1 Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, MD 20708, USA
2 Department of Microbiology and Molecular Genetics, University of Texas Houston Medical School, Houston, TX 77030, USA
Correspondence
Thomas A. Cebula
tac{at}cfsan.fda.gov
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ABSTRACT |
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Present address: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
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INTRODUCTION |
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Notably, although spontaneous mutation rates are enhanced by mutation in at least 25 separate genetic loci (Miller, 1998; Horst et al., 1999
), the overwhelming majority of mutators found thus far among natural strains are due to defects in the mutS, mutH, mutL or uvrD gene (LeClerc et al., 1996
; Matic et al., 1997
). These genes define the methyl-directed mismatch repair (MMR) pathway, a DNA repair system that not only corrects base mismatches in newly replicated DNA but is also the main barrier for recombination of mismatched heteroduplexes in DNA (i.e. homeologous recombination) (Modrich & Lahue, 1996
). That is, MMR defects relax the recombination barrier, allowing DNA exchange between species that normally do not mate. This promiscuous property, unique to MMR mutants, may explain the emergence and persistence of MMR mutants in natural populations. Most importantly, it implicates a role for horizontal gene transfer in gaining the benefits for survival and growth. Since we reported the high incidence of MMR-defective mutators among E. coli and Salmonella pathogens (LeClerc et al., 1996
), the accounts of mutator subpopulations among pathogenic isolates of E. coli (Matic et al., 1997
; Denamur et al., 2002
), Pseudomonas aeruginosa (Oliver et al., 2000
), Neisseria meningitidis (Richardson et al., 2002
) and Streptococcus pneumoniae (Negri et al., 2002
) bolster the view that these mutators are positively selected in nature, most likely because of the beneficial mutations they carry.
Since the earliest observations of mutators in natural populations (Jyssum, 1960; Gross & Siegal, 1981
), several features of their incidence in the feral setting have been puzzling. That is, although laboratory experiments have shown that a high mutation rate can be advantageous, as evidenced by the successful competition of mutators against their non-mutator counterparts in chemostat cultures (Gibson et al., 1970
; Nestman & Hill, 1973
; Cox & Gibson, 1974
; Tröbner & Piechocki, 1981
; Chao & Cox, 1983
) and the invasion of non-mutator clones by mutators in continuous growth experiments (Sniegowski et al., 1997
), mutators have not overtaken natural populations of bacteria. Moreover, most organisms appear to have evolved a seemingly constant mutation rate (Drake, 1991
; Ochman et al., 1999
). Finally, since deleterious mutations arise at rates roughly 104 times those of beneficial mutations (Cebula & LeClerc, 2000
), a heightened mutation rate should merely hasten the mutator to extinction in a stable environment. Yet, our studies demonstrated that MMR mutator alleles persist among natural clones at frequencies 10- to 1000-fold greater than expected for deleterious mutations (LeClerc et al., 1996
). Our results suggested, therefore, that the mutator state must be a temporary situation; i.e. a mutator allele rises to prominence with a beneficial trait that it spawned (i.e. hitch-hikes) and then falls when selection again sweeps the population (see Shaver et al., 2002
). Haploidy dictates, after all, that if the trait is to be maintained in perpetuity, the bacterium must lose (by reversion or recombination) or otherwise quiet (by suppression) the mutator allele.
A transient mutator state might also result, however, because of an altered expression of MMR proteins. As downregulation of MutS, and to a lesser extent MutH, is known to occur in stationary phase and in starved bacteria (Feng et al., 1996), and the amount of MutS in E. coli K-12 is already near limiting for MMR in exponentially growing cells, the decreases of MMR proteins observed in stationary-phase cells and in nutrient-deprived cells are of especial interest. If similar downregulation occurs in natural isolates, as observed in laboratory cultures of E. coli K-12, the decreased amounts of MutS and MutH could contribute significantly to increased mutagenesis and homeologous recombination as cells enter and leave the stationary phase or otherwise stressed environments.
