Mutations in poll but not mutSLH destabilize Haemophilus influenzae tetranucleotide repeats
2002; Springer Nature; Volume: 21; Issue: 6 Linguagem: Inglês
10.1093/emboj/21.6.1465
ISSN1460-2075
AutoresChristopher D. Bayliss, Tamsin van de Ven, E. Richard Moxon,
Tópico(s)Viral Infections and Immunology Research
ResumoArticle15 March 2002free access Mutations in poll but not mutSLH destabilize Haemophilus influenzae tetranucleotide repeats Christopher D. Bayliss Corresponding Author Christopher D. Bayliss Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU UK Search for more papers by this author Tamsin van de Ven Tamsin van de Ven Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU UK Search for more papers by this author E. Richard Moxon E. Richard Moxon Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU UK Search for more papers by this author Christopher D. Bayliss Corresponding Author Christopher D. Bayliss Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU UK Search for more papers by this author Tamsin van de Ven Tamsin van de Ven Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU UK Search for more papers by this author E. Richard Moxon E. Richard Moxon Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU UK Search for more papers by this author Author Information Christopher D. Bayliss 1, Tamsin van de Ven1 and E. Richard Moxon1 1Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1465-1476https://doi.org/10.1093/emboj/21.6.1465 Correction(s) for this article Mutations in polI but not mutSLH destabilize Haemophilus influenzae tetranucleotide repeats15 August 2002 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Haemophilus influenzae (Hi), an obligate upper respiratory tract commensal/pathogen, uses phase variation (PV) to adapt to host environment changes. Switching occurs by slippage of nucleotide repeats (microsatellites) within genes coding for virulence molecules. Most such microsatellites in Hi are tetranucleotide repeats, but an exception is the dinucleotide repeats in the pilin locus. To investigate the effects on PV rates of mutations in genes for mismatch repair (MMR), insertion/deletion mutations of mutS, mutL, mutH, dam, polI, uvrD, mfd and recA were constructed in Hi strain Rd. Only inactivation of polI destabilized tetranucleotide (5′AGTC) repeat tracts of chromosomally located reporter constructs, whereas inactivation of mutS, but not polI, destabilized dinucleotide (5′AT) repeats. Deletions of repeats were predominant in polI mutants, which we propose are due to end-joining occurring without DNA polymerization during polI-deficient Okazaki fragment processing. The high prevalence of tetranucleotides mediating PV is an exceptional feature of the Hi genome. The refractoriness to MMR of hypermutation in Hi tetranucleotides facilitates adaptive switching without the deleterious increase in global mutation rates that accompanies a mutator genotype. Introduction High frequency gain and loss of the expression of virulence determinants is an intrinsic feature of many bacterial pathogens. This reversible switching of surface antigens, referred to as phase variation (PV), was originally coined to describe the switching of Salmonella flagella antigens (Andrewes, 1922). This process is stochastic and driven by highly mutable loci that generate repertoires of phenotypic variants from which the fittest variant is selected. These hypermutable loci have been called contingency loci to explain their role in facilitating bacterial adaptation to the dynamic and unpredictable changes in the host environment (Moxon et al., 1994). There are many mechanisms of hypermutation in contingency loci, but one of the commonest