Mercuric resistance genes in gram-positive oral bacteria

FEMS Microbiology Letters 236 (2004) 213–220

www.fems-microbiology.org

Mercuric resistance genes in gram-positive oral bacteria

Paul Stapleton a,*, Rachel Pike b, Peter Mullany b, Victoria Lucas b, Graham Roberts b,Robin Rowbury a, Michael Wilson b, Hilary Richards a

a Department of Biology, University College London, Gower Street, London, UKb Department of Microbiology, Eastman Dental Institute, University College London, Grays Inn Road, London, UK

Received 7 April 2004; received in revised form 23 May 2004; accepted 25 May 2004

First published online 9 June 2004

Abstract

Mercury-resistant bacteria isolated from the oral cavities of children carried one of two types of merA gene that appear to have

evolved from a common ancestor. Streptococcus oralis, Streptococcus mitis and a few other species had merA genes that were very

similar to merA of Bacillus cereus strain RC607. Unlike the B. cereus RC607 merA gene, however, the streptococcal merA genes were

not carried on Tn5084-like transposons. Instead, comparisons with microbial genomic sequences suggest the merA gene is located on

a novel type II transposon. Coagulase-negative staphylococci and Streptococcus parasanguis had identical merA genes that represent

a new merA variant.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Mercury resistance; Oral bacteria; Viridans group streptococci; Coagulase-negative staphylococci

1. Introduction

The predominant mechanism of mercury resistance in

bacteria is the enzymatic reduction of mercury, Hg2þ to

Hg0 by mercuric reductase enzyme. The mercuric re-

ductase is encoded by the merA gene, which usually

forms part of a mer operon consisting of genes coding in

addition for proteins involved in mercury transport

(MerC, MerE, MerF, MerP and MerT) [1–3], the reg-ulation of mer gene expression (MerR and MerD) [4,5],

and in some cases mercuric lyase enzymes conferring in

combination with MerA, organomercuric resistance

(MerB1, MerB3, and MerB3 in Bacillus cereus RC607)

[6,7]. For comprehensive reviews on mercury resistance

see [8–11].

In Gram-positive bacteria a merA gene identified in

B. cereus RC607 isolated in Boston Harbour, USA[12,13] is widely distributed amongst the Bacillaceae

* Corresponding author. Present address: Microbiology Group,

School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX,

UK. Tel.: +44-20-7753-5848; fax: +44-20-7753-5942.

E-mail address: [email protected] (P. Stapleton).

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.05.041

[7,12–17] and has also been found in a Clostridium bu-

tyricum isolate. Nucleotide sequence similarities of these

genes with the B. cereus RC607 merA gene vary between

76.8% and 100%. All are chromosomally located with

the exception of the merA gene from Exiguobacterium

sp. TC38-2b, which is located on plasmid pKLH3 [14].

Two other merA variants from Gram-positive bacteria

have also been described; one originally identified on

plasmid pI258 in Staphylococcus aureus [18] and anotherlocated on the chromosome of Streptomyces lividans

[19]. Staph. aureus and Streptomyces lividans merA genes

share 58.8% and 35.2% nucleotide sequence similarities

with the RC607 merA gene, respectively. Mercuric re-

ductase genes amongst Gram-positive bacteria are not

consistent in length; nucleotide sequences vary from

1641 to 1896 bp for the Bacillaecae group, 1644 bp for

Staph. aureus, and 1425 bp for S. lividans. Variations inmerA gene lengths are primarily caused by alterations at

the 50end of the gene coding for an N-terminal domain,

of approximately 70 amino acids, that shares homology

with the mercury transport protein, MerP [20]. Shorter

gene sequences such as the merA from Steptomyces

lividans code for a protein that lacks the N-terminal

. Published by Elsevier B.V. All rights reserved.

mail to: [email protected]

214 P. Stapleton et al. / FEMS Microbiology Letters 236 (2004) 213–220

domain, intermediate length sequences code for proteins

with one N-terminal domain (e.g. plasmids of Staph.

aureus), while long sequences code for MerA proteins

that have a duplication of the N-terminal domain (e.g.

chromosome of B. cereus RC607). Function of theN-terminal domain remains to be resolved but a metal

binding motif within the domain is thought to bind

mercury and assist translocation of mercury to the cat-

alytic site [11].

