas K62 that MerP and MerT are not required for transport of methylmercury into the cell [39]. The transport of organomercurials into the cell may occur by a different mechanism to that of inorganic mercury. Expression of the mer operon is inducible and under the control of the product of the merR gene. MerR binds to the mer operator/promoter (O/P) and, in the absence of Hg2+ ions, represses expression. On addition of Hg2+, the MerR-merO/P complex undergoes a conformational change permitting RNA polymerase to transcribe the structural genes (for a more detailed discussion see the review by Summers) [40]. In S. lividans 1326, MerR behaves only as a repressor, preventing transcription by binding to an operator within each promoter. Mercuric ions prevent MerR from binding to DNA hence allowing transcription of the operon [41]. In many operons an additional gene product, MerD is thought to down regulate the operon by competing with MerR for binding to the mer O/P [42].

2The distribution and ubiquity of Hg2+ reduction

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

Plasmid encoded mercury resistance was first reported in Escherichia coli[39], but since then has been found to be present in many Gram-negative and Gram-positive genera and to possess a global geographical distribution (Table 1). A number of other plasmid borne traits e.g. resistance to antibiotics (reviewed in [44]) and ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid [45]are similarly found across a diverse range of bacteria and have widespread geographical distributions. Their high levels of carriage may be a reflection of the recent widespread use of such compounds by man.

Table 1.  Species range of bacteria resistant to organomercurial and/or inorganic mercury compounds
Bacterial speciesOrigin*Plasmid and/or transposon locationReference
  1. *aqMER, R. Mersey aquatic isolates, UK; CB, Chesapeake Bay, USA (aquatic and sediment isolates); DU, clinical isolates from a urological unit, Dublin; EPS, Environmental plasmid survey collection (Gram-negative isolates from human and animal sources, Boston area, USA); RY, River Yare sediment, Norfolk, UK; SB, soil from Peak District National Park, UK; SE, R. Mersey sediment, UK; SO, soil from banks of R. Mersey, UK; T2, Tipperary copper and mercury mine, Eire.Indicates where HgR determinants have been definitely linked to a particular plasmid and/or transposon.

Aeromonas sp.EPS [55]
Aeromonas hydrophilaT2 [52]
Acinetobacter calcoaceticusSE, T2 [52]
Acinetobacter sp.CBa, Khaidarkan mercury and antimony mine, former USSRb, aqMERc, RYdbpKLH2 cpMER637a[131], b[46], c[27], d[47]
Alcaligenes sp.aqMERa, SEbapMER334, pMER610a[27], b[52]
Alcaligenes denitrificans pHG27, pHG29-c[132]
Alcaligenes eutrophussoil isolate, zinc contaminated site, BelgiumpMOL28, pMOL50 (both carry Tn4378)[133]
Alcaligenes faecalisSE [52]
Azotobacter sp.  [134]
Bacillus sp.CBa, Boston Harbor, USAb, Minimata Bay sediment, Japanc, Minas Basin sediment, Canadad a[131], b[135], c[136], d[137], e[48]
Bacillus cereusSoil isolate, Japan [138]
Bacillus pasteuriiDurgapur steel plant effluent, India [139]
Bacteriodes fragilisHuman and marmoset isolates [140]
Bacteriodes ruminicolaSewage sludge, Detroit, USA [141]
Beijerinckia sp.  [134]
Chromobacterium sp.  [136]
Citrobacter sp.Soil isolate, Christchurch, NZa, EPSbaTn3402a[25], b[55]
Clostridium perfringensClinical isolate, Detroit, USA [141]
Enterobacter sp.EPS [55]
Enterobacter aerogenesT2 [52]
Enterobacter cloacaeJapana, DUb, aqMERc, SOdaTn2101, pDU1362, cpMER1a[142], b[143], c[27], d[52]
Enterobacter faecalisClinical isolates, Santiago, Chile, and Houston, USAplasmid borne[144]
Erwinia sp.  [136]
Escherichia coliClinical isolates, London, UKba and bseveral plasmid borne, cpBH100a[43], b[53], c[145]
Flavobacterium sp.RY [47]
Klebsiella sp.aqMERa, SEbapMER627a[27], b[52]
Klebsiella pneumonia aTn2608, bsome plasmid bornea[98], b[146]
Moraxella sp. pUO1[147]
Morganella sp.EPS [55]
Mycobacterium scrofulaceumCBpVT1[148]
Paracoccus sp.  [139]
Planococcus sp.  [139]
Pseudomonas aeruginosaAustraliaaaTn501, bFP2, pRLW103 cTn1406a[149], b[150], c[151]
Pseudomonas cepaciaSpainpAMJ6[152]
Pseudomonas fluorescensaqMERa, SE, SBbapMER05, pMER330, pMER327, pMER419a[27], b[52]
Pseudomonas putida aOCT plasmid; bseveral HgR plasmidsa[153], b[154]
Pseudomonas putrefaciensaqMERpMERPH[28]
Pseudomonas stutzeri pPB[155]
Pseudomonas testosteroniSE [52]
Proteus mirabilisUSAa, JapanbaTn2613; bTn1831a[98], b[156]
Proteus morganiiDUpDU1360[143]
Proteus rettgeriClinical isolate, South AfricaR391[93]
Proteus vulgarisDUpDU1359[143]
Rhodococcus erythropolis pBD2[157]
Salmonella typhimuriumGermanyTn2410, Tn2411[158]
Serratia marcescensDUpDU1358[143]
Shigella flexneriJapanTn21[159]
Shigella sonneiJapanaTn2424, Tn2425a[160], b[156]
Staphylococcus aureus bpI258[161]b[162]
Streptococcus agalactiae  [163]
Streptomyces lividans insertion element (AUD2)[163]
Thiobacillus ferrooxidansJapanb a[164], b[165]
Xanthomonas sp. Tn5053[57]
Yersinia enterocolitica aTn3926a[166]b[167]