To evaluate mechanisms that may be involved in the emergence of a mutator phenotype, we determined the molecular basis for MMR deficiencies in natural isolates. To do this, we analysed a wild-type isolate of enterohaemorrhagic E. coli O157 : H7 for levels of MutS, MutH and MutL proteins in growing and stationary-phase cultures. We also determined the molecular defects in stable mutators identified among pathogenic isolates of E. coli: two E. coli O157 : H7 isolates, EC503 and EC535; a diarrhoeagenic E. coli of the O55 : H7 serotype, DEC5A; and a uropathogenic E. coli from the E. coli reference (ECOR) strain collection, ECOR48. Genetic complementation studies showed that normal mutability could be restored in each of these mutator strains by plasmid copies of a wild-type mutS gene (LeClerc et al., 1996), hence molecular analyses were carried out to define the nature of their MutS defects.
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METHODS |
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Growth conditions.
For routine growth, cells from frozen stocks of each bacterial strain were grown in LB medium (Miller, 1972) with shaking at 37 °C. For RNase protection assays and Western blotting experiments, bacterial stocks were added to 5 ml enriched minimal salts/glucose [EMMG; Vogel-Bonner (1xE) minimal salts, 0·01 mM FeSO4, 0·4 % (w/v) glucose and 0·5 % (w/v) vitamin-assay Casamino acids (Difco Laboratories)]. Cultures were grown overnight with shaking (300 r.p.m.) at 37 °C, and the overnight cultures were diluted 200-fold with fresh EMMG and grown with shaking. Samples for RNase T2 protection assays were collected during exponential growth at a turbidity of 50 Klett (660 nm) units (
5x108 cells per ml determined for MG1655). Samples for Western analysis were collected from cultures grown to mid-exponential phase (a turbidity of 5065 Klett units), transitional phase (200 Klett units) and stationary phase (24 and 48 h after inoculation).
Western blotting.
Samples for Western blotting were prepared as described previously (Feng et al., 1996) or with the following modifications. Portions of cultures (20, 7 and 3 ml) of strains MG1655 and EC536 were removed at culture turbidity values of 5065 Klett units and 200 Klett units, or at 24 and 48 h after inoculation. After centrifugation at 5000 g for 7 min at room temperature, pellets were washed once with 20 ml of 1xE salts and washed a second time in 1 ml of 1xE salts in microfuge tubes. The final pellets were resuspended in 300 µl buffer containing 0·125 M Tris/HCl, pH 6·8, and 4 % SDS and boiled for 10 min. Total protein concentrations were determined by using the Bio-Rad Dc protein assay kit on samples diluted 20-fold in water and on BSA standards in the same diluted buffer. For analysis on SDS-PAGE gels, samples were diluted in a solution containing 2 M DTT, 40 % (v/v) glycerol and 0·1 % bromophenol blue, so that final protein samples were resuspended in Laemmli buffer (Laemmli, 1970
; 2 % SDS, 100 mM DTT, 10 % glycerol, 62·5 mM Tris/HCl, pH 6·8).
Quantitative Western blotting assays to detect MutS, MutL and MutH were carried out as described previously (Tsui et al., 1997) or with the changes described below. Antisera against hexahistidine (His-6)-tagged E. coli MutS, MutL and MutH proteins were produced as described previously (Feng & Winkler, 1995
) and affinity-purified with CNBr-activated Sepharose 4B (Pharmacia Biotech) coupled to His-6-tagged E. coli MutS, His-6-tagged E. coli MutL or His-6-tagged E. coli MutH as described previously (Harris et al., 1997
). In gel analysis, the His-6 tag (about 2 kDa) did not appreciably change the migration of the tagged proteins. To further reduce background, the antibodies were pre-adsorbed to lysates prepared from mutS, mutL or mutH insertion mutants (Feng et al., 1996
). Alkaline-phosphatase-conjugated secondary antibodies or ECL Western blotting detection reagents (Amersham) were used for detection.
RNase T2 protection assays of chromosomal transcripts.
RNA was prepared by adding portions of bacterial cultures directly to lysis solutions without intervening steps as described previously (Feng et al., 1996). RNase T2 protection assays of transcripts from the bacterial chromosome were conducted as described by Tsui et al. (1994)
. RNA probes for detecting mutS transcripts (see Fig. 2
) were synthesized using the following phage RNA polymerases and linearized plasmid templates: SP6 and EcoRI-treated pTX414 (5' end of mutS transcripts); T7 and HincII-treated pTX541 (3' end of mutS transcripts); and SP6 and EcoRI-treated pTX541 (antisense transcripts at the 3' end of mutS) (Tsui et al., 1997
). A series of labelled, undigested probes of known length were used as size standards to determine the lengths of the protected fragments (standard errors of 510 %) (Tsui et al., 1994
). Each hybridization reaction contained 25 µg total RNA from cells grown in EMMG. Radioactivity in bands was quantified by using an InstantImager (Packard).