The widespread distribution of homologous merA

genes amongst the Bacillaceae is due in part to their

location on type II transposons (i.e. transposons flanked

by short inverted repeat sequences rather than insertionsequence elements) [7,14,21]: movement of the transpo-

son from the chromosome onto plasmids facilitates

distribution of mercury resistance between bacterial

species [21]. All of the transposons carrying RC607-like

merA genes are homologous and consist of 38 bp in-

verted repeat sequences, a res site, and transposase

(tnpA) and resolvase genes encoding enzymes involved

in translocation of the element [21,22]. Usually the genesinvolved in transposition lie upstream from the mer

operon. The genetic organisation of the mer determinant

of B. cereus RC607 carried on transposon Tn5084

consists of two operons (given in brackets); merB1,

(merR1, merE, merT, merP, merA), (merR2, merB2,

merB3), where merB1, merB2 and merB3 are genes en-

coding organomercurial lyases with different substrate

specificities, and merR1 and merR2 encode regulatoryproteins that control expression of genes within the two

operons [13,22,23].

Dental amalgam fillings are a major source of human

exposure to mercury. Oral bacteria resistant to mercury

have been reported [24]. However, the mechanism of

mercury resistance in this group has not been elucidated.

The aim of this study was to determine the nature of

mercuric resistance genes in oral bacteria.

2. Materials and methods

2.1. Bacterial strains

Mercury-resistant oral bacteria, from the saliva and

plaque samples of 100 children (age range: 5–15 years),were isolated on Mueller–Hinton agar containing 40 lMHgCl2 [25,26]. Isolates were identified to species level

with the API identification system (bioM�erieux UK

Limited, Basingstoke, United Kingdom) and by carbo-

hydrate substrate analysis [27].

2.2. DNA techniques, PCR and DNA–DNA hybridisation

DNA extraction was performed with a Gram-positive

DNA isolation kit (Flowgen, Ashby de la Zouch, United

Kingdom). Southern blotting to Nylon N+ membranes

(Amersham Biosciences) and DNA–DNA hybridisation

with digoxygenin-labelled probes was carried out ac-

cording to the manufacturer’s instructions (Roche Mo-

lecular Biochemicals, Lewes, United Kingdom).

Digoxygenin-labelled merA probes were prepared fromStaph. aureus NCTC8325 (carrying pI258) and B. cereus

RC607 DNA by PCR by the incorporation of alkali-

stable digoxygenin-labelled dUTP (Roche Molecular

Biochemicals) in the amplification mixture. PCR am-

plification was performed as described by Ausubel et al.

[28] with Dynazyme II thermostable polymerase

(Flowgen) and an annealing temperature of 50 �C. Prim-

ers used for the specific detection of themerA genes were:BamerAF1 [50-CATCATCGGTTCTGGTGGAG-30]and BamerAR1 [50-AGTTGTCCTAATTCCATGCC-

30] for the detection of B. cereus RC607 merA gene (537

bp product; DNA sequence positions 2737–3273 of

GenBank Accession Number AF138877; merA from

RC607), and SamerAF1 [50-CGACGATAAGACGA-

TACAAG-30] and SamerAR1 [50-TCCAAATTGA-

ACCGCTAACG-30] for the detection of the merA genefrom Staph. aureus (927 bp product; DNA sequence

positions 3744–4671 of GenBank Accession Number

L29436; merA from pI258). Primers capable of ampli-

fying both the Bacillus spp. and staphylococcal merA

genes were also used in this study, these were: UmerA-F

[50-CTGGTTGTGAAGAACAT-30] and UmerA-R [50-TCCTTCTGCCATTGTTA-30]. Due to a sequence

repeat at the 50 end of the RC607 merA gene theseprimers give two products (1556 and 1792 bp; DNA

sequence positions 2259–4051 and 2496–4051 of Gen-

Bank Accession Number AF138877; merA from RC607)

with RC607 but give a single product with merA genes

that lack the repeat (e.g. merA on pI258 from S. aureus

gives a 1535 bp product; DNA sequence positions 3194–

4728 of GenBank Accession Number L29436).

