Department of Microbiology and Immunology, University of Illinois, M/C 790, 835 S. Wolcott Ave, Room E-703, Chicago, IL 60612-7344, USA1
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton T6G 2H7 Alberta, Canada2
Author for correspondence: Amit Gupta. Tel: +1 630 305 1735. Fax: +1 630 305 2982. e-mail: agupta{at}uic.edu
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ABSTRACT |
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Keywords: IncH plasmids, E. coli chromosome, plasmid incompatibility group
The GenBank accession numbers for the sequences reported in this paper are AY009372AY009396.
a Present address: ONDEO-Nalco Company, One Nalco Center, Naperville, IL 605 3, USA.
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INTRODUCTION |
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Silver compounds and products are increasingly common as microbicidal agents in hygiene, agriculture and industry, in addition to clinical uses (reviewed by Gupta & Silver, 1998 ; Silver et al., 1999a
, b
). In hospitals, silver sulfadiazine is used in burn dressings (George et al., 1997
) to prevent nosocomial infections. The most familiar human exposure to silver is with dental amalgams that contain 35% Ag(0) and 50% Hg(0) (Dunne et al., 1997
). The slow release of Hg(0) from amalgams is known to select for mercury-resistant bacteria in the gut (Lorscheider et al., 1995
). Ag(0) is also released (Lygre et al., 1999
), but whether it has any antimicrobial activity that selects for resistance (perhaps in the mouth) has never been tested.
Ag+ ions are highly toxic to all micro-organisms, perhaps due to poisoning of the respiratory electron transport chains and components of DNA replication (Modak & Fox, 1973 ; Russell & Hugo, 1994
). Although bacterial Ag+ resistance has been periodically reported, the basis was not studied before our recent efforts (Gupta & Silver, 1998
; Gupta et al., 1999
; Silver et al., 1999a
, b
). Human exposure to silver compounds has no serious adverse effect (Russell & Hugo, 1994
). Prolonged silver use occasionally results in argyria, a condition with an irreversible grey to blue-black colouring of the skin and mucous membranes due to Ag(0) or Ag2S deposits. Argyria, however, is mostly of cosmetic concern (Russell & Hugo, 1994
).
Bacterial resistances to different toxic metal ions are encoded by genes located mostly on plasmids, but sometimes on bacterial chromosomes (reviewed by Silver, 1998 ; Silver & Phung, 1996
; Silver et al., 2000
). These resistances are selected frequently when metal salts are used as antiseptics. Ag+-resistant bacteria have been reported periodically from sources such as hospitals and burn wounds where silver toxicity might be expected to select for resistance (Annear et al., 1976
; Bridges et al., 1979
; Hendry & Stewart, 1979
; McHugh et al., 1975
; Pruitt et al., 1998
). The physiological, biochemical and molecular basis of bacterial Ag+ resistance were not described prior to our studies (Gupta et al., 1999
). The first report on the genetic and molecular basis for Ag+ resistance concerned a Salmonella typhimurium isolate, from the Massachusetts General Hospital, that killed several patients and required the closing of the burn ward in 1975 (Gupta et al., 1999
; McHugh et al., 1975
). Plasmid pMG101 from this strain confers resistances to Ag+, Hg2+ and tellurite, as well as to several antibiotics, for example ampicillin, chloramphenicol, tetracycline and streptomycin (Gupta et al., 1999
; McHugh et al., 1975
).
The Ag+ resistance determinant from pMG101 contains nine ORFs (Fig. 1a) in 12·5 kb of sequence (GenBank accession no. AF067954; Gupta et al., 1999
). The ORFs are arranged in three transcriptional units (Fig. 1a
; Gupta et al., 1999
). Functions for the seven named genes were assigned on the basis of homologies to known proteins for other metal resistances; the two unnamed ORFs lack such homologues. The first gene identified, silE, encodes a 123 aa periplasmic metal-binding protein (Gupta et al., 1999
). Upstream from silE and in the same orientation is a presumed two-component gene pair, silRS, encoding a transcriptional regulatory responder protein and a membrane sensor kinase, homologous to other members of the two-component family (Hoch & Silhavy, 1995
).