Bacteria resistant to mercury and its compounds have been found in a diverse range of habitats including soils and sediment, water, both freshwater and marine [25–27, 46–52], and in addition have been extensively found in clinical samples [53–56]. It is likely that plasmid and transposon associated dissemination has played a major role in this dispersal, in particular the carriage of HgR determinants by both Class II transposons and broad host range plasmids. Significantly, identical HgR sequences have been isolated across immense geographical distances, suggesting either international dissemination of such sequences, possibly by air travel, or alternatively, that such sequences may have ancient origins and following an early dispersal, perhaps millions of years ago, have since remained in their particular habitat, e.g. the HgR determinants of pKLH2 and T217 from mercury mines in the former USSR and Eire, respectively [52, 57, 58]. It is likely that the distribution of bacterial mercury resistance, both globally and across such a wide species range, is a combination of three major factors, namely long ancestry, coupled with localised selection pressures in the form of mercury compounds, and the dissemination of mer sequences by a powerful array of broad host range plasmids and transposons.

Analysis of sediment cores is beginning to provide evidence that bacterial resistance to mercury is in fact an ancient phenomenon [59](I.P. Miskin, personal communication). In a study of lakewater sediment, merRTΔP sequences were amplified using the polymerase chain reaction (PCR), with primers designed to consensus regions in the HgR determinants of Tn501, Tn21 and pMER419, from total DNA extracted directly from sediment bacteria without prior culture from 6m down the core (deposited about 10000 years ago). Radosevich and Klein [59]isolated a number of both aerobic and anaerobic bacteria from a deep sediment core (213 m deep). Most of the aerobic bacteria were capable of volatilising Hg2+ whilst only negligible volatilisation was found by anaerobic bacteria.

Meanwhile, short-term persistence of specific mer sequences in natural environments has been demonstrated by the identification of pMER419-like sequences identified in culturable isolates from River Mersey sediment some eight years after the original isolation of bacteria containing pMER419 [58]. Similarly pMERPH-like sequences have been amplified from both cultured and non-cultured bacteria from a River Mersey sediment sample collected six years after the original isolation of the Pseudomonas putrefaciens strain bearing pMERPH [60].

3Genetic diversity within mercury resistance operons and genes

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

3.1Genetic organisation of mer operons

Bacterial mercury resistance by enzymatic reduction of Hg2+ is encoded by the genes of the mer operon. Analysis of the sequences of a number of these operons cloned from a diverse range of bacterial species has revealed considerable similarity of genetic organisation among the Gram-negative HgR determinants of Tn501, Tn21, pKLH2, pDU1358, pMER419, Tn5053, pMERPH and pPB (Fig. 2). Most of these operons contain a regulatory gene, merR at one terminus, which is divergently transcribed from the structural genes from a mer O/P region. Proximal to the mer O/P region lie a number of genes encoding transport functions: all have merT and merP, and in some operons merC and orfF which have been postulated as encoding transport functions due to their homology to merT. However, Hamlett et al. [61]have demonstrated, in strains containing Tn21, that the MerC protein is required neither for Hg2+ transport nor for functional mercury resistance. In contrast, in the chromosomally encoded HgR determinant from Thiobacillus ferrooxidans a role in transport for merC genes is proposed, as was suggested by a slightly hypersensitive phenotype in E. coli strains containing the merC gene cloned in the absence of merA[62]. More recently MerC has been shown to be located in the membrane, and both hypersensitivity to Hg2+ ions and increased uptake of 203Hg2+ has been shown to be dependent upon merC induction in E. coli cells, supporting the hypothesis of MerC as a membrane bound Hg2+ transporter [63]. Downstream of the genes encoding transport functions lies merA, which encodes mercuric reductase, and merD, encoding the presumptive down regulatory protein MerD. In the broad spectrum resistance operon of pDU1358 merA and merD are separated by merB, encoding organomercurial lyase. In pPB merB is found between the merR and merT genes (Fig. 2) [64]. The mer operon of pMERPH is notable for the absence of merR and merD genes [60]. Of the mer operons from Gram-negative bacteria that have been sequenced to date only that from T. ferrooxidans E15 differs significantly in structure. In this system two merR genes, together with duplicate copies of the merC gene, are found to be physically separated from the merA gene, which is found together with a third merC gene (Fig. 2) [65].

image

Figure 2. Schematic representation of Gram-negative and Gram-positive mer operons, derived from DNA sequence data: Tn501[94, 100, 168]; Tn21[86]; pKLH2 [57]; pDU1358 [34, 60, 87]; Tn5053[77, 104]; pMER419 [73], pMERPH [60]; pPB [64]; Thiobacillus ferrooxidans E15 [65, 105, 169]; RC607 [36]; pI258 [35]and Streptomyces lividans 1326 [37]. In the paper of Reniero et al., [64]they hypothesize that pPB bears a merC gene. However, given the similarity between the merR (see Fig. 3) and merT genes of pPB and pMER419, it is possible that pPB in fact possesses orfF instead of merC, which is the arrangement in pMER419 and Tn5053.

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The mer operons isolated from Gram-positive bacteria differ in a number of ways from the Gram-negative sequences. The merR genes of pI258 and RC607 are transcribed in the same direction as the structural genes, rather than being divergently transcribed (Fig. 2) [35, 36]. In the Streptomyces lividans operon, the merR gene encodes a protein product bearing homology to the ArsR regulatory protein from the arsenical resistance determinant on the E. coli plasmid R773 [37, 41]. This regulatory gene together with the genes proposed to encode transport functions are unidirectionally transcribed and in the opposite direction to that of the genes merA and merB (Fig. 2) [37].