2.3. Ligation-assisted PCR

Ligation-assisted PCR was carried out on HindIII

digested DNA cloned into the HindIII site of pMOS-

Blue (Amersham Biosciences). Primers, INVF (50-TCGGCATGGAATTAGGACAAC-30) located within

the merA gene (DNA sequence positions 3252–3272 in

GenBank Accession Number AF138877; merA forRC607) and T7P (50-TAATACGACTCACTATAG-

GG-30) located within the vector (T7 promoter site) were

used to amplify the region downstream from the merA

gene.

2.4. DNA sequencing and sequence analysis

DNA sequencing was performed with the aid of anApplied Biosystems automated fluorescent sequencer

(model 373A). Sequence homology searches were per-

formed with the BLAST algorithms provided by the

P. Stapleton et al. / FEMS Microbiology Letters 236 (2004) 213–220 215

National Center for Biotechnology Information (http://

www.ncbi.nlm.nih.gov). Preliminary sequence data for

Streptococcus mitis and Streptococcus gordonii was ob-

tained from The Institute for Genomic Research

through their website at http://www.tigr.org. Clustal Vsequence alignments were performed with the MegAlign

program (DNASTAR, Inc).

The Accession numbers for the DNA sequences

determined in this study are: AJ582645 (Streptococcus

oralis 1601A); AJ582646 (S. mitis 26410); AJ582647

(Streptococcus parasanguis 18110); AJ582648 (S. para-

sanguis 17910); and AJ582649 (Staphylococcus sp.

1863A).

3. Results

3.1. Mercury-resistant bacteria isolated from the oral

cavity

415 non-duplicated mercury-resistant bacteria werecollected. The predominant group of bacteria to be

isolated were viridans group streptococci (83%), of

which, S. oralis was the most common species. Coagu-

lase-negative staphylococci were the next most frequent

bacterial group isolated (3%) followed by Staph. aureus

(1%).

3.2. Mercury resistance in viridans group streptococci

3.2.1. Detection and sequencing of the merA gene

PCR amplification with primers specific for the merA

genes from Staph. aureus plasmid, pI258 (SamerAF1and SamerAR1) and Bacillus spp. (BamerAF1 and Ba-

merAR1) (Fig. 1(a)) were used to screen the mercury-

resistant isolates. Of 76 mercury-resistant viridans group

streptococcal isolates screened, 59 (mostly S. oralis and

S. mitis) gave positive amplification products with

primers specific for the merA from Bacillus spp. No

amplification products were obtained with primers spe-

cific for the merA from Staph. aureus. DNA sequencingof the PCR products (536 bp) from 6 isolates indicated

that the strains carried a merA gene that was identical in

this region to the merA gene from B. cereus RC607.

Primers capable of amplifying a larger proportion of

the merA gene (UmerA-F and UmerA-R; Fig. 1(a)) were

used to amplify the merA gene from two isolates,

S. mitis 26410 and S. oralis 1601A. These primers are

capable of amplifying the merA genes from both RC607and pI258. Due to a sequence repeat at the 50 end of the

RC607 merA gene these primers give two products (1555

and 1792 bp) with RC607 but give a single product

(�1555 bp) with merA genes that lack the repeat (e.g.

merA on pI258 from S. aureus) (Fig. 1(a)). Both S. mitis

26410 and S. oralis 1601A gave a single amplification

product. DNA sequence analysis revealed that the merA

core sequences of S. mitis 26410 and S. oralis 1601A had

99% and 100% identity, respectively, with the merA se-

quence of RC607 (Fig. 1(a)). S. mitis 26410 differed from

that of RC607 at three nucleotide positions; 4025G!T,4026C!G and 4078T!C (RC607 numbering scheme,

