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Arsenical resistance in the IncHI2 plasmids
Plasmid 47 (2002) 234–240 www.academicpress.com
Arsenical resistance in the IncHI2 plasmids David Ryana,* and Emer Colleranb a
Department of Applied Biology and Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland b Environmental Research Unit, Department of Microbiology, National University of Ireland, Galway, Ireland Received 13 November 2001, revised 18 April 2002
Abstract The IncHI2 plasmid R478, like other arsenic resistance IncH plasmids, provides increased levels of resistance to sodium arsenate (up to 100 mM) and sodium arsenite (up to 10 mM) to the host cell. An arsenic resistance fragment of R478 was cloned and sequenced revealing four arsenic resistance associated gene homologues, arsR, arsB, arsC, and arsH. Two other open reading frames in the cloned fragment were found to be homologues of sulphate transport associated genes. Both the four gene arsenic resistance operons and the two gene sulphate transport operons have been previously shown to be transposon associated. However, no evidence of transposability was found associated with these operons in R478. Both the R478 associated arsenic and sulphate transport operons were shown to be common to all arsenic resistance IncH plasmids examined by Southern hybridisation and PCR analysis. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Incompatibility group H plasmids; R478; Arsenic resistance; Sulphate transport
1. Introduction Resistance to arsenical compounds in bacteria can be plasmid or chromosomally mediated. Resistance operons have been documented in a wide variety of chromosomes including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Thiobacillus ferroxidans (Butcher et al., 2000; Cai et al., 1998; Rosen, 1999; Sofia et al., 1994). The physiological role of the chromosomal arsenic operon (ars operon) appears to be the provision of low level arsenical resistance (Carlin et al., 1995; Silver et al., 1981). Plasmids which mediate for arsenic resistance include the Staphylococcus
*
Corresponding author. Fax: +353-503-70500. E-mail addresses: [email protected] (D. Ryan), [email protected] (E. Colleran).
aureus plasmid pI258, the E. coli plasmids R773 and R46, the Staphylococcus xylosus plasmid pSX267, the P. aeruginosa plasmid pUM310, the Yersinia enterocolitica virulence plasmid pYV, and the Acidiphilium multivorium AIU301 plasmid, pKW301 (Brown and Willetts, 1981; Bruhn et al., 1996; Cervantes and Chavez, 1992; Gotz et al., 1983; Hedges and Baumberg, 1973; Neyt et al., 1997; Novick and Bouanchaud, 1971; Suzuki et al., 1998). Three prototype ars operons have been well documented and these are the three-, four-, and five-gene arsenic resistance determinants, although novel resistance mechanisms have also been described. The arsR gene encodes a repressor protein that controls operon expression. The arsB gene protein product is a transmembrane efflux channel. The arsC gene encodes a soluble arsenate reductase which reduces intracellular arsenate to arsenite for efflux from the cell
0147-619X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 1 4 7 - 6 1 9 X ( 0 2 ) 0 0 0 1 2 - 4
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(Ji and Silver, 1992). The arsD gene encodes an inducer independent regulatory protein which controls the upper level of operon expression (Wu and Rosen, 1993a). The arsA codes for a unique ATPase which binds to the ArsB membrane protein forming an anion transporting arsenite pump. Resistance to arsenic in the Y. enterocolitica virulence plasmid pYV requires an additional gene, arsH, the function of which is unknown. The incompatibility group H plasmids encode multiple antibiotic and heavy metal resistances and are of particular interest because of their common association with pathogenic bacteria of the family Enterobacteriaceae (Anderson, 1975; Summers and Jacoby, 1977; Taylor and Summers, 1979). Arsenic resistance is widespread amongst the IncHI2 plasmid subgroup (Taylor and Summers, 1979). R478 is the prototype IncHI2 plasmid and was first isolated in 1969 in the US from a clinical isolate of Serratia marcesens. It is a 272-kb plasmid encoding a variety of antibiotic and heavy metal resistances including resistance to arsenate, arsenite, antimony, mercury, tellurite, tetracycline, chloramphenicol, and kanamycin (Whelan, 1992). This paper describes the analysis of the method of arsenical resistance utilised by R478 and other plasmids of the IncHI2 group.
2. Materials and methods 2.1. Growth conditions Sodium arsenite (Sigma) was routinely used at a working concentration of 5 mM in tryptone soya broth (Oxoid) or tryptone soya agar (Oxoid), while sodium arsenate heptahydrate (Sigma) was used at a concentration of 20 mM in LB agar or broth. 2.2. Molecular analysis Plasmid DNA was isolated according to the method of Birnbiom and Doly (1979) followed by CsCl ethidium bromide density ultracentrifugation. DNA manipulations, restriction digests, ligations, transformations, and agarose electrophoresis were carried out according to standard procedures (Sambrook et al., 1989). DNA isolated from low melt point (LMP) agarose was purified using the Wizard PCR Preps Kit (Promega) as per manufacturer’s instructions. The QIAprep Miniprep (QIAGEN) was also routinely used for plasmid DNA isolation as per manufacturer’s instructions.