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For sequencing the mutS gene of strain ECOR48, the ECOR48 mutS gene clone was grown in LB medium at 37 °C overnight and subjected to a Wizard Plus Midiprep DNA Purification System (Promega), according to the manufacturer's instructions. Plasmid DNA was further purified by phenol extraction and ethanol precipitation. The 3·9 kb DNA fragment, covering the mutS gene and its flanking sequences, was sequenced from plasmid DNA using a commercial service (Lark Technologies).
Nucleotide sequencing and analysis.
To determine the exact end points of mutS deletions in strains EC503, EC535 and DEC5A, PCR products containing the junction fragments were sequenced. DNA sequencing was performed using the T7 Sequenase Version 2.0 DNA Sequencing Kit (Amersham) according to the manufacturer's instructions. Programs from the Wisconsin Sequence Analysis Package (Genetics Computer Group) were used for sequence analyses.
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RESULTS |
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Mapping and measurement of mutS transcripts
To determine whether the transcriptional organization of the mutS gene in E. coli O157 : H7 is similar to that in MG1655, we mapped the transcripts by RNase T2 protection assays using RNA probes specific for the 5' or 3' end of the mutS gene (Fig. 3a). EC536, the wild-type O157 : H7 strain, showed similar patterns of 5' and 3' ends of mutS transcripts as in MG1655 (Fig. 3b
). Three major protected transcripts of 520, 480 and 428 nt were seen using the 5'-end mutS probe on RNA from MG1655 and EC536. The 5' ends of the 520 and 480 nt protected species were mapped to positions approximately 74 and 34 nt, respectively, upstream from the mutS start codon of MG1655 (Tsui et al., 1997
). The 428 nt protected species is a partial mutS transcript (Tsui et al., 1997
). Two major protected transcripts of 990 and 920 nt were seen using the 3'-end mutS probe on RNA from MG1655 and EC536. These bands correspond to mutS transcripts with 3' ends at approximately 97 and 29 nt downstream from the mutS stop codon. Together, these results show that mutS in EC536 (O157 : H7) is in a single-gene operon, as was reported previously for MG1655 (K-12) (Tsui et al., 1997
), and that the transcription starts, stops and possible processing sites appear similar for the enterohaemorrhagic and laboratory strains of E. coli.
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PCR and sequence analysis of EC503, EC535 and DEC5A mutant DNAs
In EC536, mutS is adjacent on its 5' side to fhlA and on its 3' side to the o218yclDyclCyclBslyArpoS gene cluster (cf. Fig. 3). Attempts to PCR-amplify an intact mutS gene from the EC503, EC535 and DEC5A mutators failed, suggesting that primer-annealing sites were disrupted by deletions or rearrangements of the gene in these strains. To determine the extent of possible mutS deletions, the mutants were probed under low stringency conditions using a series of 32P-end-labelled oligonucleotides designed to anneal at 1·5 kb intervals throughout a 12 kb region surrounding mutS from either E. coli K-12 or O157 : H7. The results of colony hybridization indicated that EC503 contained a deletion of the 3' end of the mutS gene and open reading frames (ORFs) between the mutS and rpoS genes; EC535 and DEC5A carried extensive deletions encompassing mutS and neighbouring 5'-fhlA gene and extending at least 3 kb to the rpoS gene (data not shown).
Analysis of the EC503 mutant by PCR amplification of DNA from the 5' end of the mutS gene to the middle of the rpoS gene followed by sequencing of the PCR product confirmed that the 3' end of the mutS gene and ORFs between the mutS and rpoS genes were deleted in this mutant. Initial PCR analysis of the more extensive deletions in EC535 and DEC5A involved amplification of short products on each side of the deletions in order to determine their approximate extent. Primers, established for productive PCR, were then used in long PCRs for amplification of products containing the deletion junctions. Sequence analysis of these PCR products defined the deletions in mutant DNAs. Table 2 gives the sizes and positions of the deletions and shows wild-type sequence present at the deletion end points based on the K-12 genome. Sequences of 68 base pairs are repeated at the end points of the deletions, leaving one intact copy in the cases of EC503 and EC535 or deleting both copies in DEC5A. Fig. 4
shows the PCR products amplified from primers at fixed sites in the three mutant DNAs. In the experiment of Fig. 4
, an amplification product of 14·1 kb is expected to be produced on template prepared from a wild-type strain of E. coli O157 : H7 (EC536), while products of 10·6, 4·2 and 0·8 kb are observed on EC503, DEC5A and EC535 DNAs, respectively.
