1 Department of Microbiology and Immunology, Dalhousie University, Sir Charles Tupper Medical Building, College Street, Halifax, Nova Scotia, Canada B3H 4H7
2 Department of Medical Microbiology and Immunology, 1-28 Medical Science Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
3 Division of Infectious Diseases, Department of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, College Street, Halifax, Nova Scotia, Canada B3H 4H7
Correspondence
Paul S. Hoffman
phoffman{at}tupdean2.med.dal.ca
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AF168355.
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INTRODUCTION |
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At least five genetically distinct chromosomal and plasmid-borne bacterial tellurite resistance systems have been described (Taylor, 1999; Turner et al., 1999
; Taylor et al., 2002
). The emergence of several unrelated Ter determinants among a wide range of bacterial species, including human pathogens, suggests that these determinants provide some selective advantage in natural environments, which may be unrelated to the Ter phenotype (Hill et al., 1993
; Walter & Taylor, 1992
). For example, IncHI2 and IncHII conjugative plasmids carrying the ter locus (terZABCDEF) confer constitutive, high level Ter to E. coli, as well as resistance to bacteriophage and colicins (Taylor, 1999
; Whelan et al., 1995
; Taylor et al., 2002
). Homology searches have found putative ter genes in other bacteria, including Yersinia pestis and Deinococcus radiodurans, and variability in gene complement and number of copies among clinical isolates of E. coli O157 (Taylor, 1999
; Taylor et al., 2002
).
In this study we have characterized a chromosomal cluster of six genes from the urinary tract pathogen Proteus mirabilis that are highly homologous to members of the ter-gene clusters found on large conjugative IncHI2 plasmids (Jobling & Ritchie, 1987, 1988
; Whelan et al., 1995
) and in the chromosome of E. coli O157 : H7 strains (Taylor et al., 2002
). The transposon insertion into the carboxyl coding sequences of the terC gene resulted in increased susceptibility to K2TeO3 (Tes), which could be reversed (Ter) in trans by a multicopy plasmid carrying terC. In contrast to the plasmid-borne ter loci, the ter operon of P. mirabilis was induced in response to K2TeO3 and to a lesser extent by hydrogen peroxide and superoxide anions, suggesting that the mechanism of activation might involve thiol oxidation. Finally, unlike the well-characterized plasmid-borne ter loci, the inducible ter operon of P. mirabilis is contained in the chromosome, an apparent feature of this genus.
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METHODS |
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DNA manipulations.
Recombinant DNA techniques employed standard protocols (Sambrook et al., 1989). Plasmid DNA was isolated by alkaline extraction (Birnboim & Doly, 1979
) and by centrifugation of total DNA in caesium chloride/ethidium bromide gradients (Sambrook et al., 1989
). Restriction endonucleases, T4 DNA ligase, RNase, T4 polynucleotide kinase and DNA polymerase I were purchased from Gibco-BRL or New England Biolabs and used according to the manufacturer's directions.
Construction of P. mirabilis genomic libraries.
Genomic libraries from P. mirabilis strains S2R and S2R-6/39 were constructed following partial cleavage of chromosomal DNA with Sau3AI and ligation into cosmid pHC79 as described previously (Hoffman et al., 1989). A library of
3 kbp EcoRIPstI fragments of P. mirabilis chromosomal DNA was created in E. coli JF626 following separation of the DNA fragments by agarose gel electrophoresis and ligation into pBluescript. Transformants were selected for appropriate antibiotic resistance on solid medium and colonies were picked to 96-well plates for screening. Libraries were screened for the desired genes by colony Southern blot procedures as described previously (Hoffman et al., 1989
).
DNA sequencing and sequence analysis.
DNA sequencing was performed manually with a Sequenase (version 2.0) kit from USB and [35S]dATP from NEN/Life Science Products (Sanger et al., 1977), or automated on a LiCor 4000L sequencer by the Dalhousie UniversityNRC Institute for Marine Biosciences Joint Laboratory. Sequence assembly and analysis were performed using the Genetics Computer Group suite of software (Devereux et al., 1984
). Relatedness between predicted polypeptides was detected with the BLAST (Altschul et al., 1990
) and FASTA (Pearson, 1990
) programs of the National Center for Biological Information (NCBI), Bethesda, MD, USA, and CLUSTAL W (Thompson et al., 1994
) was used for multiple alignments of putative amino acid sequences.