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2.3. Southern blotting Hybridisation reactions were carried out at high and low stringencies (65 and 55 °C, respectively), using a-32 P-labelled pDJR6 DNA isolated from R478; a-32 P-labelled pUM3 DNA isolated from the IncF1 plasmid R773; a-32 P-labelled pGJ103 DNA isolated from the S. aureus plasmid pI258. 2.4. DNA sequences and PCR analyses Sequencing of the plasmid clone pDJR6 was carried out by MWG-Biotech through a series of primer walking reactions, which were initiated from each end of the pUC119 cloning vector, using standard M13/pUC forward and reverse primers. Sequence analysis of the pDJR6 5.505-kb nucleotide sequence was carried out using the nucleic acid and amino acid basic local alignment sequence tool (BLAST) analysis programs (Altschul et al., 1990), CLUSTALW (Thompson et al., 1994), and DNA Strider 1.0. PCR primers were designed using the Primer3 output program (Rozen and Skaletsky, 1998). Primers were synthesised and supplied by Genosys. Primer pair sequences and target genes were as follows (50 –30 ): R478 arsC-CTTCTCACCGTCCTCTTTCG-, -ATATG GG GATCACGGTACGG- (product size 244 bases), R478 arsH-GAAGGACGAACAGCACC TTC-, -CGCATCTGATTCACAGCATT- (product size 276 bases), R478 arsB-GCATCGCGAT AACGATAGGT-, -GTCTTCTGATCGACGA ATCC- (product size 175 bases), R478 sfpA-CC TGCTTGAGTCGATGATGA-, -CCGGGATA CTTTAGTGGCAA- (product size 466 bases).
3. Results and discussion Subcloning of the IncHI2 plasmid R478 using the cloning vector pUC119 resulted in a 5.5-kb arsenic resistant subclone, pDJR6. This clone displayed lower levels of resistance to arsenic when compared to the parent plasmid. Hybridisation analysis indicated homologies between this R478 arsenic resistance determinant and the determinants of other arsenic resistant IncHI2 plasmids, as well as with the E. coli plasmid R773 but little hybridisation was noted with other plasmids examined (results not shown). The R478 subclone pDJR6 was sequenced and the full 5505bp sequence is available online (Accession Nos. AJ 288983 and AJ288984). Subclone pDJR6 was found to contain six ORFs. ORFs 5, 4, and 3,
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were orientated in the same direction. These three ORFs encoded putative proteins which were found to belong to three characteristic families of arsenic resistance proteins, ArsR, ArsB, and ArsC. The pDJR6 ars genes were in the same orientation and sequence as those of previously described three gene ars operons. Putative ribosomal binding sequences were noted just prior to the arsR and arsB genes of pDJR6. Sixteen base pairs downstream of the arsC, a 22-bp-long palindrome sequence, was found which may form an mRNA hairpin structure which it is thought may act as an operon transcription terminator region. ORFs 1, 2, and 6, were orientated in the opposite direction. The ORF6 putative protein product showed a high degree of similarity to the arsH gene of the Y. enterocolitica virulence plasmid pYV ars operon. ORF1 and ORF2 encoded putative proteins which showed a high degree of homology with two ORFs from an avirulent, foodborne Y. enterocolitica biogroup 1A plasmid (Hoffmann et al., 1998). The proteins were a sulphate permease encoded by ORF1 (sfpA) and a hypothetical sulphate transport associated protein encoded by ORF2 (sfpB) (Fig. 1). Primers were designed to detect the R478 arsB, arsC, arsH, and sfpA genes. A wide range of IncH
plasmids were screened using these primers (IncHI1: R27; IncHI2: R476-b, R828, R478, R826, R477-1, MIP235; IncHI3: MIP233; IncHII: pHH1457/2, pHH1457/3, pHH1508a) and only the arsenic resistance IncHI2 plasmids and the arsenic sensitive IncHI2 plasmid R27 resulted in amplicon production with all four primer pairs (Fig. 2). This indicated that all arsenic resistant IncHI2 plasmids tested utilised a four gene arsenic resistance mechanism as well as suggesting either a highly conserved arsenic/sulphate transport region or a possible role for the R478 encoded sulphate transport mechanism in arsenic resistance. The putative arsR gene of pDJR6 is 321-bases in length and encodes a predicted ArsR regulatory protein of 107 amino acids which shows a 70% amino acid homology with the ArsR regulatory protein of the Y. enterocolitica virulence plasmid pYV. The E. coli chromosomal ars operon has a region of inverted repeats of the sequence 50 ACTTA-30 , separated by 9 bp, upstream of the )35 region. This arsR operator sequence is conserved as an inverted repeat of 50 -NCNTA-30 separated by 12 bp from )59 to )64 in the R773 ars operon (Cai and DuBow, 1996). In the case of the R478 plasmid, the sequence 50 -NCNTA-30 occurs as an inverted repeat at position )25 to
Fig. 1. Percent amino acid homology of the predicted proteins encoded by the R478 subclone pDJR6 with Tn2501 and Tn2502 associated proteins. (A) 6.5 kbp HindIII fragment of the Y. enterocolitica avirulent plasmid p15673 (Hoffmann et al., 1998). (B) EcoRI fragment of the IncHI2 plasmid R478, pDJR6 (this study). (C) Section of the Y. enterocolitica virulence plasmid pYV (Tn2502) (Neyt et al., 1997). Arrows indicate the direction of the reading frame of each gene. IRR is a transposon associated inverse repeat region.