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DISCUSSION |
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No antisense transcript to mutS was detected in EC536 during exponential or early stationary phase (data not shown). Curiously, an antisense transcript was observed in the EC503 deletion mutant. Although antisense transcription from rpoS and other downstream genes from mutS could provide an exquisite mechanism for effecting a transient mutator phenotype, there is no evidence that an antisense mechanism operates to mediate MutS amount under the conditions tested so far.
To date, studies have investigated increased mutagenesis caused by decreases of MutS in stationary-phase cells (Schaaper & Radman, 1989; Zhao & Winkler, 2000
), though increased homeologous recombination caused by MutS depletion may be the more significant occurrence in resting or non-dividing cells in nature. Examining recombination in starved bacteria may be especially important not only because MutS expression is compromised severely under nutrient-deprived conditions (Feng et al., 1996
) but also because cells in the natural habitat constantly experience a feast or famine existence. Such conditions may signal the need for a transient hypermutable and/or hyper-recombinagenic phenotype.
Analysis of molecular defects in the stable E. coli mutators EC503, EC535 and DEC5A revealed large deletions incapable of direct reversion to the wild-type allele. This outcome is compatible with findings on defects in natural P. aeruginosa mutators, which were frameshift, insertion, deletion or multiple substitution mutations (Oliver et al., 2002). While the nonsense mutation in the mutS gene of the ECOR48 mutator is susceptible to simple reversion, other base changes in the gene the expected consequence for an inactive gene in a mutator background also likely lead to its inactivation. The question then remains how an adaptive mutant can break its linkage with the mutator locus and thereby escape the deleterious effects of a heightened mutation rate. Besides reversion at the mutator locus, the mutator phenotype may be suppressed by mutation at other loci, as evidenced by results from long-term growth experiments using a mutT strain of E. coli (Tröbner & Piechocki, 1984
). In these studies, isolates taken after 2200 generations of chemostat growth had lower mutation frequencies than the starting culture, but transduction analyses showed that the mutT allele itself had not changed. The lower mutation rates of the isolates were likely due to secondary mutations that suppressed the mutator phenotype (Tröbner & Piechocki, 1984
).
Another option, one particularly feasible in promiscuous MMR mutators in feral settings, is repair of the mutator defect by homeologous recombination. An intact mut locus could be inherited from a Mut+ cell after horizontal transfer of sequence from either closely or distantly related species. Phylogenetic analysis of mutS-positive alleles from naturally occurring E. coli strains (including pathogens and strains of the ECOR collection) showed that mutS alleles indeed have recombined, evidenced by a phylogeny of mutS alleles different from that of the E. coli strains in which they reside (Denamur et al., 2000; Brown et al., 2001a
, b
). Such differences between gene evolution and strain evolution are indicative of horizontal exchange, a conclusion also supported by evidence for specific transfer events in the mutS region of the E. coli chromosome (LeClerc et al., 1999
; Culham & Wood, 2000
; Reid et al., 2000
). The identification of mutH among a class of genes inherited from horizontal transfer (Médigue et al., 1991
) suggests that defective mutH alleles may also have been repaired by homeologous recombination.
The occurrence of strains carrying deletions that encompass multiple loci, as many as 15 genes and ORFs in the EC535 mutant, is surprising in view of the environmental challenges of natural conditions. We have argued that the preponderance of mutS mutants among mutators in nature is indicative of the benefits of adaptive change generated by increased recombination and mutagenesis (LeClerc et al., 1996). The deletion mutants analysed here also affect the rpoS gene, however, and many genes needed for survival during environmental stress are regulated by the RpoS sigma factor. There are rpoS mutants of E. coli that have a selective advantage over wild-type cells as they emerge from stationary- to exponential-phase growth (Zambrino et al., 1993
) and rpoS mutants are common in aged cultures (Sutton et al., 2000
). It is possible that other, compensatory changes in these mutator strains overcome the limitations of growth in the absence of RpoS regulation.
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ACKNOWLEDGEMENTS |
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Received 31 December 2002;
accepted 22 January 2003.
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