3.2Genetic diversity of mer genes and operons from different environments

Early studies of genetic diversity among HgR determinants was largely based on DNA–DNA hybridisation analysis. In the earliest study Barkay et al. [66]demonstrated that a 2.6 kb probe spanning most of the Tn21 mer operon hybridised to a number of Gram-negative HgR determinants but not to a series of six Gram-positive HgR bacteria. This suggested significant DNA sequence variation between the mer genes from Gram-negative and Gram-positive bacteria.

Diversity within mer operons from Gram-negative bacteria was investigated with a series of seven probes derived from Tn21 and Tn501 which were used to screen HgR bacteria from the Environmental Plasmid Survey (EPS) and E. coli Reference (ECOR) collections [55]. Of the 96 bacteria tested, approximately two thirds showed significant homology to probes spanning the majority of both the Tn501 and Tn21 operons, leaving a significant minority bearing a HgR determinant significantly different to that of these well characterised mer operons. A third of this collection of bacteria which hybridised to probes from both Tn501 and Tn21 possessed sequences which hybridised to the merC gene of Tn21. It is apparent from this study and from the more detailed data available from DNA sequencing of entire operons (see Fig. 2) that mer operons from Gram-negative bacteria can be classified on the basis of the presence or absence of merC. This widespread distribution of merC in HgR determinants from Gram-negative bacteria, when considered with the suggestion that merC genes are more common in bacteria from mercury polluted sediments than in non-polluted sediments [47], points to the possibility that merC confers enhanced resistance when levels of environmental mercury are elevated.

The use of a single mer probe or of probes which bear significant cross homology e.g. those from Tn501 and Tn21 gives only a very simple illustration of the genetic diversity present within natural populations. To overcome this limitation Rochelle et al. [49]used a series of probes constructed from the merR, merA and merB genes from Tn501, pDU1358 and pI258 to screen bacteria isolated from mercury-polluted and pristine soils and lake waters and identified mer sequences homologous to each of the probes. Although giving a more complete picture of mer gene diversity in natural bacterial populations, they stressed the importance of using other approaches in addition to phenotypic expression and probe analysis to study bacterial diversity and in particular mer gene variation.

In many of the earlier studies a number of HgR bacteria bore sequences that did not hybridise to mer probes. Such bacteria may bear mer sequences which are significantly divergent from characterised sequences or may employ an alternative mechanism of resistance to Hg2+ ions. A number of sequences from Gram-negative bacteria that did not hybridise to a Tn21 mer probe were identified in a study by Barkay et al. [67]. Subsequent hybridisation analysis at a lower stringency which permitted hybrid formation between the merA gene from Tn501 and the merA genes from Bacillus RC607 and Staphylococcus aureus pI258 detected merA-homologous sequences from all the isolates which had previously failed to hybridise to the Tn21 mer probe at high stringency [68]. They identified volatilisation as the resistance mechanism, and concluded that Hg2+ volatilisation was a more common mechanism for resistance to mercury than could be estimated by hybridisation to a single probe at high stringency.

It has long been established that the culturable population may represent between as little as 3 and 0.001% of the total bacterial community in natural environments ([69]and references therein). Barkay et al. [70]attempted to address this issue by investigating the distribution of both Gram-negative and Gram-positive mer determinants in extracts of bacterial DNA from the total microbial biomass without prior cultivation. In a freshwater community a 29-fold enrichment of Tn501-homologous sequences was observed upon addition of Hg2+ ions, whilst no enrichment was observed in the number of Gram-positive mer sequences. In an estuarine community such an addition resulted in only slight enrichment (3- to 5-fold), but of both Gram-negative and Gram-positive mer sequences. Similarly Prabu and Mahadevan [51]identified by DNA–DNA hybridisation experiments Tn501-homologous sequences in both cultivated bacteria and whole community genome DNA isolated from the marine coastal waters of the Bay of Bengal. These experiments demonstrated the potential use of whole community genome DNA for the analysis of specific bacterial genes in the total population in natural environments. Such studies are of course still limited in that they screen only for known resistance determinants, albeit in the total population as opposed to the less representative culturable fraction.

Studies involving DNA hybridisation, whilst useful for identifying and determining the distribution of important genotypes within a population of HgR bacteria, can only be successfully used to reveal gross genetic differences. Restriction fragment length polymorphism (RFLP) analysis has been used to permit a more detailed study of genetic diversity within mer operons. RFLP analysis of a series of mer determinants that had been cloned from conjugative plasmids [27, 71]identified a number of closely related determinants that were quite distantly related to the mer operons of Tn21 and Tn501 on the basis of restriction analysis. However, analysis of the polypeptides produced by these cloned determinants identified proteins equivalent in size to the MerT, MerP and MerA of Tn501, whilst hybridisation studies [72]showed that their gene sequences bore homologies of at least 70% to that of Tn501. These studies emphasise the importance of using multiple approaches when investigating sequence diversity. In spite of bearing sequences that hybridised to a mer probe from Tn501, their DNA sequences [73, 74]reveal significant variation, a fact that underlines the diversity seen in the earlier RFLP studies.

A more recent study, which employed restriction endonuclease and subsequent Southern hybridisation analysis of a series of closely related, chromosomally located mer determinants from Bacillus isolates from Minimata Bay sediment [75], has shown them to be broadly similar to that of the Bacillus RC607 HgR determinant isolated in Boston Harbor, USA. RC607-like sequences also been found in soil and sediment from the banks of the River Mersey, U.K. (A.M.O., M.C. Hart and G.N. Elliott, unpublished results), suggesting that such sequences may have a global distribution similar to that of the HgR determinants from Gram-negative bacteria, e.g. Tn21, Tn501, pKLH2 and pMER419 [58].