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Fig. 2. Agarose gels (1.0%) showing the PCR products obtained using arsH primers (gel A) and sfpA primers (gelB). Lane A: Lambda marker; Lane B: R27; Lane C: R476/b; Lane D: R828; Lane E: R478; Lane F: R826; Lane G: R477-1; Lane H: MIP235; Lane I: pHH1457/2; Lane J: pHH1457/3; Lane K: pHH1508a; Lane L: MIP233; Lane M: Lambda marker (Gel B only).
)35, and again, as a direct repeat separated by 21 bp between positions )42 and )73, upstream of the arsR gene. These regions are thought to be the sites of ArsR protein binding and regulation of the ars operon. In the case of R478, these sequences interrupt the provisional polymerase binding promoter region and so would provide an effective control of ars operon transcription. The region between Leu-7 and Ile-77 of R478 ArsR is conserved in the E. coli chromosomal, pYV, R46, and R773 ArsR proteins. Within these regions, metal binding sites conserved as ‘‘ELCVCDL’’, and DNA binding sites conserved as helix-turnhelix motifs, and including the essential residues Ser-48 and His-50, have been described (Shi et al., 1994, 1996). In the R478 ArsR protein, the amino acid sequence of this region is EMC30 VC32 DIC35 : The putative arsB gene of R478 is 1.29 kbp in length and the predicted ArsB protein shows a high degree of homology with previously described ArsB proteins. ArsB regulatory structures, described by Suzuki et al. (1998), are not evident between arsR and arsB on the R478 operon. Overexpression of the ArsB protein has been described as toxic to the cell and both the ArsD protein and the arsB mRNA hairpin loops have been described as controls to prevent this overexpression (Cai and DuBow, 1996; Owolabi and Rosen, 1990; Suzuki et al., 1998; Wu et al., 1992). The fact that these controls are absent from the R478 system indicates that the toxicity of the
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R478 arsB gene is controlled by another mechanism. Southern blotting analysis using the R773 arsA gene as a probe against R478 did not reveal the presence of an arsA homologue on any arsenic resistant IncHI2 plasmid examined (results not shown). For these reasons, it is considered likely that the ArsB protein of the R478 ars operon also acts to transport arsenite using energy from the electrochemical gradient. The pDJR6 arsC gene is 423-bases in length and is located just 11 bases downstream from the arsB stop codon. Despite the short length of this intergenic region, the 11-base sequence contains a putative ribosome binding site which reads GAATAN5 ATG. The R478 arsC gene encodes a predicted ArsC arsenate reductase of 141 amino acids in length, the same number of amino acids and an almost identical molecular weight as other ArsC proteins described with the notable exception of the pI258 ArsC. Plasmid R478 was originally found in the enteric bacteria S. marcesens and is housed routinely in an E. coli host strain, coupled with the high degree of sequence homology between R478 ArsC and the R773 ArsC. It is likely that the R478 ArsC protein accepts its reducing potential from NADPH via glutathione and glutaredoxin in a similar fashion to that used by the R773 system. A fourth gene (arsH) was located at 86 bases upstream of the pDJR6 arsR. This arsH homologue was orientated clockwise, in the opposite direction to arsRBC. The arsH gene was first described by Neyt et al. (1997), as an essential component in a new four gene arsenical resistance transposon associated operon, arsHRBC. Two other arsH homologues have since been described, which show a significant degree of homology to the R478 and pYV arsH genes, the chromosomal arsH genes of Thiobacillus ferroxidans and of Cyanobacterium synechocystis (Butcher et al., 2000; Kaneko et al., 1996). The ArsH proteins of the Y. enterocolitica plasmid pYV and the predicted protein of the IncHI2 plasmid R478 show an 82% amino acid sequence identity in their first 195 amino acids. However, the carboxy terminal of the R478 ArsH protein was missing as evidenced by the lack of a stop codon on the corrected amino acid sequence. Based on the sequence of the pYV ArsH, it was estimated that approximately 16% of the C-terminal region of the R478 ArsH was missing from the clone pDJR6 meaning that the R478 ArsH protein may be nonfunctional in pDJR6. However, Neyt et al. (1997) stated that the ArsH protein was
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fundamental to arsenic resistance in the four gene operon. The complete removal of the R478 arsH gene creating the clone, pDJR3, resulted in a total loss of resistance to arsenic. Perhaps this was the reason that pDJR6 showed slightly lower levels of resistance to sodium arsenate and sodium arsenite when compared to the parent plasmid indicating that at least some portion of the R478 arsH gene is required for resistance. As is the case for the pYV ArsH, no signal sequence, no ATP-binding motif, and no other known motif or domain could be found on amino acid sequence analysis of the putative R478 ArsH protein. The pDJR6 arsH gene may be under the regulatory control of the ArsR protein as the ArsR protein binding inverted repeat sequence, 50 NCNTA-30 , occurs frequently in the 86 bases between the arsR and the arsH genes on the coding strand for both genes. Putative RNA polymerase binding sequences have been noted in the sequence upstream of the arsR start codon. It appears that arsH may be under the control of the ArsR protein but may be transcribed separately from the other ars genes. The arsH gene represents something of an anomaly. Whether the ArsH protein product is essential for arsenic resistance in the plasmid R478 remains to be determined. The four gene ars operons do not provide obvious evidence of mechanisms controlling the upper limit of operon expression, yet provide high level arsenic resistance