is mediated by changes in the number of repeats within simple sequence repeat tracts (microsatellites) located in either the promoter or open reading frame (ORF) of a gene (Moxon et al., 1994; Bayliss et al., 2001). These loci, referred to as simple sequence contingency loci, are found in comparatively high numbers in the genomes of many pathogenic bacteria, including Haemophilus influenzae (Hi), Neisseria meningitidis (Nm), Helicobacter pylori (Hp) and Campylobacter jejuni (Cj) (Hood et al., 1996; Saunders et al., 1998, 2000; Parkhill et al., 2000). A critical role in pathogenesis has been proposed for some of these loci (see Saunders et al., 1998; Bayliss et al., 2001; Linton et al., 2001). The rate of PV of contingency loci is predicted to be a major factor controlling the genetic diversity of bacterial populations (De Bolle et al., 2000) and this diversity may have a major influence on fitness and virulence (Bayliss et al., 2001). Thus, an understanding of the processes that control the mutation rates of simple sequences in these organisms is central to unravelling the contribution of phase-variable genes to the biology of these bacteria. Hi is a ubiquitous commensal of the human upper respiratory tract, but also includes strains with the potential to cause invasive diseases such as meningitis. Hi can phase vary a number of its surface determinants including many of the carbohydrate epitopes of its lipopolysaccharide (LPS). LPS is a major pathogenic determinant of this bacterium and the PV of parts of this molecule is likely to have an impact on the ability of Hi to cause disease (Hood and Moxon, 1999). The genome of strain Rd contains 12 tetranucleotide repeat tracts of >6 repeat units, all of which are within ORFs and most of which modulate expression of surface components (Hood et al., 1996). Additionally, some Hi strains have loci containing penta- and heptanucleotide repeat tracts (van Belkum et al., 1997; Dawid et al., 1999). Surprisingly, only one locus, hif, has a dinucleotide repeat tract (van Ham et al., 1993) and none have mononucleotide repeat tracts. In contrast, many of the simple sequence contingency loci of other phase-variable bacteria, notably Neisseria sp. and Hp (Saunders et al., 1998, 2000), contain mononucleotide repeat tracts. PV rates are known for a number of the simple sequence contingency loci of Hi, and rates appear to correlate with the length of the repeat tract (De Bolle et al., 2000). Few studies, however, have looked at the effect of trans-acting factors on PV rates in Hi and the only finding to date is that recA mutations do not have an effect (Dawid et al., 1999). Studies of simple sequence contingency loci in other PV bacteria have indicated roles for Rho transcription termination factor (Lavitola et al., 1999) and mismatch repair genes, mutS and mutL (Richardson and Stojiljkovic, 2001), in controlling PV rates of Nm. Mutation rates of simple sequences, termed microsatellites, have been examined in detail in yeast, using both artificial and native sequences, and in Escherichia coli (Ec), using reporter constructs containing artificial microsatellites. These studies have implicated roles for mismatch repair (MMR), proof-reading by the replicative DNA polymerase, processing of Okazaki fragments, and other pathways, in modulating the mutation rates of microsatellites with unit lengths of 1–3 nucleotides (Sia et al., 1997; Strauss et al., 1997; Tran et al., 1997; Kokoska et al., 1998; Morel et al., 1998). In the few studies of tetranucleotide repeat tracts, mutation rates of these tracts are increased by MMR mutations in yeast (Sia et al., 1997), but not in Ec (Eckert and Yan, 2000). We have