Accession Number AF138877). In contrast to the core

sequences, the 50 ends of the merA genes had much lower

sequence similarity (68% identities) with the B. cereus

RC607 merA gene (Fig. 1(b)). The deduced amino acid

sequence for this region had 63% sequence similarity

when compared with the MerA protein sequence of

B. cereus RC607.Comparison of the merA sequences of S. mitis 26410

and S. oralis 1601A with the DNA sequences of unfin-

ished microbial genomes determined at The Institute of

Genomic Research revealed 99% identity with the se-

quences of S mitis NCTC 12261 (contig 101; nucleotide

positions 5362–7020) and S. gordonii (contig 2383; nu-

cleotide positions 2325–4220). The complete length of

the merA gene from S. gordonii is predicted to be thesame length as the B. cereus RC607 merA gene (1896

bp), while the merA genes from S. mitis NCTC 12261

and the isolates identified in this study are predicted to

be 1659 bp in length. The 50 ends of the S. mitis NCTC

12261 and S. gordonii merA genes are identical to those

from S. mitis 26410 and S. oralis 1601A (Fig. 1(a)

and (b)).

3.2.2. merA is not carried on Tn5084 or related transpo-

sons in viridans group streptococci

The mer operon in B. cereus RC607 is carried on a

class II transposon (Tn5084) [22]. Since the merA geneof the isolates in this study shared high sequence ho-

mology with the merA from B. cereus RC607, DNA

from 28 isolates was screened by DNA–DNA hybridi-

sation for the presence of the Tn5084 transposase (tnpA)

and resolvase (tnpR) genes (data not shown). None of

these isolates carried these genes, indicating that the

merA gene in the isolates was not carried on Tn5084. To

investigate whether the regions upstream and down-stream from the merA gene was consistent with the mer

operon structure of B. cereus RC607, both the merR1

and merB3 genes were examined. The merR1 gene is the

principle regulatory gene the controls the expression of

merA and is invariably located upstream from the merA

gene. The merB3 gene codes for an organomercurial

lyase that confers resistance to organomercurial com-

pounds such as methylmercury chloride and ethylmer-cury chloride. This gene does not form part of all mer

operons from Gram-positive bacillus spp but is located

downstream from merA on Tn5084 in B cereus RC607.

Hybridisation studies showed that the 28 isolates in-

vestigated above did not carry merR1 and merB3 genes

homologous to those found on Tn5084 (data not

http://www.ncbi.nlm.nih.gov

http://www.ncbi.nlm.nih.gov

http://www.tigr.org

Fig. 1. (a) Comparison of the merA sequences from B. cereus RC607, oral bacteria, and plasmid pI258 from S. aureus. The merA from B. cereus

RC607 consists of a core sequence and a repeat sequence at the 50 end of the gene (denoted I and II). Diagonal hatching indicates 50 ends with lower

identities (68%) to RC607. S. parasanguis, S. agalactiae, and coagulase-negative staphylococci have core merA sequences (indicated in black) with

low similarities (66%) to the RC607 sequence. The percentage similarities of the merA genes are given in the table. Binding positions of the PCR

primers used in this study and lengths of the corresponding amplification products are also given (indicated by arrows). Nucleotides positions for

RC607 and S. aureus pI258 are from GenBank Accession Numbers AF138877 and L29436, respectively. (b) Clustal V nucleotide sequence alignment

of the 50 ends of the merA genes from Bacillus spp. and streptococci. Nucleotides identical to the merA sequence from B. cereus RC607 are shaded

black. The numbers above the sequence alignment correspond to the RC607 merA sequence (Accession Number AF138877). The Accession numbers

for the other nucleotide sequences are: Y09024 (B. cereus TA32-5); Y10855 (Bacillus licheniformis FA6-12); Y09906 (Bacillus macroides TC47-5);

Y09907 (B. megaterium MK64-1); and AE014249 (S. agalactiae 2603V/R). S. mitis NCTC 12261 and S. gordonii merA operons were con-

structed from sequence data provided by TIGR (S. mitis, contig 101; nucleotide positions 5401–6956 and S. gordonii, contig 2383; nucleotide

positions 2325–4220).


shown). The presence of other mer genes on Tn5084

(merE, merT, merP, merR2, merB1 and merB2) was not

investigated.