Complementation of the terC mutant.
A XhoI/BamHI DNA fragment containing terC was subcloned from pBSK-HP into pBOC20 and the resulting plasmid (pATC12) was introduced into the terC : : lacZ mutant strain S2R-6/39 by conjugation from E. coli S17-1 pir. Exconjugants were selected on non-swarming LB agar supplemented with Km and Cm to maintain pATC12 : terC in trans or following selection on the non-swarming LB agar. Ter was determined by viable count on non-swarming medium containing various concentrations of K2TeO3 (0350 µg ml-1).
RNA manipulations.
Total RNA was extracted by the hot SDS/acid phenol method (Hoffman et al., 1992) from P. mirabilis strains grown in the presence or absence of K2TeO3 or following treatments with hydrogen peroxide or methyl viologen. Northern blot hybridization assays were optimized as generally described (Kroczek & Siebert, 1990
). [32P]dCTP-radiolabelled DNA probes were generated by random priming of restriction fragments obtained from cloned DNA sequences from regions of the ter operon, including terZA, terCD or from a 1·6 kb BamHI fragment of ureR, a gene constitutively expressed in the absence of urea (unpublished data). Primer extension was performed as described previously (Hoffman et al., 1992
) by hybridizing a 5'-end-labelled oligonucleotide primer TERZ2R (see Table 1
) to P. mirabilis total RNA. End-labelling of TERZ2R was done using T4 polynucleotide kinase with subsequent purification using a Sep-Pack C18 cartridge (Millipore). RT-PCR was also used to monitor mRNA levels of the ter operon. Total RNA was treated with RQ1 DNase (Promega) and then purified by standard protocols. cDNA was generated with M-MLV reverse transcriptase (Gibco-BRL), according to the manufacturer's instructions, in a 20 µl reaction containing 1·5 µg RNA and 1 µg random primers, pd(N)6 (Amersham Pharmacia Biotech). Standard PCR procedures were used to generate amplicons from 1 µl of the RT reaction using primer pairs (see Table 1
) designed for the 5' region of terZ, the first gene in the putative operon, and ureR. The minimum number of cycles required for detection of amplicons was determined empirically (generally 2227 cycles). PCR samples were examined by agarose gel electrophoresis and photographed under UV light using a Fotodyne MP-4 photographic system (Fotodyne). Digital images were then created using an Epson ES-1200C scanner (Seiko Epson) and Adobe PhotoShop 4.0 software. Analysis of the gel bands was done with Gel-Pro Analyser software (Media Cybernetics).
Determination of plating efficiency on K2TeO3 medium following induction of the ter operon.
P. mirabilis strain S2R was grown overnight in LB broth and following adjustment of the OD660 to 0·5, either 50 µg K2TeO3 ml-1 or 100 µM H2O2 was added and the cultures (including a control) were shaken at 37 °C. At 30 and 60 min, samples were diluted and plated on LB agar (-NaCl) with or without 200 µg K2TeO3 ml-1. The plating efficiency was determined for each treatment by comparing the number of c.f.u. ml-1 of the treated or untreated groups relative to the number of c.f.u. obtained on K2TeO3-containing medium. The means and standard deviations for triplicate platings were computed.
-Galactosidase assays and inhibitory effect of K2TeO3.
P. mirabilis mutant S2R-6/39 containing mini-Tn5lacZ was grown in LB broth and aliquots were removed at intervals and assayed for -galactosidase activity by the method of Miller (1972)
. All assays were done in triplicate and the mean and standard deviation were computed. The inhibitory effect of K2TeO3 on
-galactosidase activity was determined by measuring the activity of the terC : lacZ fusion in the absence and presence of K2TeO3 (50200 µg ml-1). As a control for these experiments, a ureR : lacZ fusion (mini-Tn5) inserted at a locus other than the urease locus in P. mirabilis strain S2UR1 was similarly challenged with K2TeO3. UreR is a positive activator of the urease locus and in the absence of the inducer urea, is expressed constitutively at low level (unpublished data). The percentage inhibition was determined from the mean of three determinations.