and it is possible that ArsH may somehow protect the host cell from ars-polypeptide-induced toxicity in a similar manner to the ArsD protein in the five gene system. No significant amino acid homology was found between the ArsH protein of R478 and the ArsD proteins of R773, R46, or pKW301 upon CLUSTALW analysis. However, no homology can be found between the ArsR and ArsD regulatory proteins of five gene ars operons, yet the ArsR and ArsD proteins still bind to the same region of the ars operon promoter element in the five gene system (Chen and Rosen, 1997). So arsH or ArsH may provide a secondary regulatory control function in the four gene arsenic resistance operon either for the entire operon acting as a binding site for another regulatory protein or for the control of a particular gene such as arsB. The R478 sfpA and sfpB genes were separated by 18 bases and preceded by a putative polymerase binding region. As sfpB did not appear to have an independent polymerase binding site it may be co-transcribed with sfpA in a mechanism similar
to that employed by the ars operon. The nucleic acid sequence of R478 sfpB is 39 bases shorter than ORF5 of the p15673 plasmid, resulting in a truncated R478 protein which is 13 amino acids shorter than its p15673 counterpart (Fig. 1). After the R478 sfpB stop codon, the sequences of both plasmids continue to be closely homologous for just 7 bases and then abruptly become completely unrelated. The avirulent plasmid p15673 sfp and ORF genes are followed by an inverted repeat region (IRR) which is believed to be the terminal repeat sequence of transposon Tn2501. Upstream of sfp and ORF5 on the p15673 plasmid, Hoffmann et al. (1998) found a sequence which corresponded to the C-terminal region of the resolvase gene tnpR of the transposon Tn2501, supporting the theory that the ORF5 and sfp genes of p15673 are, or have been, transposon associated. No evidence of a tnpR gene or transposon associated IRR was found in the region upstream of sfpA in the pDJR6 sequence. This region shows close sequence homology with the corresponding p15673 region for 95 bases before becoming completely unrelated (Fig. 1). sfpB and arsC on pDJR6 are separated by 59 bases which bear little or no resemblance to the downstream region of ORF5 of the Y. enterocolitica avirulent plasmid p15673 or to the downstream region of the arsC gene of the Y. enterocolitica virulence plasmid pYV. No evidence of the IRR of Tn2501, or of the tnpR gene of Tn2502, was found in the intergenic region between arsC and ORF2 of pDJR6. It appears therefore that the R478 subclone, pDJR6, shows close homology to transposon associated regions of two different Y. enterocolitica plasmids. However, these regions of R478 do not appear to be transposon associated. Protein gels derived from E. coli J53-2 cells containing R478, and which had been exposed during culture to sodium arsenite, revealed the expression of a protein, approximately the same size as the predicted pDJR6 sulphate permease protein with a molecular weight of 52.8 kDa (results not shown). The ArsR binding sequences— NCNTA—have also been found upstream of the pDJR6 sulphate permease start codon. It is tempting to speculate that the sulphate permease and ORF2 proteins of pDJR6 are also under ArsR regulation and that these two proteins are part of a larger arsenic resistance mechanism which was divided by insertion sequences thereby becoming the sulphate permease transposon, T2501, and the arsenic resistance transposon, Tn2502. Sulphate permease mediated sulphate
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transport into the cell could have resulted in an increased internal membrane negative charge. This, in turn, would help in the efflux of negatively charged arsenite ions through the ArsB membrane channel in the absence of an ArsA ATPase in a type of symporter mechanism. Both Tn2501 and Tn2502 originated in different Yersinia species (Michiels et al., 1987; Neyt et al., 1997). The presence of the arsenic resistance Tn2502 transposon on the pYV virulence plasmid of Y. enterocolitica was considered unusual as this plasmid is not normally associated with resistance determinants. The ars operon was found on all Y. enterocolitica virulence plasmids examined (Neyt et al., 1997). The entire pYV plasmid has been sequenced (NCBI, 1999; NCBI, Accession No. AF102990). It does not contain a sulphate permease gene or any other transposon associated regions. IncH plasmids, including the IncHI2 plasmid R478, are associated with a wide variety of resistance determinants. Many of these are transposon associated. Analysis of subclone pDJR6 indicates that the plasmid, R478, carries Tn2501 associated sulphate permease genes and Tn2502 associated arsenic resistance genes, but the transposon IRR sequences and transposase and resolvase genes are not evident. Transposability experiments, carried out during the course of this research, failed to obtain transposition of any of the resistance determinants of R478 (results not shown). The R478 plasmid may contain transposon hotspots similar to those described on the IncP-1a plasmids of Pseudomonas species (Thornsted et al., 1998). Subsequent removal of the transposon associated mechanism would result in ‘fixing’ of a particular resistance determinant on the plasmid. This may help explain the large size and high number of resistance determinants and ‘housekeeping genes’ associated with R478 and other IncH plasmids. Alternatively, the R478 plasmid may be the source of a variety of transposons through the insertion of composite transposons or integrons with subsequent excision carrying R478 resistance determinants which then insert into other recipient plasmids.
Acknowledgments Several of the plasmids and clones used in the course of this research were kindly provided by Diane Taylor, Cecile Neyt, and Simon Silver.