Genetic diversity within mer determinants from four sites in the British Isles has been investigated by RFLP analysis of individual merRTΔP regions amplified by the polymerase chain reaction from both culturable bacteria [52]and from total community DNA isolated from soil bacteria, without prior recourse to culture [76]. PCR amplification using primers designed to consensus sequences in the merR and merP genes of Tn501, Tn21 and pMER419 yielded products of two sizes, and subsequent RFLP and phylogenetic analysis confirmed this subdivision. Subsequent sequence analysis of representatives from each of the PCR/RFLP derived classes has shown that the difference in amplification product length was due to sequence variation at the 3′-end of merR[58]. Furthermore, this sequence analysis has revealed that the larger PCR products contained merR genes related to pKLH2 [58]and the virtually identical sequences of Tn5053 and pMER419 [73, 77]

These results also underlined the importance of analysing both culturable and total sequences, as the vast majority of mer sequences identified were found either in cultured or in non-cultured bacteria, but not in both [76]. It is quite clear from these results that the culturable population was not representative of the total population and, in addition, that analysis of the total population by PCR amplification, resolved by cloning individual mer sequences, fails to reveal all the diversity within a particular environment. This may reflect the numerical dominance of certain sequence types or alternatively, an inherent bias in the PCR for particular sequences as has been suggested by Suzuki and Giovannoni [78].

RFLP analysis of amplification products from the 3′-end of the mer operons (merP to merA, including in some operons, merC or orfF) (K.D.B, unpublished results) and from genes encoding transposition functions tnpR and tnpA[79, 80]from the culturable bacteria used in our earlier study is providing evidence that some of these mer determinants have undergone recombination, and that subtle variants of determinants such as those of pKLH2, pMER419 and Tn501 exist in natural environments.

3.3Determination and comparison of DNA sequences: conservation versus divergence

The most revealing studies of mer gene and operon diversity have been those based on the DNA sequence of entire or partial mer operons. Since 1983 eleven operons have been sequenced in their entirety (see Fig. 2), whilst many more have been partially characterised e.g. the merRBT region from pPB [64]and the merR-mer O/P regions from ten Gram-negative HgR soil and sediment bacteria [58]. Comparison of these sequences has revealed extensive diversity in operon organisation (see above) and has permitted the construction of phylogenetic trees that offer a valuable insight into the assessment of evolutionary relationships between bacterial mercury resistance operons [81, 58, 60, 64]. A Neighbor-Joining tree derived from merA sequences has recently highlighted the presence of a distinct group of closely related mer determinants from Gram-negative bacteria (Tn501, Tn21, pMER419, pKLH2 and pDU1358). The merA genes of all three of the sequenced Gram-positive HgR determinants and of pMERPH from Pseudomonas putrefaciens are seen to be clearly divergent from this group, and to a much larger degree from each other [60]. A similar phylogeny has also been established for the merR gene (Fig. 3 and [81, 58, 64]). The MerR phylogeny is based on a much greater number of sequences, and in addition includes a number of merR homologues e.g. yhdM[82, 83]and soxR[84]. Within both the merA and merR gene families a number of features are conserved and represent intrinsic features that have been maintained by natural selection. In MerA there is considerable conservation of amino acid sequence in both the active site and at the carboxyl terminus of the protein, whilst in MerR, conserved helix turn helix domains and the presence of a number of cysteine residues implicated in the binding of Hg2+ ions are indicative of a system that is maintained by strong selective pressure to perform a particular role. This sequence conservation within a particular family of mer genes or Mer polypeptides is not limited to merR and merA. Alignment of MerT protein sequences reveals conservation of a cysteine pair involved in Hg2+ sequestration and an overall conservation of three membrane spanning regions in the MerT proteins from all but pI258, S. lividans 1326 and the putative transporter protein orfF encoded by pMER419 [85].

image

Figure 3. Unweighted Pair Group Matrix Analysis (UPGMA) dendrogram derived from the deduced protein sequences encoded by the merR genes and merR homologues: E. coli yhdM[82]; H. influenzae yhdM and merR[83]; Tn5467 (T. ferrooxidans plasmid pTF-FC2) [101, 170]; pDU1358 [87]; pMER419/Tn5053[73, 77]; pPB [64]; pKLH2 [57]; Tn501[168]; Tn21[86]; SE3, SE11, SE12, SE20, SE31, SO1, T217, T238, SB3 and SB4 [58]; E15 merR1 and merR2 (T. ferrooxidans E15 chromosomal determinant) [65]; pI258 [35]; RC607 [36]and soxR[84]. Bootstrap values, which provide confidence intervals for the phylogeny, are shown to the left of the node being considered. MerR sequences were aligned using the PILEUP program on UWGCG [171]. Distance values were calculated using the program PROTDIST on PHYLIP 3.5C [172]. Bootstrap analysis [173](100 replicates) was performed using SEQBOOT and a consensus tree produced using the UPGMA option on NEIGHBOR, and CONSENSE (PHYLIP 3.5C).

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Comparison of MerR protein sequences not only identifies a number of conserved features but in addition reveals considerable diversity at the carboxyl terminus. Some of this may be a result of a reduced selection pressure acting in this region, allowing some genetic drift to occur. In Tn21 it has been demonstrated that replacement of the 14 amino acids at the carboxyl terminus of MerR with 19 different amino acids had no effect upon induction of the operon by Hg2+[86]. However, this same region in other MerR proteins is subject to strong selection pressure in the maintenance of the organomercurial sensing region. This was first identified at the carboxyl terminus of pDU1358 [87]and has subsequently been identified in both pMER419 [73]and pPB [64], and illustrates not only the intricate subtleties within mer genes but also the power of natural selection to modify and subsequently maintain such systems.