investigated the effect of trans-acting factors on mutation rates of tetranucleotide repeat tracts in Hi, an organism in which such tracts are the major mechanism of PV. We show that mutations in polI, but not seven other genes whose products are involved in DNA metabolism, destabilize tetranucleotide repeat tracts of PV reporter constructs and increase deletions of repeats in these tracts. Results Construction and analysis of Hi DNA metabolism mutants Potential trans-acting factors for PV in Hi were identified by considering effects of trans-acting mutations on the mutation rates of microsatellites in other systems. Based on this analysis, we constructed mutations in the Hi homologs (Table I) of the following Ec genes: MMR genes (mutS, mutL, mutH and dam), a nucleotide excision repair gene (uvrD), a transcription-coupled repair factor (mfd), a recombinase (recA), Pol I (polI or polA), a replication/repair-associated DNA helicase (rep), the proof-reading subunit of Pol III (dnaQ) and the SOS repressor (lexA). Insertion mutations were made in recA and uvrD by inserting an antibiotic cassette into a restriction site in the central part of the cloned gene (Table I). Insertion/deletion mutations were made in the other genes by cloning 5′ and 3′ fragments of the genes that were not co-linear and inserting an antibiotic cassette between them (Table I). Hi strain Rd was transfected with these clones and transformants were isolated in which the wild-type (wt) chromosomal gene was replaced with the mutated gene for eight of the genes, but not for rep, dnaQ and lexA, possibly because mutations in these genes are lethal in Hi. Table 1. Oligonucleotide primers used to construct mutations in DNA metabolism genes of Hi HI gene numbera E.coli homologue (% similarity)a Insertion site of antibiotic cassetteb (size of deletion) Sequencesc 0707 mutS (84%) 1288–1345 (57 bp) 5′CCGAATTCCAACAACATACGCCAATG [19–39] 5′CGAAGCTTCAGCAATCACGCCACC [1288–1270] 5′CGAAGCTTGGAAAACCTAGAAAAACG [1345–1364] 5′CGCCAAGGCTTGTTTAGGGC [2553–2534] 0067 mutL (67%) 904–929 (25 bp) 5′CCGGATCCTATTAAAATTCTTTCCCC [4–23] 5′CGAAGCTTGACATCTACATCGTGCGG [904–883] 5′CGAAGCTTTCATCAACAACGCCTC [929–948] 5′CGGAATTCGTCTAATAAAGGCTGCC [1884–1865] 0403 mutH (81%) 355–475 (120 bp) 5′CGGAATTCTCAAGCACATGCAAATC [(−)664–(−)645] 5′CGAAGCTTCAATGGGCATCCAAAG [355–337] 5′CGAAGCTTGGCSAAATTAGATCAAATTAC [475–497] 5′CCGGATCCAATTTTACGCCCTTTGTTAG [1495–1474] 0209 dam (71%) 530–551 (21 bp) 5′CCGGATCCCTCGTGTTAAACGCAATTC [(−)45–(−)25] 5′CGAAGCTTTCACAGCATAAAAACACCGC [530–511] 5′CGAAGCTTGCCGATAAGGATTCGGTG [551–570] 5′TTTTATGGGGCAGAATTCGC [1419–1400] 0856 polI (77%) 1049–1715 (666 bp) 5′CGGAATTCACAAGTACAGCCCACTC [151–169] 5′CGAAGCTTTTCAATCCAGCGGG [1049–1031] 5′CAATCTAACGAAATTGCCTC [1672–1691] 5′CCGGATCCCAATTTTGTCCAACACC [2781–2761] 1188 uvrD (83%) 1106d 5′CCGAATTCCGCCTAAACAGGCTTG [425–443] 5′CCGGATCCACATTAATCACTGTGCC [2087–2068] 0600 recA (86%) 521d 5′CGGGATCCCCATCCTCTTTACTGAA [(−)123–(−)104] 5′TTATTGCGGAGCTCGAATTC [1220–1201] 1258 mfd (83%) 1915–1987 (72 bp) 5′GCCGGAGCAAACGCTGTTTG [831–850] 5′CGAAGCTTCAGTTTTACCAAAACCC [1915–1896] 5′CGAAGCTTAGCCCAACAGCATTATG [1987–2005] 5′CGGAATTCTGCTTTACTCTCTGC [3122–3103] 0137 dnaQ (77%) 250–951 (701 bp) 5′CGGGATCCAATCGCCAAAACGGACG [(−)719–(−)698] 5′CGAAGCTTGGGCAACTTCTTTGAATTC [250–229] 5′CGAAGCTTATCTCGGAGTGGTAGTTC [951–971] 5′CGCATCATCAATTAATGCCG [2070–2051] 0649 rep (83%) 818–834 (16 bp) 5′CGGGATCCTCAACAACAACAAGCC [11–30] 5′CGAAGCTTAATCACGTTCAAACG [818–799] 5′CGAAGCTTCAACCCAGCGTATTTTGC [834–853] 5′TGCCCTCCTCCATACCAATC [1732–1713] 0749 lexA (85%) 37–276 (239 bp) 5′CCGAATTCTGAGCCATGTTCAACTACC [(−)802–(−)782] 5′CTAAGCTTCACTTCTTGTTGGCGTGCGG [33–14] 5′CGAAGCTTGCAGAGCAGCATATTGAAGC [277–299] 