3.2.3. Analysis of the region downstream of themerA gene

Ligation-assisted PCR was used to amplify the region

downstream from the merA gene of two isolates, S. mitis


26410 and S. oralis 1601A. Genomic DNA was cut with

the restriction endonuclease HindIII, which cuts within

the merA gene and the flanking DNA region. The DNA

fragments were ligated into the HindIII site of plasmid

vector pMOSBlue, which was subsequently used as atemplate. PCR was used, with primers INVF (located

within the merA gene) and T7P (located within the

vector), to amplify the region downstream from the

merA gene. With this technique 388- and 500-bp were

amplified beyond the end of the merA gene for S. oralis

1601A and S. mitis 26410, respectively (Fig. 2). DNA

sequence analysis indicated that homology of S. oralis

1601A and S. mitis 26410 with the B. cereus RC607 mer

operon ended at the translational stop codon (TAA) of

merA. No mer-related genes were found downstream

from merA and the DNA sequence identity between

strains 26410 and 1601A ended after 108-bp (Fig. 2). In

strain 1601A, a gene coding for a novel transposase

could be identified 159-bp downstream from merA.

Examination of finished and unfinished microbial ge-

nome sequences revealed that the same transposase genewas present at identical positions downstream from the

merA genes of S. mitis NCTC12261, S. gordonii and

Streptococcus agalactiae 260 V/R. Comparison of the

transposase sequence of S. mitis NCTC12261 with the

DNA sequence amplified from S. mitis 26410 indicated

Fig. 2. DNA sequences downstream from the merA genes of (a), S. oralis 16

Number AJ582646). The filled triangles indicate the positions where sequenc

associated with the deletion point is enclosed in a box. Positions of the right

genes are also underlined. Letters above the DNA sequence are one-letter a

that the first part of the transposase gene was present

downstream from merA but a deletion had occurred

removing most of the gene (Figs. 2 and 3).

3.2.4. merA gene from S. parasanguis

Strains that were PCR-negative with primers specific

for the B. cereus RC607 and Staph. aureus merA genes

were subjected to further PCR amplification with

primers UmerA-F and UmerA-R. All S. parasanguis

isolates that were previously PCR-negative gave positive

amplification products with these primers (6 isolates,

35% of previously PCR-negative isolates). The remain-

ing isolates, mainly Streptococcus vestibularis and

Streptococcus salivarius were PCR-negative. DNA se-

quencing of the merA gene from two S. parasanguis

isolates, 18110 and 17910 revealed that both isolates had

identical merA genes. The 50 ends of the genes wereidentical to those found in the other streptococcal iso-

lates (Fig. 1). However, the merA gene had low overall

sequence similarity with the B. cereus RC607 merA gene

(67%) (Fig. 1(a)). Comparison of the S. parasanguis se-

quences with the merA sequences from the recently

published genome sequence of S. agalactiae [29] (Gen-

Bank Accession Numbers AE014249, AE014282, and

AE014283) revealed an identical match.

01A (Accession Number AJ582645) and (b), S. mitis 26410 (Accession

e identity is lost between the two sequences. The sequence, CGACAT,

-hand inverted repeat (IR-R), and the positions of start and end of the

bbreviations for the deduced amino acids coded for by the sequence.