3.4Non-functional vestigial HgR determinants

A number of studies are beginning to suggest that the conservation of both operon structure and individual gene sequence can break down when selection pressures are not maintained. Among a collection of Gram-positive bacteria isolated from natural environments in Russia, a number were identified which were sensitive to Hg2+ ions but still bore functional merR and merA genes [88]. The mercury sensitive phenotypes of these isolates was probably due to the absence or inactivation of genes encoding transport functions. More recently, partial characterisation of several sequences amplified and subsequently cloned from total community DNA from a soil where the mercury levels were below detection (<0.012 ppm mercury) has suggested that such vestigial sequences may be common in this environment [76]. Further evidence for the occurrence of vestigial mer sequences is perhaps suggested by the presence of three clusters of mer-related genes in the chromosome of H. influenzae Rd: (i) merP and the merR-homologue ydmM, (ii) merT and merP, and (iii) merR. These clusters are separated by over 600 kb of DNA [83]. These sequences may have been deposited during separate transposition events or perhaps of greater consequence may represent the remnants of a functional HgR system, and hence offer clues to the earlier origins of the mer operon.

4The evolution of mer operons and associated transposons

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

We have shown that mer sequences are widely distributed with respect to geographical location, environment and perhaps most significantly across an extensive and diverse range of bacterial species. This raises several questions about both the ubiquity of the mer system and about its evolution. How did the mer operon become so widely disseminated? Do the mer determinants we find today have a common origin? If so, is this an ancient resistance mechanism or is the widespread distribution of mer a more recent phenomenon, mediated by a vast array of horizontal transfer mechanisms? At present it may not be possible to provide absolute answers to these questions. In this section we suggest a hypothesis to explain how the mer system might have evolved and established its present ubiquity.

When discussing potentially long-term evolutionary events, it is important to consider the original environment in which bacteria had to survive. As discussed earlier, mercury is found in every class of igneous rock across the globe. Studies of the close association between mercury mineralization and alkaline basaltic volcanism at the mercury mining site of Almadén, Spain and the mercury deposit at Nikitovka, Ukraine [89]may imply that the levels of available mercury in the environment were once higher than they are today, due to increased levels of volcanic activity. Given that bacterial life has existed on this planet for at least 3.4 billion years [90], early bacteria may well have encountered significant amounts of mercury and mercury compounds in their environment, and consequently required a mechanism for detoxification. It is also important not to treat the mer system in isolation; four other potential resistance mechanisms have been proposed (see above) and, in combination with environmental processes, result in the biogeochemical cycling of mercury that is evident today.

The observed sequence similarity between the deduced MerR proteins from Tn21 and Tn501 from Gram-negative bacteria with that from the Gram-positive Bacillus RC607, led Helmann et al. [91]to suggest that resistance to mercuric ions may have developed prior to the divergence of Gram-negative and Gram-positive bacteria. This hypothesis was further supported by immunological data which showed that MerA proteins from Gram-positive bacteria were immunologically distinct from those from Gram-negative bacteria suggesting that no exchange of these mercury resistance genes had occurred between Gram-positive and Gram-negative bacteria [92].

Data from sediment cores, albeit only from the last 10 000 years (I.P. Miskin, personal communication) have at the very least suggested that the mer system has a certain antiquity. This is further supported by the data concerning the distribution of mer sequences. The global distribution of mercury resistance genes may have been achieved in response to recent environmental pressures, with the genes being widely disseminated by plasmids and transposons, often linked to antibiotic resistance genes; or alternatively, it could be a legacy of a much earlier evolutionary response. Studies from environments which are pristine with respect to mercury are perhaps revealing, when we consider that we can identify not only functional sequences but also vestigial sequences in these environments. Potential candidate vestigial sequences have also been identified following analysis of the complete genomic sequence of H. influenzae[83]. Two possible explanations for this phenomenon arise; namely, that these sequences represent the remains of a previously functional mer operon, or, that bacterial genomes are the subject of a parasitic invasion by insertion sequences that deposit genes randomly around the genome. If the latter is true, this has important implications for how we study the evolution of bacterial genomes.

The ubiquity in both species diversity and geographical distribution (Table 1) indicates that the mer system is clearly very successful in evolutionary terms and is reflected in the considerable conservation of both protein sequence and operon organisation across a wide range of bacterial species. The closely related mer determinants from Gram-negative bacteria may represent recent plasmid- and transposon-mediated dissemination, although it is apparent that there are important differences between them. However, when considering this, we then need to ask how pKLH2-like determinants came to be found in a mercury mine in the former USSR [57], in a mercury mine in the Republic of Ireland, in the River Mersey, UK [58]and in the sediments of Lake Windermere, U.K. (A.M.O., unpublished results). Similarly what series of events connect the plasmid pMERPH found in the River Mersey [28, 60], to the closely related R plasmid R391 from a strain of Proteus rettgeri isolated in South Africa, 18 years earlier [93]? Furthermore, sequences virtually identical to each of the mer determinants of pMER419, Tn21 and Tn501 have also been found in more than one geographical location [58].

Probably the most important example of mer sequence conservation is that seen within the group of mer operons which are closely related to a chromosomally encoded determinant isolated from Bacillus sp. RC607 [36]. This bacterium was isolated from Boston Harbor in the USA, but since then virtually identical sequences have been identified on the chromosomes of no fewer than 74 Bacillus isolates from the sediments of Minimata Bay [75], and again in the River Mersey, U.K. (A.M.O., M.C. Hart and G.N. Elliott, unpublished results). In the Minimata Bay study, the numbers of bacteria may represent a clonal expansion in response to the original contamination of the bay with methylmercury. However, the chromosomal location of the mer operon would suggest that this is not a product of a gene transfer event. The occurrence of virtually identical sequences around the world may be the remnants of a select number of surviving genotypes, which were once ubiquitous.