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Arsenical resistance in the IncHI2 plasmids David Ryana,* and Emer Colleranb a
Department of Applied Biology and Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland b Environmental Research Unit, Department of Microbiology, National University of Ireland, Galway, Ireland Received 13 November 2001, revised 18 April 2002
Abstract The IncHI2 plasmid R478, like other arsenic resistance IncH plasmids, provides increased levels of resistance to sodium arsenate (up to 100 mM) and sodium arsenite (up to 10 mM) to the host cell. An arsenic resistance fragment of R478 was cloned and sequenced revealing four arsenic resistance associated gene homologues, arsR, arsB, arsC, and arsH. Two other open reading frames in the cloned fragment were found to be homologues of sulphate transport associated genes. Both the four gene arsenic resistance operons and the two gene sulphate transport operons have been previously shown to be transposon associated. However, no evidence of transposability was found associated with these operons in R478. Both the R478 associated arsenic and sulphate transport operons were shown to be common to all arsenic resistance IncH plasmids examined by Southern hybridisation and PCR analysis. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Incompatibility group H plasmids; R478; Arsenic resistance; Sulphate transport
1. Introduction Resistance to arsenical compounds in bacteria can be plasmid or chromosomally mediated. Resistance operons have been documented in a wide variety of chromosomes including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Thiobacillus ferroxidans (Butcher et al., 2000; Cai et al., 1998; Rosen, 1999; Sofia et al., 1994). The physiological role of the chromosomal arsenic operon (ars operon) appears to be the provision of low level arsenical resistance (Carlin et al., 1995; Silver et al., 1981). Plasmids which mediate for arsenic resistance include the Staphylococcus
*
Corresponding author. Fax: +353-503-70500. E-mail addresses: [email protected] (D. Ryan), [email protected] (E. Colleran).
aureus plasmid pI258, the E. coli plasmids R773 and R46, the Staphylococcus xylosus plasmid pSX267, the P. aeruginosa plasmid pUM310, the Yersinia enterocolitica virulence plasmid pYV, and the Acidiphilium multivorium AIU301 plasmid, pKW301 (Brown and Willetts, 1981; Bruhn et al., 1996; Cervantes and Chavez, 1992; Gotz et al., 1983; Hedges and Baumberg, 1973; Neyt et al., 1997; Novick and Bouanchaud, 1971; Suzuki et al., 1998). Three prototype ars operons have been well documented and these are the three-, four-, and five-gene arsenic resistance determinants, although novel resistance mechanisms have also been described. The arsR gene encodes a repressor protein that controls operon expression. The arsB gene protein product is a transmembrane efflux channel. The arsC gene encodes a soluble arsenate reductase which reduces intracellular arsenate to arsenite for efflux from the cell
0147-619X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 1 4 7 - 6 1 9 X ( 0 2 ) 0 0 0 1 2 - 4
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(Ji and Silver, 1992). The arsD gene encodes an inducer independent regulatory protein which controls the upper level of operon expression (Wu and Rosen, 1993a). The arsA codes for a unique ATPase which binds to the ArsB membrane protein forming an anion transporting arsenite pump. Resistance to arsenic in the Y. enterocolitica virulence plasmid pYV requires an additional gene, arsH, the function of which is unknown. The incompatibility group H plasmids encode multiple antibiotic and heavy metal resistances and are of particular interest because of their common association with pathogenic bacteria of the family Enterobacteriaceae (Anderson, 1975; Summers and Jacoby, 1977; Taylor and Summers, 1979). Arsenic resistance is widespread amongst the IncHI2 plasmid subgroup (Taylor and Summers, 1979). R478 is the prototype IncHI2 plasmid and was first isolated in 1969 in the US from a clinical isolate of Serratia marcesens. It is a 272-kb plasmid encoding a variety of antibiotic and heavy metal resistances including resistance to arsenate, arsenite, antimony, mercury, tellurite, tetracycline, chloramphenicol, and kanamycin (Whelan, 1992). This paper describes the analysis of the method of arsenical resistance utilised by R478 and other plasmids of the IncHI2 group.
2. Materials and methods 2.1. Growth conditions Sodium arsenite (Sigma) was routinely used at a working concentration of 5 mM in tryptone soya broth (Oxoid) or tryptone soya agar (Oxoid), while sodium arsenate heptahydrate (Sigma) was used at a concentration of 20 mM in LB agar or broth. 2.2. Molecular analysis Plasmid DNA was isolated according to the method of Birnbiom and Doly (1979) followed by CsCl ethidium bromide density ultracentrifugation. DNA manipulations, restriction digests, ligations, transformations, and agarose electrophoresis were carried out according to standard procedures (Sambrook et al., 1989). DNA isolated from low melt point (LMP) agarose was purified using the Wizard PCR Preps Kit (Promega) as per manufacturer’s instructions. The QIAprep Miniprep (QIAGEN) was also routinely used for plasmid DNA isolation as per manufacturer’s instructions.