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The product of the last gene of the Ag+ resistance determinant, SilP, is predicted to be a P-type ATPase, a member of the family of heavy-metal resistance ATPases (Rensing et al., 1999 ; Silver & Phung, 1996
). The Ag+ resistance determinant is unique to date in encoding both a metal-binding protein and two biochemically different efflux mechanisms.
With the characterization of the first Ag+ resistance determinant, it is important to understand how widely such systems are to be found in clinical isolates (exposed or not exposed to silver) and in those from silver-stressed non-clinical environments. To begin, this study focused on the occurrence of sil genes on different groups of plasmids, rather than on the molecular mechanism of Ag+ resistance. Different laboratory stock plasmids of the IncH and IncP incompatibility groups known to carry multiple antibiotic resistance markers were tested for the presence of sil genes. These plasmids were originally isolated from varying geographic locations (see supplementary data at http://mic.sgmjournals.org or http://www.uic.edu/depts/mcmi/individual/gupta/index.htm). The identification and diversity of non-plasmid, chromosomally located sil homologous determinants in genome sequences from different bacteria is also discussed. The data presented are starting points for molecular epidemiological studies with clinical and non-clinical bacterial isolates.
A wide distribution of sil homologous determinants, localized on plasmids or on the bacterial chromosomes might pose a threat toward effective use of silver compounds as antiseptics, analogous to the development of antibiotic-resistant bacteria when antibiotic usage increases (Liu, 1999 ; Salyers & Amabile-Cuevas, 1997
).
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METHODS |
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Bacterial strains.
In this study, a series of E. coli strains carrying IncH plasmids from the University of Alberta collection were used (see supplementary data): J53(R476b), J53-2(R826), J53-2(R826-1), J53-2(R828), RG486(MIP233), RG486(pWR23), RG486(MIP235), RG192(TP116), RG1763(R478), RG192(pAS-251-2), RG192(pJT1), J53-1(R1022), J53(R27); J53-1(pHH1532b-1), JE2571(pHH1457), J62-1(pHH1457-1), J53(MG223), J53(MG224) and J53(MG225). E. coli J53 without a plasmid and J53(pMG101) (Gupta et al., 1999 ; McHugh et al., 1975
) were used as Ag+-sensitive and -resistant controls, respectively.
Identification and characterization of sil gene homologues
Dot-blot hybridization.
An aliquot (50 µl) of each overnight culture was placed on a nylon hybridization membrane using a dot-blot apparatus (Schleicher & Schuell). The bacterial cells were lysed. The liberated DNA was denatured, neutralized and fixed to the membrane (Ausubel et al. 2001 ). A 32P-labelled silA probe was hybridized to the filter-immobilized DNA. The DNA for the silA probe was generated by PCR amplification using silA gene-specific oligonucleotide primers and radiolabelled with [32P]dCTP by using the MegaPrime DNA labelling kit (Amersham Life Science). Hybridization signals were visualized by exposure to X-OMAT AR film (Eastman Kodak).
PCR.
The homologues of pMG101 sil genes were amplified from the boil-lysis supernatants (Ausubel et al., 2001 ) of overnight culture aliquots, using sil gene-specific primers and PlatiTaq DNA polymerase (Life Technologies). The amplification products were separated on a 0·7% agarose gel and visualized under UV after staining with ethidium bromide.
Cloning and DNA sequence analysis.
PCR products of the sil genes were cloned into the pGEM-Teasy vector (Promega) and transformed into E. coli DH5 by electroporation of competent cells (Ausubel et al., 2001
; Shigekawa & Dower, 1988
). Plasmid DNA was isolated (Ausubel et al., 2001
) and cloned DNA was sequenced by using M13 universal forward and reverse sequencing primers by the dideoxy chain termination method using an ABI automated sequencer at the University of Illinois Sequencing Facility. DNA sequences were compared and analysed using CLUSTAL X version 1·64b and DNA Sequencher version 3.1.1 software.