Fig. 3. Schematic representation of the genetic organisation of mer operons and flanking sequences of streptococci. Diagrams of S. mitis NCTC

12261 and S. gordonii mer operons were constructed from sequence data provided by TIGR (S. mitis, contig 101; nucleotide positions 5401–6956 and

S. gordonii, contig 2383; nucleotide positions 2325–4220). Sequence data for S. oralis 1601A and S. mitis 26410 was determined in this study (Ac-

cession Numbers AJ582645 and AJ582646, respectively). The diagrams of S. agalactiae 260 V/R were drawn from information provided in GenBank

data files (Accession Numbers AE014249, AE014282, and AE014283). The sequences of the left (IR-L) and right (IR-R) inverted repeat sequences of

a putative type II transposon proposed to carry the merA genes are indicated. Direct-repeat sequences associated with the movement of the

transposon are boxed. The directions of the arrows associated with the genes indicate the relative directions of gene transcription.


3.2.5. Mercury-sensitive isolates

It was noted that some mercury-sensitive isolates

(15%) gave positive hybridisation with the Bacillus spp.

merA probe. Only one strain, 20210 gave a PCR productafter PCR amplification with primers specific for the

Bacillus spp. merA gene. The product was approxi-

mately 150 bp smaller than the expected size and DNA

sequencing confirmed that PCR product was a trun-

cated form of the merA gene. None of the mercury-

sensitive isolates gave amplification products with the

primers UmerA-F and UmerA-R, or primers specific for

the staphylococcal merA gene.

Fig. 4. Southern blot and hybridisation with a merA probe of AccI

digested genomic DNA from staphylococci and related bacteria pro-

bed with a merA probe prepared from Staph. aureus NCTC8325. Lane

1, molecular size markers; lane 2 Staph. aureus NCTC6571; lane 3,

Staph. aureus 2763O; lane 4, coagulase-negative staphylococcus

2733O; lane 5, coagulase-negative staphylococcus 1863A; lane 6, co-

agulase-negative staphylococcus 1864A; lanes 7, Staph. aureus 1864O;

lanes 8, CNS 1881A; lane 9, Stomatococcus mucilagenosus 2784O.

3.3. Coagulase-negative staphylococci

The merA genes from mercury-resistant oral coagu-

lase-negative staphylococcal isolates were investigated

by PCR and DNA–DNA hybridisation. DNA–DNA

hybridisation indicated that the coagulase-negative

staphylococcal isolates did not carry merA genes highlyhomologous to the merA from Staph. aureus (Fig. 4).

Amplification with the primers UmerA-F and UmerA-R,


followed by DNA sequencing revealed that coagulase-

negative staphylococcus 1863A (GenBank Accession

Number AJ582649) had a merA gene identical to those

found in S. parasanguis and S. agalactiae. This sug-

gested that horizontal gene transfer had occurred be-tween these two species, although the mechanism of

transfer remains to be determined.

4. Discussion

Analysis of the merA sequences from bacteria iso-

lated from the oral cavity of children has shown thatthey carry one of two types of merA gene variants.

The merA genes from S. parasanguis and coagulase-

negative staphylococci (CNS) isolated in this study are

identical to the merA genes (merA-1 and merA-2)

found in S. agalactiae 2603V/R for which the com-

plete genome sequence is available [29]. This variant

of the merA gene will be referred to as ‘‘variant-1’’.

The second gene variant, ‘‘variant-2’’, typically foundin S. oralis and S. mitis, has high DNA sequence

similarity (99–100% identity) with core sequence the B.

cereus RC607 merA but the 50 end of the gene has

lower sequence similarity (68% identity) suggesting the

gene has a mosaic structure. Interestingly, the 50 endsof variants 1 and 2 are identical despite their core

merA sequences having low similarities (66% identity)

and both variants have similar sequences flanking themerA genes, suggesting that both genes have evolved

from a common ancestor. The absence in the oral

isolates of merR and merB3 genes homologous to

those found within RC607 suggests that they do not

carry RC607-like mer operons. Consequently, gene

variant-2 probably evolved from variant-1 by DNA

recombination with RC607-like merA DNA.

A mosaic merA gene has been described previouslyin Bacillus megaterium MK64-1 [21], where the 50 and30 ends of the RC607-like merA gene have undergone

recombination with DNA from Bacillus firmus [21].