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2.3. Southern blotting Hybridisation reactions were carried out at high and low stringencies (65 and 55 °C, respectively), using a-32 P-labelled pDJR6 DNA isolated from R478; a-32 P-labelled pUM3 DNA isolated from the IncF1 plasmid R773; a-32 P-labelled pGJ103 DNA isolated from the S. aureus plasmid pI258. 2.4. DNA sequences and PCR analyses Sequencing of the plasmid clone pDJR6 was carried out by MWG-Biotech through a series of primer walking reactions, which were initiated from each end of the pUC119 cloning vector, using standard M13/pUC forward and reverse primers. Sequence analysis of the pDJR6 5.505-kb nucleotide sequence was carried out using the nucleic acid and amino acid basic local alignment sequence tool (BLAST) analysis programs (Altschul et al., 1990), CLUSTALW (Thompson et al., 1994), and DNA Strider 1.0. PCR primers were designed using the Primer3 output program (Rozen and Skaletsky, 1998). Primers were synthesised and supplied by Genosys. Primer pair sequences and target genes were as follows (50 –30 ): R478 arsC-CTTCTCACCGTCCTCTTTCG-, -ATATG GG GATCACGGTACGG- (product size 244 bases), R478 arsH-GAAGGACGAACAGCACC TTC-, -CGCATCTGATTCACAGCATT- (product size 276 bases), R478 arsB-GCATCGCGAT AACGATAGGT-, -GTCTTCTGATCGACGA ATCC- (product size 175 bases), R478 sfpA-CC TGCTTGAGTCGATGATGA-, -CCGGGATA CTTTAGTGGCAA- (product size 466 bases).
3. Results and discussion Subcloning of the IncHI2 plasmid R478 using the cloning vector pUC119 resulted in a 5.5-kb arsenic resistant subclone, pDJR6. This clone displayed lower levels of resistance to arsenic when compared to the parent plasmid. Hybridisation analysis indicated homologies between this R478 arsenic resistance determinant and the determinants of other arsenic resistant IncHI2 plasmids, as well as with the E. coli plasmid R773 but little hybridisation was noted with other plasmids examined (results not shown). The R478 subclone pDJR6 was sequenced and the full 5505bp sequence is available online (Accession Nos. AJ 288983 and AJ288984). Subclone pDJR6 was found to contain six ORFs. ORFs 5, 4, and 3,
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were orientated in the same direction. These three ORFs encoded putative proteins which were found to belong to three characteristic families of arsenic resistance proteins, ArsR, ArsB, and ArsC. The pDJR6 ars genes were in the same orientation and sequence as those of previously described three gene ars operons. Putative ribosomal binding sequences were noted just prior to the arsR and arsB genes of pDJR6. Sixteen base pairs downstream of the arsC, a 22-bp-long palindrome sequence, was found which may form an mRNA hairpin structure which it is thought may act as an operon transcription terminator region. ORFs 1, 2, and 6, were orientated in the opposite direction. The ORF6 putative protein product showed a high degree of similarity to the arsH gene of the Y. enterocolitica virulence plasmid pYV ars operon. ORF1 and ORF2 encoded putative proteins which showed a high degree of homology with two ORFs from an avirulent, foodborne Y. enterocolitica biogroup 1A plasmid (Hoffmann et al., 1998). The proteins were a sulphate permease encoded by ORF1 (sfpA) and a hypothetical sulphate transport associated protein encoded by ORF2 (sfpB) (Fig. 1). Primers were designed to detect the R478 arsB, arsC, arsH, and sfpA genes. A wide range of IncH
plasmids were screened using these primers (IncHI1: R27; IncHI2: R476-b, R828, R478, R826, R477-1, MIP235; IncHI3: MIP233; IncHII: pHH1457/2, pHH1457/3, pHH1508a) and only the arsenic resistance IncHI2 plasmids and the arsenic sensitive IncHI2 plasmid R27 resulted in amplicon production with all four primer pairs (Fig. 2). This indicated that all arsenic resistant IncHI2 plasmids tested utilised a four gene arsenic resistance mechanism as well as suggesting either a highly conserved arsenic/sulphate transport region or a possible role for the R478 encoded sulphate transport mechanism in arsenic resistance. The putative arsR gene of pDJR6 is 321-bases in length and encodes a predicted ArsR regulatory protein of 107 amino acids which shows a 70% amino acid homology with the ArsR regulatory protein of the Y. enterocolitica virulence plasmid pYV. The E. coli chromosomal ars operon has a region of inverted repeats of the sequence 50 ACTTA-30 , separated by 9 bp, upstream of the )35 region. This arsR operator sequence is conserved as an inverted repeat of 50 -NCNTA-30 separated by 12 bp from )59 to )64 in the R773 ars operon (Cai and DuBow, 1996). In the case of the R478 plasmid, the sequence 50 -NCNTA-30 occurs as an inverted repeat at position )25 to
Fig. 1. Percent amino acid homology of the predicted proteins encoded by the R478 subclone pDJR6 with Tn2501 and Tn2502 associated proteins. (A) 6.5 kbp HindIII fragment of the Y. enterocolitica avirulent plasmid p15673 (Hoffmann et al., 1998). (B) EcoRI fragment of the IncHI2 plasmid R478, pDJR6 (this study). (C) Section of the Y. enterocolitica virulence plasmid pYV (Tn2502) (Neyt et al., 1997). Arrows indicate the direction of the reading frame of each gene. IRR is a transposon associated inverse repeat region.