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RESULTS |
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Isolation of a mini-Tn5-lacZ insertion in terC
The genetic nature of the Ter phenotype of P. mirabilis was investigated by transposon mutagenesis by screening for -galactosidase-positive mutants exhibiting a Tes phenotype. One clone (S2R-6/39) exhibited decreased levels of
-galactosidase activity on LB agar (X-Gal) containing tellurite and on LB agar supplemented with X-Gal the concentric rings of swarming bacteria appeared white while the non-swarming vegetative bacteria (central inoculum) appeared blue. Swarmer cells and vegetative cells were harvested from LB agar (Hoffman & Falkinham, 1981
) and assayed for
-galactosidase activity (Miller, 1972
). Vegetative S2R-6/39 bacteria contained 345±50 Miller units while swarm cells contained 73±48 units of activity, suggesting that the transposon had inserted into a locus that was downregulated during swarmer differentiation. This mutant was more susceptible to K2TeO3 (MIC
100 µg ml-1) than was the wild-type S2R strain (>300 µg ml-1) and colonies of S2R-6/39 required an additional day to appear on tellurite-containing medium.
The mini-transposon and flanking DNA from this mutant (P. mirabilis S2R-6/39) was cloned into pBR322 (pAT9501) and DNA sequence analysis revealed the transposon to have inserted into the C-terminal coding region of terC, an orthologue of the Ter systems of plasmids R478 (Whelan et al., 1995) and pMER610 (Jobling & Ritchie, 1988
). Western blot analysis with antibody against
-galactosidase eliminated the possibility that the insertion produced a TerC-LacZ chimera (data not shown). Since Ter genes are often located on plasmids (Jobling & Ritchie, 1987
, 1988
; Whelan et al., 1995
), we used caesium chloride gradient centrifugation (plasmid R478 as a control) to examine P. mirabilis for IncHI related plasmids. No evidence for plasmid sequences could be demonstrated in these studies (data not presented), suggesting that the ter genes of P. mirabilis were located in the chromosome [confirmed in the unpublished genomic sequence of P. mirabilis strain 210 (P. S. Hoffman, SmithKline Beecham)].
The S2R-6/39 mutant displayed Tes (efficiency of plating at 100 µg ml-1=10-2 compared with wild-type at 300 µg ml-1, efficiency of plating=1), which could be restored to high-level Ter following introduction of terC harboured on pATC12 (data not presented). Interestingly, the complemented mutant displayed higher resistance to K2TeO3 (efficiency of plating=1 at 350 µg ml-1) than the wild-type strain, perhaps resulting from increased expression of terC from a multicopy plasmid. These studies suggest that mutation in terC is responsible for the increased susceptibility of S2R-6/39 to K2TeO3, consistent with findings reported for terC of E. coli O157 (Kormutakova et al., 2000).
Sequence analysis of the ter gene cluster
Fig. 1 displays the physical map of the P. mirabilis Ter locus and for comparison, those from other well characterized Ter systems. The nucleotide sequence of the Ter locus revealed seven ORFs within a 6120 bp region that corresponded to orf3, terZ, -A, -B, -C, -D and -E of the plasmid-borne ter locus (Whelan et al., 1995
). Interestingly, no terF homologue was detected within the P. mirabilis genome either by sequence analysis of over 1 kb of chromosomal DNA extending 3' of the terE gene, or by PCR amplification using TEREF-F and TEREF-R primers (Table 1
) designed to amplify a putative terEF intergenic region (terF sequences are not present in the P. mirabilis strain 210 genome sequence). Also absent from the P. mirabilis Ter locus was an ORF of unknown function between orf3 and terZ that was found in other Ter systems. The highest degree of relatedness between the P. mirabilis-encoded and plasmid-borne Ter gene clusters was detected for the TerD polypeptides, with 92 and 90 % of 192 aa being identical with the orthologues of R478 and pMER610 plasmids, respectively. The lowest degree of relatedness was detected for the predicted TerA polypeptides with 66 % identity (aa) with both R478- and pMER610-encoded polypeptides. The predicted TerA polypeptide was larger by 44 aa at the N-terminal region than its plasmid-encoded counterparts, which begin at position 45 in the P. mirabilis gene (Whelan et al., 1995
). A BLAST search of the TerA N-terminal stretch of 44 aa residues showed high similarity of this polypeptide to TerD proteins, suggesting an intergenic recombination (duplication/deletion) might have occurred between these highly conserved genes.