Transcript analysis.
Total RNA was isolated from E. coli strain J53(R476b) and J53(pMG101) cells exposed to 0 or 25 µM Ag+ for 2 h at 37 °C during growth in LB broth. RNA was isolated using the RNeasy total RNA preparation kit (Qiagen) and treated with RNase-free DNase (Life Technologies). For reverse transcriptase (RT)-PCR, 1 µg RNA was used for cDNA synthesis at 42 °C using Superscript II RT according to the manufacturers protocol (Life Technologies). Subsequent PCR was performed using PlatiTaq DNA polymerase (Life Technologies). For quantitation of fluorescence intensities of the RT-PCR products, the ethidium-bromide-stained agarose gel was recorded using a Sony CCD (charge-coupled device) camera attached to the Nucleotech gel documentation system. The fluorescence intensities were measured using GelExpert 97 version 2.0 software.
Construction of agrA deletion mutant and growth measurements.
Chromosomal agrA was deleted by insertion of an antibiotic (kanamycin) resistance marker. The kanamycin cassette DNA contained flanking sequences homologous to agrA and was incorporated into the chromosome using homologous recombination as described by Datsenko & Wanner (2000) . DH5
agrA(pAG59) carries a pUC57T (MBI Fermentas) derivative containing a PCR DNA fragment for the agrABC genes. Growth was measured for E. coli strains K-12 DH5
, DH5
agrA and DH5
agrA(pAG59) by inoculation of LB broth (-NaCl) containing different concentrations of AgNO3 with an exponential-phase culture at 0·2 Klett turbidity units. The cultures were incubated with shaking for 16 h at 37 °C prior to measurements with a Klett turbidity meter.
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RESULTS |
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Sequences of plasmid-located sil genes
The silP, silS and silE genes amplified by PCR from E. coli strains J53(R476b), RG486(MIP233), RG486(pWR23), RG486(MIP235) and RG1763(R478) were cloned and the DNA for each new sil gene was sequenced. The sequences were compared with pMG101 sequences (Table 1). The DNA and protein alignments from which the detailed data in Table 1
were calculated are given in supplementary data. The silE genes from plasmids in strains J53(R476b), RG486(MIP233) and RG486(MIP235) were identical in DNA sequence (Table 1
) to pMG101 silE; silE from strains RG1763(R478) and RG486(pWR23) differed from silE of pMG101 at 19 or 1 positions, respectively (Table 1
). The 10 predicted histidine residues of SilE that are involved in Ag+protein binding (Gupta et al., 1999
; Silver et al., 1999a
) are conserved in the SilE products from the additional IncHI plasmids (see supplementary data).
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The order of sil genes is the same on different plasmids
In pMG101, the genes silP(ORF105)AB(ORF96)C are oriented divergently from silRSE. The sil gene order and orientation on the five new IncHI plasmids were determined by PCR amplification using one primer located in the 5' region of one gene and a second primer located toward the 3' end of a downstream gene. With primers in the 5' region of silR and the 3' end of silS, a PCR product of approximately 2184 bp was obtained from the six sil-positive plasmids (Fig. 3a), showing that silR and silS are contiguous and similarly oriented. With primers in the 5' region of silR and the 3' end of silE (see Fig. 1a
), a PCR product of approximately 2812 bp was obtained from all six plasmids (Fig. 3b
), indicating that silRS was contiguous with and in the same orientation as silE. PCR products of the expected size (6424 bp) were obtained with primers in the 5' region of silA and the 3' end of silP (Fig. 3c
). These results show that silA and silP are contiguous and similarly oriented in all six plasmids. The organization of the sil genes on the IncHI plasmids from E. coli strains J53(R476b), RG486(MIP233), RG486(pWR23), RG486(MIP235) and RG1763(R478) is therefore considered to be the same as the sil genes on pMG101.