The divergence within the mer operons from Gram-positive organisms is also revealing. The mer operons from Gram-negative bacteria, of which most confer narrow spectrum resistance, have a broadly similar operon organisation to each other. However, the determinants from RC607, pI258 and S. lividans 1326, which all confer broad spectrum resistance, have quite different operon organisations from each other (Fig. 2). Furthermore, they exhibit a much greater divergence in DNA sequence for any particular gene in the operon than is seen in those from Gram-negative bacteria (Fig. 3) [58, 60]. This suggests sequence divergence over a long period of time.

To summarise, we propose that mer is an ancient system which evolved at a time when levels of available mercury were much higher in natural environments, possibly as a consequence of increased volcanic activity, and that the origins of mer predates the division of Gram-negative and Gram-positive bacteria. The next part of the review discusses the evolution of the mer operon and first addresses the origin and evolution of the genes involved in mercury resistance, before considering the more recent, though not necessarily modern, evolution of the mer operons commonly found on class II transposons.

Fig. 4 illustrates a proposed scheme of evolution of the narrow spectrum mer operons found in many Gram-negative bacteria (adapted from Summers) [31]. The earlier stages involving initial formation of the operon may be representative of the evolution of an ancestral determinant, prior to subsequent divergence following the development of the Gram-barrier. Narrow spectrum mercury resistance has three components: enzymatic, transport and regulatory. The essential component for resistance is mercuric reductase (MerA) and probably would have represented the initial point in the evolution of the operon. Evidence for the origins of MerA come from analysis of its DNA sequence, in that it bears significant homology to that of a family of pyridine nucleotide-disulfide oxidoreductases e.g. glutathione reductase and lipoamide dehydrogenase [94]and it is likely that merA evolved from the common ancestor of this gene family. There is increasing evidence that merR also belongs to a family of regulatory genes which control transcriptional processes (see Fig. 3), which includes soxR[84], yhdM[82, 83], bltR, bmrR[95]and glnR[96].

image

Figure 4. Proposed evolution of Gram-negative mer operons. Modified from [31]. See text for description.

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With the enzymatic and regulatory components perhaps already in existence, then only the genes encoding transport functions would be required for the construction and subsequent evolution of an ancestral mer operon. In Fig. 4, step 1 shows the preliminary stage, with the progenitors of the mer genes physically separated on the chromosome. Step 2 involves the duplication of the regulatory gene, followed by inversion and subsequent divergence to the merR and merD genes. This step may have occurred much later in the evolutionary process, and involved a translocation event resulting in the movement of merD to a position 3′ of merA. In step 3, merT and merP insert between merR and merD followed by the insertion of the gene encoding the reductase (step 4) This is followed by duplication of merT and merP (step 5) and subsequent fusion of the promoter distal merP to the gene encoding the reductase (stage 6) resulting in the formation of merA. Comparison of the sequence of the amino terminus of MerA to that of MerP has suggested that this is a likely event [97]. Step 6 also shows the divergence of the second merT gene to merC. Again, analysis of the protein sequences of MerT and MerC provides strong evidence for this gene duplication with subsequent divergence [85]. The operons shown in stage 6, have the genetic structure of both pKLH2 and Tn21. Step 7 shows the deletion of merC which results in an operon with the structure of Tn501. As has already been observed, the presence or absence of merC is one of the defining feature of the mer determinants from Gram-negative bacteria (Figs. 5 and 2). This division may be an important one and allows the division into a subgroup containing Tn21, Tn3926, pKLH2 and pMERPH and a second group containing Tn501, pMER419, Tn5053, pDU1358 and pPB. It is likely that all of these determinants have evolved from a common ancestor and that their evolution has been closely tied to that of the class II transposons. It has been proposed that Tn2613 may represent the closest extant relative to the common ancestor of the mer operons borne by class II transposons [98, 29], although it should be stated that there are no data concerning the mer genes present on Tn2613 to substantiate this claim. Determination of the nucleotide sequence of this determinant would either further confirm or disprove this hypothesis. Of those mer operons bearing merC, that borne on Tn3926 may itself represent one of the earliest Class II transposon-borne determinants. It is likely that the mer operon of pKLH2 has evolved from a common ancestor to Tn3926 following deletion of sequences which lie 3′ of the res site during a resolution event [99], as pKLH2 and Tn3926 bear considerable sequence homology in the region which lies 5′ of the res site. The multi-antibiotic resistant transposon Tn21 has probably evolved from the common ancestor by the insertion of an In2 element containing SmR and SuR genes, indicated by the presence of 5 bp inverted repeats at the integron's ends [100].

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Figure 5. Hypothetical pathway (not to scale) describing the evolution of mer operons from Gram-negative bacteria either carried on class II transposons or that are closely related to these determinants. For description see text. The + and − indicate insertions and deletions, respectively. Also see legend for Fig. 2.

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The pMERPH mer operon is the most different of the Gram-negative mer operons identified to date [60]and has probably diverged a considerable time ago, perhaps from the common ancestor of pKLH2, Tn3926 and Tn21. Not only does it exhibit significant DNA sequence divergence in the structural genes that it still shares with its distant cousins, but, more significantly, it no longer carries adjacent regulatory genes normally present in other mer operons. Functional studies suggest that a regulatory element is found elsewhere on the plasmid. A particular riddle yet to be solved is the presence of a 20 bp spacer region between the −35 and −10 sequences in the promoter region, an arrangement that had previously only been found in the mer O/P regions from Gram-positive bacteria [60]. In mer operons from Gram-negative bacteria the usual spacer is 19 bp.