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Fig. 2. Agarose gels (1.0%) showing the PCR products obtained using arsH primers (gel A) and sfpA primers (gelB). Lane A: Lambda marker; Lane B: R27; Lane C: R476/b; Lane D: R828; Lane E: R478; Lane F: R826; Lane G: R477-1; Lane H: MIP235; Lane I: pHH1457/2; Lane J: pHH1457/3; Lane K: pHH1508a; Lane L: MIP233; Lane M: Lambda marker (Gel B only).
)35, and again, as a direct repeat separated by 21 bp between positions )42 and )73, upstream of the arsR gene. These regions are thought to be the sites of ArsR protein binding and regulation of the ars operon. In the case of R478, these sequences interrupt the provisional polymerase binding promoter region and so would provide an effective control of ars operon transcription. The region between Leu-7 and Ile-77 of R478 ArsR is conserved in the E. coli chromosomal, pYV, R46, and R773 ArsR proteins. Within these regions, metal binding sites conserved as ‘‘ELCVCDL’’, and DNA binding sites conserved as helix-turnhelix motifs, and including the essential residues Ser-48 and His-50, have been described (Shi et al., 1994, 1996). In the R478 ArsR protein, the amino acid sequence of this region is EMC30 VC32 DIC35 : The putative arsB gene of R478 is 1.29 kbp in length and the predicted ArsB protein shows a high degree of homology with previously described ArsB proteins. ArsB regulatory structures, described by Suzuki et al. (1998), are not evident between arsR and arsB on the R478 operon. Overexpression of the ArsB protein has been described as toxic to the cell and both the ArsD protein and the arsB mRNA hairpin loops have been described as controls to prevent this overexpression (Cai and DuBow, 1996; Owolabi and Rosen, 1990; Suzuki et al., 1998; Wu et al., 1992). The fact that these controls are absent from the R478 system indicates that the toxicity of the
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R478 arsB gene is controlled by another mechanism. Southern blotting analysis using the R773 arsA gene as a probe against R478 did not reveal the presence of an arsA homologue on any arsenic resistant IncHI2 plasmid examined (results not shown). For these reasons, it is considered likely that the ArsB protein of the R478 ars operon also acts to transport arsenite using energy from the electrochemical gradient. The pDJR6 arsC gene is 423-bases in length and is located just 11 bases downstream from the arsB stop codon. Despite the short length of this intergenic region, the 11-base sequence contains a putative ribosome binding site which reads GAATAN5 ATG. The R478 arsC gene encodes a predicted ArsC arsenate reductase of 141 amino acids in length, the same number of amino acids and an almost identical molecular weight as other ArsC proteins described with the notable exception of the pI258 ArsC. Plasmid R478 was originally found in the enteric bacteria S. marcesens and is housed routinely in an E. coli host strain, coupled with the high degree of sequence homology between R478 ArsC and the R773 ArsC. It is likely that the R478 ArsC protein accepts its reducing potential from NADPH via glutathione and glutaredoxin in a similar fashion to that used by the R773 system. A fourth gene (arsH) was located at 86 bases upstream of the pDJR6 arsR. This arsH homologue was orientated clockwise, in the opposite direction to arsRBC. The arsH gene was first described by Neyt et al. (1997), as an essential component in a new four gene arsenical resistance transposon associated operon, arsHRBC. Two other arsH homologues have since been described, which show a significant degree of homology to the R478 and pYV arsH genes, the chromosomal arsH genes of Thiobacillus ferroxidans and of Cyanobacterium synechocystis (Butcher et al., 2000; Kaneko et al., 1996). The ArsH proteins of the Y. enterocolitica plasmid pYV and the predicted protein of the IncHI2 plasmid R478 show an 82% amino acid sequence identity in their first 195 amino acids. However, the carboxy terminal of the R478 ArsH protein was missing as evidenced by the lack of a stop codon on the corrected amino acid sequence. Based on the sequence of the pYV ArsH, it was estimated that approximately 16% of the C-terminal region of the R478 ArsH was missing from the clone pDJR6 meaning that the R478 ArsH protein may be nonfunctional in pDJR6. However, Neyt et al. (1997) stated that the ArsH protein was
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fundamental to arsenic resistance in the four gene operon. The complete removal of the R478 arsH gene creating the clone, pDJR3, resulted in a total loss of resistance to arsenic. Perhaps this was the reason that pDJR6 showed slightly lower levels of resistance to sodium arsenate and sodium arsenite when compared to the parent plasmid indicating that at least some portion of the R478 arsH gene is required for resistance. As is the case for the pYV ArsH, no signal sequence, no ATP-binding motif, and no other known motif or domain could be found on amino acid sequence analysis of the putative R478 ArsH protein. The pDJR6 arsH gene may be under the regulatory control of the ArsR protein as the ArsR protein binding inverted repeat sequence, 50 NCNTA-30 , occurs frequently in the 86 bases between the arsR and the arsH genes on the coding strand for both genes. Putative RNA polymerase binding sequences have been noted in the sequence upstream of the arsR start codon. It appears that arsH may be under the control of the ArsR protein but may be transcribed separately from the other ars genes. The arsH gene represents something of an anomaly. Whether the ArsH protein product is essential for arsenic resistance in the plasmid R478 remains to be determined. The four gene ars operons do not provide obvious evidence of mechanisms controlling the upper limit of operon expression, yet provide high level arsenic resistance and it is possible that