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Quantitation of the PCR products for silC indicated 1·8-times more silC transcript with RNA from induced strain J53(R476b) cells than from uninduced cells (Fig. 5b, lanes 3 and 4; analysis not shown) and equivalent amounts of silC RNA with uninduced and Ag+-induced cultures of J53(pMG101) (Fig. 5b
, lanes 1 and 2). The diffusion of the band in Fig. 5(b)
lane 1 is thought to be an artifact of the gel and equivalent total fluorescence was obtained for lanes 1 and 2. Furthermore, the amount of silC PCR product from induced strain J53(R476b) cells was similar to that from pMG101. This indicates that silC transcription from strain J53(R476b) is low, but can be induced, while in J53(pMG101) silC transcription is constitutive and high. These data represent a first step toward explaining the differences in Ag+ resistance levels between the two strains.
The PCR products for silE show a lower but 1·32-fold inducible level of transcription with strain J53(R476b) (Fig. 5c, lanes 3 and 4; analysis not shown) compared with a higher and constitutive level of transcription with strain J53(pMG101) (Fig. 5c
, lanes 1 and 2). The transcript level from induced cells of strain J53(R476b) (Fig. 5c
, lane 4) was 73% of that from J53(pMG101) cells, perhaps consistent with the lower resistance level of J53(R476b) (Fig. 4b
).
Chromosomal homologues of the sil determinant
The sil determinant from pMG101 contains seven named genes (and two ORFs of unassigned function) (Fig. 1a; Gupta et al., 1999
). The closest homologues for silAB(ORF96)CRS in the GenBank database are from the published E. coli K-12 and O157:H7 chromosomes (Blattner et al., 1997
; Perna et al., 2001
). These ORFs are listed in GenBank as hypothetical genes and are designated ybdE, ylcD, ylcC, ylcB, ylcA and ybcZ, respectively (for E. coli K-12, GenBank accession no. AE000162, amino acid accession nos 17867881786783; for E. coli O157:H7: GenBank accession nos AE005236 and AE005237, amino acid accession nos AAG54903·1AAG54908·1; respectively) (Blattner et al., 1997
; Perna et al., 2001
). Based on the data presented in this paper, we propose to name these genes agrA (ybdE), agrB (ylcD), ORF110 (ylcC), agrC (ylcB), agrR (ylcA) and agrS (ybcZ) (agr for Ag+ resistance; Fig. 1b
). The mnemonic agr is introduced rather than calling the chromosomal genes sil, as it has not yet been demonstrated whether they are paralogous or only orthologous in function. The E. coli chromosomal sil gene homologues are present on what is being called the common E. coli backbone of 2·6 Mb shared between the two strains (Perna et al., 2001
). Between the two chromosomal sets, only the SilR homologues (AgrR) are 100% identical while the other homologue pairs vary between 0·74 and 2·3% at the amino acid level and to similar degrees at the nucleotide level. The chromosomal genes are oriented in the same direction as the plasmid sil genes and the spacing between genes in most cases is rather similar (Fig. 1b
). The amino acid identities between translation products of the plasmid sil determinants and the putative paralogous E. coli chromosomal products are shown in Fig. 1(b)
and detailed alignments between the sil and the chromosomally encoded products are available in the supplementary data.
The E. coli chromosomal genes are proposed to encode a three-polypeptide cation efflux transporter (AgrABC, equivalent to SilABC), plus a two-component kinase/responder transcriptional regulatory pair (AgrRS, equivalent to SilRS). These five genes and their products are related to those of the czcABCRS (for Cd2+, Zn2+ and Co2+ resistances) plasmid system of Ralstonia (Nies, 1995 ). Between ORFs agrC and agrB is ORF110, which is homologous to ORF96 in the sil determinant (Fig. 1a
and b
). An equivalent ORF is not present in the homologous CzcABCRS system. A deletion in agrA (homologous to silA) on the E. coli chromosome resulted in Ag+ hypersensitivity (Fig. 6
), but did not change the sensitivity level to high Cd2+ and Cu2+. Similar observations have also been made by Franke et al. (2001)
. The introduction of an intact agrA gene on a plasmid restored resistance to silver (Fig. 6
). These results suggest that the chromosomal genes also function for Ag+ resistance.