Two other transposons have so far been identified that are related to Tn21. The vestigial transposon Tn5467 carried on the plasmid pTF-FC2 from T. ferrooxidans contains a merR gene in isolation together with fused tnpR and tnpA genes. It is possible that the merR could be involved in the regulation of a chromosomal mer determinant, such as that found in T. ferrooxidans E15 [101], or, alternatively, that Tn5467 may represent the remnants of a formerly functional mer operon. Similarly the Tn3-like transposon Tn4556 from Streptomyces fradiae bears two ORFs that show a low degree of shared sequence identity (between 30 and 40%) to mercuric reductase [102]. However, no mercuric reductase activity has been demonstrated by strains containing Tn4556.

Of those HgR determinants lacking merC, the basic operon organisation seen in Tn501 (merRTPAD) may be representative of the archetypal determinant, although Tn501 itself is a derivative transposon formed by transposon-mediated rearrangements [103]. Analysis of the DNA sequences of the narrow spectrum resistance determinants of pMER419 [73], Tn5053[104], and of the broad spectrum determinants of pDU1358 [87]and pPB [64], shows that their merR genes all encode a MerR which has an organomercurial sensing region. In Fig. 5, it is proposed that a merB gene has inserted into the operons of pDU1358 and pPB, although it is possible that pMER419 and Tn5053 have suffered a deletion of merB. We have suggested that pPB may bear orfF (Figs. 2 and 5), whilst Reniero et al. [64]have hypothesized that it contains a merC gene. Given the strong sequence similarities between the merR and merT genes of pMER419 and pPB (see Fig. 3), any additional orf between merP and merA is more likely to be that of orfF, as is found in pMER419. Final confirmation awaits determination of the DNA sequence in this region. It is consequently proposed that pPB, pMER419 and Tn5053 have a common ancestor, which underwent a duplication of merT, with subsequent divergence of this duplicated gene to become orfF. pPB subsequently evolved by the insertion of merB between the merR and merT genes [64].

Possible models for the evolution of Tn5053 and pMER419 involve the insertion of the ancestral HgR element adjacent to an ancestral Tn402-like transposon to form a hybrid transposon [104]. pMER419 may have subsequently evolved from this structure by the deletion of the 3′-end of this HgR transposon, and of the vestigial Tn402 sequences [73, 104].

One mer determinant from a Gram-negative bacterium has yet to be discussed, namely that borne on the chromosome of T. ferrooxidans E15. As is seen in Fig. 2 the organisation of its operon is dissimilar to both the other mer operons from Gram-negative bacteria and those from Gram-positive bacteria. Of particular note is the absence of merT and merP, the role of merC as the gene encoding transport functions and the spatial separation of regulatory and reductase genes [105, 65]. It is suggested, due to the presence of a Tn7 tnsA homologue adjacent to the merR and merC genes, that Tn7-mediated transposition events may be responsible for this spatial separation of the genes [65]or possibly that this determinant is an extant representative of a time when mer genes were spatially separated prior to the evolution of intact operons on plasmids and transposons.

In the mer operons from Gram-positive bacteria, similarities in operon structure and the absence of divergent transcription in both pI258 and RC607 [35, 36]suggests a common ancestry between these two determinants, whilst the mer operon from S. lividans 1326 may represent a more distant relative. Furthermore, the presence of the latter determinant on a 92 kb insertion element (AUD2) which is amplified in this strain to approximately 20 copies [106]offers implications for the horizontal transfer of this sequence.

In summary it is suggested that the bacterial mer operon has ancient origins, and is not a recent evolutionary development. A model for its evolution has been proposed, based upon divergence from a number of ancestral genes present in the chromosome of many bacteria. Transposition events appear to have been extensively involved in the evolution of mer determinants in Gram-negative bacteria, although how long ago these events took place is open to debate.

5Perspectives on bacterial evolution: gene systems for evolutionary modelling

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

The wholesale comparison of 16S rRNA gene sequences has revolutionised our conception of evolutionary relationships, not just between closely related bacterial species, but by the classification of living organisms into the three domains Bacteria, Archaea and Eucarya [107]. The rapid collection of 16S rRNA sequences, either directly from rRNA [108]and more commonly for bacterial sequences, by amplification of the 16S rRNA gene using the universal primers designed by Edwards et al. [109], has resulted in the development of the Ribosomal Database Project [110]. This has facilitated the construction of phylogenies encompassing all three domains of life as well as numerous smaller, more specific phylogenies. This type of analysis has not been limited to culturable bacteria. The determination of DNA sequences from 16S rRNA genes amplified directly from marine ecosystems [111], terrestrial ecosystems [112, 113]and marine sediments [114]has allowed the analysis of bacterial diversity in natural environments without recourse to culture.

In the virtual absence of a fossil record, molecular methods may provide the only effective means for resolving evolutionary relationships between bacteria [115]. The rRNA gene cluster consisting of 5S, 16S and 23S rRNA genes have been chosen as a model system for the determination of phylogenies due to their ubiquity in biological systems with the 16S rRNA gene being the most commonly utilised for such analysis. In addition the considerable conservation between 16S rRNA genes together with the absence of horizontal gene transfer events in their evolution has further contributed to their candidacy as the gene of choice for phylogenetic analysis. The analysis of the phylogeny of other genes, e.g. recA, has shown broad congruence with those derived from 16S rRNA gene sequences [115]. However, a number of other phylogenies based on protein sequences e.g. the 70-kDa heat shock protein (E. coli DnaK homologues) [116]; glutamine synthetase I [117]. have shown substantial differences from that derived from 16S rRNA gene sequences. This inconsistency between phylogenies based on different gene and protein sequences is an important one, and underlines the principle that living organisms, and bacteria in particular, cannot all be classified with total confidence on the basis of the phylogeny of one gene.