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TerC sequence analysis
The PSORT algorithm (Nakai & Kanehisa, 1991) predicted that TerC is associated with the bacterial inner membrane. TMpred hydropathy analysis (Hofmann & Stoffel, 1993
) identified nine potential transmembrane-spanning hydrophobic helices within TerC with lengths of 1823 aa. The TerC N terminus is predicted to extend into the periplasmic space, while the C terminus protrudes into the cytoplasm. In addition, a leucine-zipper motif (Kouzarides & Ziff, 1989
) was identified in the C-terminal region (residues 268289) of this protein, though no function has been ascribed. This motif was also present in the C-terminal regions of three TerC homologues: two plasmid-encoded TerC proteins and in an M. tuberculosis hypothetical protein. Sequence analysis revealed that the mini-transposon had inserted in the carboxyl region of mutant strain S2R-6/39 (codon position 295) of terC, thereby creating a truncated TerC protein. Together with the complementation studies, these results suggest that the decreased level of Ter noted with this mutant is most likely due to attenuation of function of TerC.
Regulation of the ter operon by K2TeO3
Northern blot hybridization analysis, with different DNA probes internal to the ter operon, revealed transcripts of various sizes in RNA extracted from the wild-type and S2R-6/39 mutant strains of P. mirabilis grown in the presence of K2TeO3 (Fig. 2a). Five transcripts of 5·0, 3·0, 2·7, 2·0 and 1·5 kb were detected (K2TeO3-induced strains) when a partial terC-terD coding region was used as a probe. Similar patterns were obtained when blots were probed with a 0·9 kbp BamHIEcoRI fragment containing DNA sequences of the terZ-terA coding region (data not presented), suggesting that the multiple bands might represent mRNA degradation products rather than distinct transcripts originating from internal promoters. The 5 kb band detected in the wild-type strain is likely to correspond to the multicistronic product of the predicted ter operon, but the identities of the other bands are more difficult to establish, because of the extensive internal sequence similarity among individual ter genes. In the absence of K2TeO3, no hybridization signals were detected in RNA, suggesting that the ter operon of P. mirabilis, unlike the locus found on IncHI plasmids, may be regulated.
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Tellurite inhibits -galactosidase activity in P. mirabilis
To address the possibility that the low -galactosidase activity measured for the fully K2TeO3-induced P. mirabilis mutant strain S2R-6/39 might be due to assay interference by K2TeO3, we measured
-galactosidase activity of S2R-6/39 and P. mirabilis strain S2UR1 containing an operon fusion with the ca 500 bp upstream ureR promoter region (ureR : lacZ) and inserted in single copy elsewhere from the urease gene cluster (unpublished data). As seen in Table 2
, K2TeO3 decreased
-galactosidase activity by 39 % for S2UR1 (fully Ter) containing a ureR : lacZ fusion. In contrast, the
-galactosidase activity of S2R-6/39 was decreased by 70 % over controls following growth in K2TeO3-containing medium. The more dramatic inhibition noted with the mutant is most likely due to both decreased production of mRNA as determined by Northern blotting (Fig. 2
) and to assay inhibition as demonstrated with S2UR1. Thus, intrinsic tellurite toxicity prevented the use of the lacZ fusion as a reporter for regulatory studies.
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DISCUSSION |
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The P. mirabilis ter operon differed from both its plasmid- and E. coli O157-borne counterparts, including (i) a larger terA product (44 aa larger) which may have resulted from a fusion of terD sequences with terA; (ii) the absence of terF, which is also absent from plasmids pMJ606 and pTE53 (Kormutakova et al., 2000; Taylor et al., 2002
); and (iii) inducibility by tellurite and to a lesser extent by agents associated with oxidative stress. Alignment of the intervening DNA sequences between orf3 and terZ of P. mirabilis (
390 bp region containing putative regulatory motifs) with those from the published sequences of other ter operons revealed no nucleotide sequence identity, while that of the flanking genes was
80 %, suggesting that the regulatory region may be unique to the P. mirabilis ter operon. A consequence of this regulation for P. mirabilis is that uninduced bacteria are susceptible to the toxicity of tellurite when plated onto medium containing sub-MIC levels of K2TeO3. In contrast, fully induced bacteria exhibited no loss in viability when plated onto K2TeO3-containing medium and by RT-PCR, ter mRNA transcripts from induced bacteria were increased >eightfold over those from the uninduced bacteria. The selective advantage, if any, for a regulated ter operon in natural environments, such as the urinary tract of humans, remains to be investigated.