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DISCUSSION |
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pMG101 conferred strong Ag+ resistance and a higher constitutive level of transcription, compared to plasmid R476b. It is unclear and remains to be tested why plasmid R476b shows lesser resistance to Ag+ and a lower level of constitutive, but inducible expression. The newly identified sil genes may not provide a high level of Ag+ resistance in the strains as isolated, but may provide a selective advantage to the bacteria that possess these genes on exposure to Ag+. Mutations that allow higher expression from these genes and increased resistance would be selected. Ag+-resistant clinical E. coli isolates selected by step-wise exposure to higher concentrations of Ag+ showed active efflux of Ag+ (Li et al., 1997 ). It is possible that the pMG101 sil system has been already selected in this regard by repeated growth on high concentrations of Ag+ during the more than 25 years of laboratory culturing since it was isolated. It is also notable that pMG101 was found in a Salmonella isolate from a burn ward where silver sulfadiazine was used to prevent nosocomial infections (McHugh et al., 1975
).
Contiguous homologues for five of the seven sil genes were identified on the E. coli K-12 and O157:H7 chromosomes. There are no ORFs on the E. coli K-12 or O157:H7 chromosomes homologous to silE, suggesting that this gene was added late to the plasmid system. E. coli K-12 has six genes for P-type ATPases, and the closest with regard to amino acid product sequence on both the K-12 and O157:H7 chromosomes to that of pMG101 SilP is ybaR (GenBank accession U58330; renamed copA; Rensing et al., 2000 ), which is involved in Cu+ efflux. copA is located approximately 60 kb away from the agrABORF110CRS system. copA may also mediate efflux of Ag+ since a deletion in copA resulted in hypersensitivity to Ag+ (A. Gupta, data not shown) as well as to Cu+ (Rensing et al., 2000
). The Ag+ efflux reported in clinical E. coli isolates (Li et al., 1997
) may involve the homologous agrAB(ORF110)CRS chromosomal regions (Fig. 1b
). The likely cation substrates for efflux by the chromosomal agr system are Ag+ and Cu+, since a shared Ag+/Cu+ efflux transporter has been identified in Enterococcus (Solioz & Odermatt, 1995
) and a deletion in agrA renders the cell hypersensitive to Ag+ (Fig. 6
). The differences between the two E. coli K-12 and O157:H7 chromosomal agr gene systems, although small (Fig. 1b
), are sufficient to suggest that a common ancestor of both strains, that are predicted to have diverged about 4·5 million years ago (Perna et al., 2001
), already contained the agr gene system. The differences between the two E. coli chromosomal systems are similar in percentage to those seen between the sil systems from different plasmids of the IncHI group, as reported in this study (Table 1
). This suggests that the plasmid sil gene determinants have also been present for a sufficiently long time to establish heterogeneity in DNA and protein sequences. With increasing numbers of bacterial genomes being sequenced and homologues of the sil system being found in increasing numbers, it is reasonable to designate the chromosomal genes as agr to allow more accurate subsequent annotation of new DNA sequences.
The availability of DNA sequences for sil genes on six IncHI plasmids and identification of close homologues on bacterial chromosomes are starting points for molecular epidemiological studies of sil genes in clinical and environmental isolates of concern that may or may not have been exposed to silver. Such molecular diversity studies of the sil genes will be helpful in addressing questions regarding nosocomial infections, toxic metal bioremediation and the effective use of Ag+ as a biocide.
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ACKNOWLEDGEMENTS |
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Received 2 April 2001;
revised 20 June 2001;
accepted 20 August 2001.