In bacteria, in addition to selection, the principle forces which are involved in evolution are recombination and horizontal transfer [118]. The mosaic nature of certain genes and the role of interspecies transfer has been ably demonstrated in the evolution of penicillin resistance in Neisseria species [119, 120]. Similarly, plasmids have a composite structure consisting of genes essential for replication, transfer and maintenance together with a diverse collection of insertion sequences and transposons [121]. The influence of horizontal transfer on the evolution of bacterial genes should not be underestimated and the roles of plasmids, phages, transposons, integrons, conjugative transposons, insertion elements and gene cassettes in this process are critical, and should not be ignored. When considering evolution it is therefore important to realise that one gene system, namely the 16S rRNA gene, although of great value for the derivation of phylogenies of both bacteria and life in general, does not provide a complete model for bacterial evolution. All living organisms are a collection of genes each evolving in a selfish manner. Moreover, horizontal gene transfer is rife in nature and is capable of having a major input in evolution. Traditional methods for constructing dendrograms have no mechanism for determining the role of horizontal transfer and Arber [122]has proposed that evolutionary trees should be drawn as a network to take account of the role of this contribution to the process. The mer operon, which has been found on the bacterial chromosome, plasmids and transposons, and has been shown to have undergone both recombinational and horizontal transfer events during evolution of the operon, would seem a highly suitable candidate system for the evaluation of methods for studying the effects of horizontal gene transfer on gene and operon evolution, if and when such methods are developed. In short, there is much more to the study of evolution, and of bacterial evolution in particular, than the compilation of a phylogeny based on 16S rRNA gene sequences. The role of recombination and horizontal gene transfer events must not be ignored, especially in bacteria where such events permit rapid adaptation to environmental pressures to occur.

6The mer operon as a model system in environmental microbiology

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

The mer operon, largely because of its well characterised genetic system, has been widely used both as a model system for investigating the evolution of genes in natural environments. In addition, the established role of horizontal gene transfer in the evolution of mer operons makes them a highly suitable system for investigating evolutionary processes. Inorganic mercury has been used to apply a selection pressure in order to study phenotypic and genotypic adaptation of aerobic heterotrophic sediment bacterial communities [123]. A strong positive correlation was found between the concentration of mercury in sediment and both the frequency of mer genes and of phenotypic resistance to mercury, suggesting that selection or genetic exchange (horizontal gene transfer) had promoted bacterial adaptation to mercury within the community. This work has in addition demonstrated the appropriateness of using mercury resistance for investigating the evolution of a phenotypic response to environmental pressure.

Resistance to inorganic mercury has also been widely used as a marker system for studying the processes of plasmid transfer. HgR plasmids have been used to investigate the effect of environmental factors upon conjugal transfer [124, 125]and to study the kinetics of retrotransfer [126]. Moreover, the in situ transfer of plasmids between Pseudomonas spp. in field release experiments has been monitored using an exogenously isolated HgR plasmid to assess the degree of plasmid transfer in the sugar beet rhizosphere [127]. Such studies are essential for assessing the mechanisms and frequencies of natural gene transfer prior to the release of genetically manipulated microorganisms into the environment.

Coupled to its use for investigating the evolution of a genetic system which responds to environmental stress has been the role of the mer operon as a generic system in the development of new techniques for the analysis of microbial populations in natural environments. mer has been used as a probe in experiments to detect bacterial sequences in whole community DNA isolated directly from lake and river sediments [70, 128]. More recently oligonucleotides designed to amplify mer regions have been used in the development of a robust method for extracting total community DNA suitable for PCR amplification [129]This has subsequently allowed an assessment of mer gene diversity in the total bacterial population of soils and sediments [76].

7Concluding remarks

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

In conclusion, mer has a global geographical distribution and is found in a plethora of bacterial species. Resistance to inorganic mercury is a highly evolved detoxification mechanism which we suggest is an ancient genetic system in which horizontal gene transfer has played an important role in its dissemination and evolution. Further analysis of bacteria from deep sediment cores, and the intriguing possibility of searching for mercury resistance genes and bacteria in the ancient waters of Lake Vostok beneath Antarctic ice [130]may strengthen the hypothesis that mer is an ancient system which has survived from a time when the levels of available mercury in the environment were much greater than today. Moreover, the widespread sequencing of entire bacterial genomes may identify further mer homologues. While much is now known about the distribution, diversity and evolution of mer in Gram-negative aerobic bacteria there is scope for further investigation of mer and of other mercury resistance mechanisms in Gram-positive aerobes, in anaerobic bacteria and the Archaea.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

This work was supported by NERC grant GR3/9502 (AMO/PS/DAR) and a NERC Postdoctoral Fellowship (GT5/TLS/94) (KDB) The authors wish to thank A.J. Pearson, M.C. Hart, G.N. Elliot, R.J. Holt and I.P. Miskin for contribution of data prior to publication. This work benefitted from the use of the SEQNET facility at Daresbury, UK.

References

  1. Top of page
  2. Abstract
  3. 1Introduction and overview
  4. 2The distribution and ubiquity of Hg2+ reduction
  5. 3Genetic diversity within mercury resistance operons and genes
  6. 4The evolution of mer operons and associated transposons
  7. 5Perspectives on bacterial evolution: gene systems for evolutionary modelling
  8. 6The mer operon as a model system in environmental microbiology
  9. 7Concluding remarks
  10. Acknowledgements
  11. References
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