ArsH may somehow protect the host cell from ars-polypeptide-induced toxicity in a similar manner to the ArsD protein in the five gene system. No significant amino acid homology was found between the ArsH protein of R478 and the ArsD proteins of R773, R46, or pKW301 upon CLUSTALW analysis. However, no homology can be found between the ArsR and ArsD regulatory proteins of five gene ars operons, yet the ArsR and ArsD proteins still bind to the same region of the ars operon promoter element in the five gene system (Chen and Rosen, 1997). So arsH or ArsH may provide a secondary regulatory control function in the four gene arsenic resistance operon either for the entire operon acting as a binding site for another regulatory protein or for the control of a particular gene such as arsB. The R478 sfpA and sfpB genes were separated by 18 bases and preceded by a putative polymerase binding region. As sfpB did not appear to have an independent polymerase binding site it may be co-transcribed with sfpA in a mechanism similar
to that employed by the ars operon. The nucleic acid sequence of R478 sfpB is 39 bases shorter than ORF5 of the p15673 plasmid, resulting in a truncated R478 protein which is 13 amino acids shorter than its p15673 counterpart (Fig. 1). After the R478 sfpB stop codon, the sequences of both plasmids continue to be closely homologous for just 7 bases and then abruptly become completely unrelated. The avirulent plasmid p15673 sfp and ORF genes are followed by an inverted repeat region (IRR) which is believed to be the terminal repeat sequence of transposon Tn2501. Upstream of sfp and ORF5 on the p15673 plasmid, Hoffmann et al. (1998) found a sequence which corresponded to the C-terminal region of the resolvase gene tnpR of the transposon Tn2501, supporting the theory that the ORF5 and sfp genes of p15673 are, or have been, transposon associated. No evidence of a tnpR gene or transposon associated IRR was found in the region upstream of sfpA in the pDJR6 sequence. This region shows close sequence homology with the corresponding p15673 region for 95 bases before becoming completely unrelated (Fig. 1). sfpB and arsC on pDJR6 are separated by 59 bases which bear little or no resemblance to the downstream region of ORF5 of the Y. enterocolitica avirulent plasmid p15673 or to the downstream region of the arsC gene of the Y. enterocolitica virulence plasmid pYV. No evidence of the IRR of Tn2501, or of the tnpR gene of Tn2502, was found in the intergenic region between arsC and ORF2 of pDJR6. It appears therefore that the R478 subclone, pDJR6, shows close homology to transposon associated regions of two different Y. enterocolitica plasmids. However, these regions of R478 do not appear to be transposon associated. Protein gels derived from E. coli J53-2 cells containing R478, and which had been exposed during culture to sodium arsenite, revealed the expression of a protein, approximately the same size as the predicted pDJR6 sulphate permease protein with a molecular weight of 52.8 kDa (results not shown). The ArsR binding sequences— NCNTA—have also been found upstream of the pDJR6 sulphate permease start codon. It is tempting to speculate that the sulphate permease and ORF2 proteins of pDJR6 are also under ArsR regulation and that these two proteins are part of a larger arsenic resistance mechanism which was divided by insertion sequences thereby becoming the sulphate permease transposon, T2501, and the arsenic resistance transposon, Tn2502. Sulphate permease mediated sulphate
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transport into the cell could have resulted in an increased internal membrane negative charge. This, in turn, would help in the efflux of negatively charged arsenite ions through the ArsB membrane channel in the absence of an ArsA ATPase in a type of symporter mechanism. Both Tn2501 and Tn2502 originated in different Yersinia species (Michiels et al., 1987; Neyt et al., 1997). The presence of the arsenic resistance Tn2502 transposon on the pYV virulence plasmid of Y. enterocolitica was considered unusual as this plasmid is not normally associated with resistance determinants. The ars operon was found on all Y. enterocolitica virulence plasmids examined (Neyt et al., 1997). The entire pYV plasmid has been sequenced (NCBI, 1999; NCBI, Accession No. AF102990). It does not contain a sulphate permease gene or any other transposon associated regions. IncH plasmids, including the IncHI2 plasmid R478, are associated with a wide variety of resistance determinants. Many of these are transposon associated. Analysis of subclone pDJR6 indicates that the plasmid, R478, carries Tn2501 associated sulphate permease genes and Tn2502 associated arsenic resistance genes, but the transposon IRR sequences and transposase and resolvase genes are not evident. Transposability experiments, carried out during the course of this research, failed to obtain transposition of any of the resistance determinants of R478 (results not shown). The R478 plasmid may contain transposon hotspots similar to those described on the IncP-1a plasmids of Pseudomonas species (Thornsted et al., 1998). Subsequent removal of the transposon associated mechanism would result in ‘fixing’ of a particular resistance determinant on the plasmid. This may help explain the large size and high number of resistance determinants and ‘housekeeping genes’ associated with R478 and other IncH plasmids. Alternatively, the R478 plasmid may be the source of a variety of transposons through the insertion of composite transposons or integrons with subsequent excision carrying R478 resistance determinants which then insert into other recipient plasmids.
Acknowledgments Several of the plasmids and clones used in the course of this research were kindly provided by Diane Taylor, Cecile Neyt, and Simon Silver.
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