5′CGGGATCCGAAACTGCACCACATAGTAC [1227–1208] 0855 b3109 (75%) 160–187 (27 bp) 5′CGGAATTCCAATCTAACGAAATTGCCTC [(−)1136–(−)1117] 5′CGGATATCTTGCTTCAATGCGTTCAGGTG [160–140] 5′CGGATATCGGATTGGGCGTTGCTGTGCC [187–206] 5′CCGGATCCAGCCCATTTGGCTATGACGC [1472–1452] a Gene numbers, homologous Ec K12 genes and percentage similarities are taken from the TIGR Microbial database (http://www.tigr.org) and their analysis of the Hi Rd genome sequence. b Numbers indicate nucleotide positions relative to the initiation codon. c Primers are ordered 1–4 from top to bottom for each gene. Most of the 1 and 4 primers contain either EcoRI or BamHI restriction sites at the 5′ end, and most of the 2 and 3 primers contain HindIII restriction sites at the 5′ end. In the cases where these sites are lacking in the primers, or only two primers were used, restriction sites in the native sequences were utilized. Numbers in parentheses indicate the position of the primers relative to the initiation codon for the gene, with negative numbers indicating a position that is 5′ to this codon. d In these cases, the tetracycline cassette was inserted at this position and no deletion was made. Growth curves were performed with these eight mutants and, apart from polI mutants, doubling times were similar to strain Rd (data not shown). RdΔpolI mutants exhibited, relative to strain Rd, up to 3-fold higher doubling times, 2- to 3-fold lower plating efficiencies (i.e. c.f.u./OD490 unit), but similar amounts of protein per OD490 unit (data not shown). These results indicated that RdΔpolI mutants were forming filaments with an average size of 2–3 cells/filament and examination under phase contrast light microscopy revealed the presence of filaments ranging up to 20 μm in length (single cells being ∼1 μm in length). A similar phenotype has been observed when Ec polI null mutants are grown on rich media (Joyce and Grindley, 1984). RdΔpolI mutants also exhibited heterogeneity in colony size and elongated growth times for colonies on plates. Global mutation rates were measured for the Hi mutants by measuring the frequency of generation of nalidixic acid-resistant variants (Table II). The mutS, mutL and mutH mutants exhibited 3- to 4-fold higher mutation rates than strain Rd. These rates were significantly different as shown by the non-overlapping confidence intervals (Table II). Higher rates for mutSLH mutants were expected because their products are involved in MMR, but the absence of an increase in rates for dam mutants was unexpected and suggests that MMR in Hi may not follow the canonical Ec model. The 2.5-fold higher rate for the polI mutant was not significant but may indicate that Pol I has a critical role in error correction during DNA replication. Table 2. Spontaneous mutation rates of Hi mutant strains Strain NalR mutation rate (× 10−9)a Rd 1.79 (1.0) {2.57–0.99} RdΔmutS 6.9 (3.9) {13.15–2.62} RdΔmutL 6.09 (3.4) {14.8–3.74} RdΔmutH 6.86 (3.8) {12.55–3.62} RdΔpolI 4.54 (2.5) {25.51–1.59} RdΔrecA 2.9 (1.6) {4.28–2.38} RdΔmfd 0.88 (0.5) {1.82–0.62} RdΔuvrD 0.98 (0.5) {2.98–0.58} RdΔdam 1.13 (0.6) {3.54–0.93} a Frequencies of nalidixic acid resistance were determined for at least 10 colonies of Rd and each of the mutant strains. Mutation rates were derived using the median frequency according to Drake (1991) and are expressed as the number of nalidixic acid-resistant bacteria produced per cell per division. Numbers in parentheses are the fold increase over wild type. Numbers in curly brackets are 95% confidence intervals calculated as described by Kokoska et al. (1998). Tetranucleotide repeat-mediated PV rates of Hi DNA metabolism mutants Most of the simple sequence contingency loci