Neither the 50 nor 30 ends of variant-1 or variant-2

shared significant homology with the corresponding

DNA regions in B. megaterium MK64-1 suggesting

that the merA genes are of independent origin.

B. cereus RC607-like merA genes are found on classII transposons (transposons flanked by inverted repeat

sequences rather than insertion sequence elements), lo-

cated on chromosomes and plasmids, and distributed

amongst Bacillus spp., Exiguibacterium spp. and Clos-

tridia [14,21,22]. Hybridisation of genomic DNA from

the oral isolates with probes for the transposase and

resolvase genes of Tn5084, which carries the mer operon

in B. cereus RC607 was unsuccessful. This suggests thatthe merA genes in the oral isolates were not carried on

Tn5084-like transposons. Analysis of the regions

downstream of the variant-2 merA genes from two iso-

lates in this study indicated that, unlike the mer operons

on Tn5084-like transposons, no additional mer-related

genes were found downstream from merA. The lack of

merR2 and merB3 genes downstream from merA is acharacteristic of the mer operon from B. megaterium

MK64-1 [21]. However, the transposase gene that mo-

bilises the mer operon in MK64-1, which differs from the

Tn5084 transposase gene, could not be identified in the

two oral isolates. Instead, part of the sequence coding

for a novel transposase belonging to the ISL3 family was

found downstream of merA in S. oralis 1601A. This

transposase gene is also pre-sent downstream from themerA genes of S. mitis NCTC 12261, S. gordonii and S.

agalactiae 260 V/R suggesting that the ISL3-like gene

may form part of a transposon capable of mobilising the

merA gene (Fig. 3). Inspection of the sequences of S.

mitis NCTC 12261 and S. agalactiae 260 V/R reveals a

24-bp inverted repeat sequence that forms the ends of

putative transposon carrying a merR gene, a merA gene

and a transposase gene. An 8-bp direct repeat sequence(GATTTTTT) consistent with movement of the trans-

poson could also be identified in the sequence from S.

agalactiae 260 V/R (Fig. 3). In S. mitis 26410 most of the

transposase gene had been deleted so it is not possible

say whether the putative transposon played a part in the

movement of the merA gene in this isolate.

In this study, the merA gene could be detected in

some mercury-sensitive isolates. Previous studies haveshown that mercury-sensitive Gram-positive environ-

mental isolates can carry functional merA and merR

genes but are thought to contain non-functional genes

coding for proteins involved in mercury transport

[10,30]. All the merA-positive isolates tested, both

mercury-sensitive and mercury-resistant, lacked the

genes coding for proteins involved in mercury trans-

port suggesting this was not the reason for the ob-served differences in mercury susceptibilities. One oral

isolate in this study carries a truncated merA gene,

which would explain why the isolate was mercury-

sensitive. The other mercury-sensitive isolates were not

investigated in detail but they may also carry trun-

cated merA genes. Absence of functional mercury

transport proteins may explain why mercury-resistant

isolates (merA-positive) in this study exhibited only a2- to 4-fold decrease in mercury-susceptibility com-

pared to mercury-sensitive isolates (merA-negative).

The presence of RC607-like DNA in merA variant-2

indicates a link between the flow of genetic information

from environmental bacteria to oral isolates. In addi-

tion, the presence of identical merA genes in oral

S. parasanguis and coagulase-negative staphylococcal

isolates suggests that horizontal gene transfer has oc-curred between them probably within the oral cavity.

Further work is necessary to establish the mechanism of

merA gene transfer between the isolates.


Acknowledgements

This work was funded by the Medical Research

Council (grants G9810729 and G9810341). The authors

wish to thank E.S. Bogdanova (Russian Academy ofSciences, Moscow, Russia) and J. Hobman (The Uni-

versity of Birmingham, Birmingham, United Kingdom)

for providing strains used as controls. Sequencing of

S. mitis NCTC12261 by TIGR was accomplished with

support from the National Institute of Health and the

National Institute of Dental and Craniofacial Research.

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Mercuric resistance genes in gram-positive oral bacteria

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