Until recently, Ter was considered a constitutive trait of IncHI2 plasmids or the closely related chromosomal counterparts in E. coli O157 : H7 strains. Moreover, since these loci are also associated with resistance to colicins and phage, constant insults in environments occupied by enteric pathogens, constitutive expression of Ter was viewed as essential for survival. Our finding that the ter operon of P. mirabilis was inducible by tellurite has since led to evaluation of Ter in E. coli O157 : H7 strains (Taylor et al., 2002). There is considerable variability in ter loci among clinical isolates with some strains containing duplications of ter genes while others show variability in the complement of ter genes (Taylor et al., 2002
). In some E. coli O157 : H7 strains, prior exposure to tellurite led to increased resistance of strains when plated on K2TeO3-containing medium and analysis of the ter genes by RT-PCR showed that mRNA of terBC and F genes was increased while that for terZD and E was not induced (Taylor et al., 2002
). Interestingly, the study found that terA was not expressed, suggesting that a potential regulatory region might exist in this sequence. In contrast, an operon structure was inferred for the P. mirabilis ter locus based on primer extension and Northern blotting analysis using probes for terZA and terCD regions. Northern blots and primer extensions were negative in the absence of prior induction by tellurite and transcript size was consistent with production of a polycistronic message originating from a
70 promoter upstream of terZ. However, we cannot rule out the possibility of internal promoters, message processing or degradation contributing to the smaller transcript sizes observed by Northern blotting.
Analysis of upstream DNA promoter sequences of the ter operon revealed several motifs that might be associated with regulation. Both inverted and direct repeats were found (see Fig. 3) in the -10 and -35 promoter region and upstream of the -35 hexamer were sequence motifs resembling OxyR-binding sites (Ochsner et al., 2000
). While the sequences were not identical to canonical OxyR-binding sites of catalase and alkylhydroperoxide reductase other genes putatively regulated by OxyR in P. mirabilis (unpublished results) the similarity may be sufficient to account for the low level stimulation of Ter by reduced oxygen intermediates that were observed in this study.
Our initial observation that non-swarming bacteria contained higher levels of -galactosidase than swarming bacteria could not be pursued further due to the intrinsic toxicity of tellurite for
-galactosidase activity. Thus, the eightfold increase in terC mRNA was not accompanied by a similar increase in
-galactosidase activity. In general, tellurite was toxic even for wild-type P. mirabilis as shown by decreased levels of mRNA of ter genes as well as for ureR.
Tellurite resistance is a biological puzzle
It seems unlikely that tellurite resistance mechanisms, which are both diverse and widespread in the microbial world, exist solely for the purpose of protection from tellurite toxicity, as tellurite is not found in appreciable levels in the intestinal and urinary habitats of many of these bacteria. Furthermore, since the ter operon of P. mirabilis is not expressed in the absence of tellurite, its participation in colicin and phage resistance also seems unlikely since constitutive expression, as shown for the plasmid-borne Ter operon (Whelan et al., 1997), would be required. In contrast to other enteric bacteria, colicin (proticine) production and susceptibility of the genus Proteus occurs during swarming as demonstrated by lysis of swarmer cells of incompatible strains at the intersection of two colliding swarming bands (Dienes test) (Senior, 1977
). Our finding that the ter operon was even further depressed during swarmer cell development might explain the inability of this locus to provide adequate protection from the proticines of incompatible strains (data not presented). We presume that the ter operon must contribute to virulence or fitness since all Proteus strains tested contained this operon and displayed high-level Ter. Perhaps the Ter genes are associated with protection from other forms of oxidative stress or agents causing membrane damage as suggested in this study.
Finally, inducible Ter appears to be an integral feature of the genus Proteus and perhaps of related genera (Morganella and Providencia spp.), suggesting that Ter may be a distinguishing characteristic and a selective advantage in the ecology of the urinary tract.
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ACKNOWLEDGEMENTS |
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Received 6 September 2002;
revised 20 January 2003;
accepted 24 January 2003.
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