in Hi contain tetranucleotide repeat tracts. The influence of trans-acting factors on this major mechanism of PV in Hi was investigated by transferring the mutations described above into strains carrying mod–lacZ fusions with tetranucleotide repeat tracts of 38 or 17 5′AGTC repeat units (R), RdGΔZ38R and RdGΔZ17R, respectively (De Bolle et al., 2000), and measuring the switching rates of these constructs (Table III). Switching from ON-to-OFF occurred at rates similar to those of the parental constructs for all the mutants, including those in MMR genes, except polI mutants which exhibited 30- and 49-fold higher rates than the parental constructs (Table III). The fact that these rates were significantly different from those of parental constructs was shown by non-overlapping confidence intervals (Table III) and a statistical analysis, which yielded P <0.0001 (using a Mann–Whitney non-parametric rank sum test) for comparisons between frequencies of variants of polI mutants and equivalent parental strains. The possibility that this increase was due to the polI mutation interfering with expression of downstream genes was investigated by constructing a mutation in hi0855 (Table I). This gene encodes a conserved hypothetical protein, and its initiation codon is 15 bp 3′ to the termination codon of polI. PV rates for the RdΔhi0855GΔZ38R mutants were similar to those of parental constructs (Table III), indicating that the increased rates observed with polI mutants were not attributable to polar effects on hi0855. Pleiotrophic effects are common in DNA replication mutants and Ec polI mutants are known to induce the SOS response (Morel et al., 1998). As RecA protein is essential for induction of the SOS response (for a review see Little, 1993), we investigated a possible role of the SOS response by attempting to inactivate recA in RdΔpolI strains. Strains were transformed with pUCΔrecAchl and two mutants were obtained, RdΔpolIΔrecAchlGΔZ17R and RdΔpolIΔrecAchlGΔZ29R. These mutants exhibited ON-to-OFF PV rates similar to the parental polI mutants (Table III) suggesting that SOS induction in these mutants is not responsible for the elevated tetranucleotide- mediated PV rates. Table 3. Influence of mutations in DNA metabolism genes on the rates of PV of tetranucleotide repeat tracts in Hi Relevant genotypea Direction of switching ON-to-OFF OFF-to-ON No. of 5′AGTC repeats Mutation rate (× 10−4)b No. of 5′AGTC repeats Mutation rate (× 10−4) No. of 5′AGTC repeats Mutation rate (× 10−4) No. of 5′AGTC repeats Mutation rate (× 10−4) Wt 38 2.03c {3.41–1.44} [1.00] 17 0.54c {0.70–0.29}[1.00] 37 1.24c {1.36–0.75} [1.00] 18 0.36c {0.48–0.18} [1.00] ΔmutS 38 2.45 {3.49–1.88} [1.21] 17 0.64 {0.83–0.46} [1.19] 37 0.84 {1.31–0.70} [0.68] ΔmutL 38 2.28 {2.85–2.69} [1.12] 17 0.41 {0.64–0.24} [0.76] 37 0.93 {1.19–0.73} [0.74] ΔmutH 38 1.88 {3.60–1.67} [0.93] 17 0.61 {0.79–0.42} [1.12] 37 1.83 {2.97–1.24} [1.48] ΔpolI 35 62.16 {72.6–45.6} [30.6] 17 26.53 {43.5–13.3} [49.1] 37 11.66 {17.5–7.23} [9.40] 21 16.01 {25.1–11.1} [44.5] 31 14.05 {19.4–12.4} [11.33] 16 4.87 {18.3–2.16} [13.5] ΔrecA 41 2.52 {3.94–1.73} [1.24] 17 1.09 {1.65–0.86} [2.01] 37 1.47 {2.12–1.09} [1.18] 35 3.17 {26.8–1.70} [1.56] Δmfd 38 2.49 {2.88–2.23} [1.23] 17 0.52 {0.83–0.37} [0.97] 37 0.91 {8.01–0.78} [0.74] Δdam 38 3.13 {3.51–2.59} [1.54] 17 0.87 {1.09–0.37} [1.60] 39 4.33 {5.22–1.85} [3.50] 36 3.89 {6.81–2.15} [3.14] ΔuvrD 38 3.59 {3.99–2.71} [1.77] 17 0.60 {0.84–0.37} [1.11] 37 1.61 {2.65–1.27} [1.30] Δhi0855 38 2.20 {18.3–1.50} [1.08] 17 0.52 {1.46–0.33} [0.96] 37 0.70 {0.88–0.58} [0.56] 35 1.33 {21.2–0.70} [0.87] ΔpolI ΔrecA 29 55.0 {145.9–45.1} [27.1] 17 34.6 {40.3–27.7} [64.1] a Phase variation reporter strains carrying mutations in other genes were constructed by transforming RdGΔZ38R or RdGΔZ17R (i.e. Hi strains carrying in-frame mod–lacZ fusions containing 38 or 17 5′AGTC repeats) with plasmid constructs, pUCΔmutS, pUCΔmutL, pUCΔmutH, pUCΔpolI, pUCΔrecA, pUCΔmfd, pUCΔdam, pUCΔuvrD or pUCΔhi0855, which all contain a tetracycline resistance cassette. PolI/recA double mutants were constructed by transforming RdGΔZ35RΔpolI or RdGΔZ17RΔpolI with pUCΔrecAchl, which contains a chloramphenicol resistance cassette. Out-of-frame or OFF variants were derived from these strains. b Mutation rates were derived from the median frequency of variants by the method of Drake (1991). Median frequencies were determined from the analysis of at least 16 colonies for most of the strains apart from the following, in which eight were analysed: 35R and 41R ΔrecA; 37R, 31R, 21R and 16R ΔpolI; 37R Δmfd; 39R and 36R Δdam; 38R, 35R and 17R Δhi0855; and 38R ΔpolIΔrecA. Numbers in curly brackets are 95% confidence intervals calculated according to Kokoska et al. (1998). Fold increase in mutation rates relative to the parental rates are given in square brackets. c Values as reported previously by De Bolle et al. (2000). Rates of switching from OFF-to-ON were also measured for variants of all the mutants (Table III) and elevated rates were only observed for OFF variants of RdΔpolIGΔZ38R, being 9- and 11-fold higher than parental rates for constructs with 37 and 31 repeats, respectively (Table III). These data indicated that the polI mutation also increased OFF-to-ON switching rates, and further evidence for this result was obtained by measuring switching from OFF-to-ON for variants derived from constructs containing 17 repeats. Rates were 13- and 44-fold higher for constructs with 16 and 21 repeats, respectively, than those derived for RdGΔZ18R (Table III). These rates for OFF-to-ON switching of polI mutants were significantly different from those of equivalent parental constructs (P <0.0005 for constructs with 37 or 31 repeats and P <0.002 for constructs with 21 or 16 repeats, calculated as above). Ratios of ON-to-OFF to OFF-to-ON PV rates for Rd mod/lacZ reporter strains with repeat tracts of equivalent lengths (38R:37R, 32R:31R, 23R:24R and 17R:18R; mutation rates were assumed to be independent of repeat number) occur within a narrow range, 1.6:1–1.5:1 (De Bolle et al., 2000). For RdΔpolI reporter strains, higher ratios were observed with OFF-to-ON switching rates derived from OFF variants, in which the insertion of a single repeat unit produced a mod/lacZ ORF (i.e. 5.3:1 for 35R:37R, 4.4:1 for 35R:31R and 5.4:1 for 17R:16R; derived from the data in Table III) whereas parental ratios were obtained if deletion of a single repeat unit produced a mod/lacZ ORF (i.e. 1.7:1 for 17R:21R). The three-fold difference between these ratios indicated that the ∼40-fold increase in ON-to-OFF switching rates of RdΔpolI reporter strains (Table III) was due to 10- and 30-fold increases, respectively, in the rates of insertions and deletions. This phenomenon was investigated further by analysing alterations in repeat tracts. Switching in Rd mod/lacZ reporter strains correlates with changes in the number of tetranucleotide repeat units; 90% of ON-to-OFF phase variants exhibiting single repeat unit changes and the ratio of deletions to insertions being 2:1 (De Bolle et al., 2000). Alterations in the repeat tracts of variants derived from mutant strains were investigated and all variant colonies had alterations in the length of the repeat tract with the proportion of one repeat unit alterations in ON-to-OFF variants being similar to the parental strains (Figure 1). However, compared with equivalent Rd strains, RdΔpolI mod/lacZ reporter strains exhibited a significantly greater number of deletions (Figure 1). The ratio of deletions to insertions for ON-to-OFF switching (Figure 1A) was 23:1 for RdΔpolI GΔZ35R, which was significantly higher than the 1.6:1 ratio for RdGΔZ38R (by Fisher's exact test P = 0.005, Odds ratio 9.47, 95% confidence intervals 1.79–50.18). The ratio for RdΔpolIGΔZ17R was 10:1 (data not shown), which was not significantly different from the 4:1 ratio of RdGΔZ17R (De Bolle et al., 2000) but indicated that 2.5-fold more deletions had occurred in this strain. This value was closer to the 3-fold increase in deletions over insertions predicted for the polI mutants from the PV rates (see above). This result may indicate either that the higher increase (14-fold) in deletions observed with constructs with 38 repeats was an overestimation or that the increase in the number of deletions may decrease as the length of the repeat tract was reduced. Values for the other mutants ranged from 1:1 to 6:1 (Figure 1A) and 2:1 to 7:1 (data not shown) for constructs with 38 and 17 repeats, respectively, and were not significantly different from those of equivalent Rd strains. Figure 1.Influence of trans-acting factors on the types of alterations occurring in tetranucleotide repeat tracts in Hi. Alterations in the 5′AGTC repeat tracts of phase-variant colonies were classified as either insertions or deletions of the repeat units (i.e. an increase or decrease, respectively, in the number of repeat units) and as changes of one, two or more repeat units. The numbers of variants in each class were expressed as a percentage of the total number examined. Relevant genotypes and total number of variants examined for (in parentheses) each strain are indicated below each column. (A) ON-to-OFF switching. Parental colonies contained 38 (wt and most of the mutants) or 35 (ΔpolI) 5′AGTC repeats. Data for the ΔrecA mutants is the combined data for parental colonies containing either 41 or 35 5′AGTC repeats. (B) OFF-to-ON switching in which the +1 reading frame produces a Mod–LacZ fusion protein. Parental colonies contained, in most cases, 37 5′AGTC repeats. Data for the wt and ΔpolI strains is the combined data for parental colonies containing either 37 or 31 repeats [in the former case, the data were reported previously (De Bolle et al., 2000)]. Download figure Download PowerPoint Ratios of deletions to insertions were also determined for OFF variants that required a +1 or a −2 repeat unit shift to produce a Mod–LacZ fusion protein (Figure 1B). The ratio was 6:1 for polI mutants (combined data for RdΔpolGΔZ34R and RdΔpolGΔZ31R), which was significantly different from the parental ratio of 1:2 (combined data for strains RdGΔZ37R and RdGΔZ31R, De Bolle et al., 2000; P = 0.0022, Odds ratio 12.25, 95% confidence intervals 2.5–61.6). Ratios for the other mutants ranged from 1:2 to 1:6 (all 37R; see Figure 1B) and were not significantly different from the parental ratio. The ratio for RdΔpolGΔZ16R, for which there was no comparable parental data, was 3:1 (data not shown). Finally, a ratio was determined for OFF variants of polI mutants containing 21 repeats that required a +2 or −1 shift. This ratio of 16:1 (data not shown) was not significantly higher than the parental ratio of 7:1 (combined data for strains RdGΔZ24R and RdGΔZ18R from De Bolle et al., 2000) but indicated that there was a 2-fold increase in deletions. Inactivation of mutS increases Hi dinucleotide repeat-mediated PV rates Since PV of the Hi pilus is driven by a dinucleotide repeat tract consisting of nine 5′TA repeats (van Ham et al., 1993) and MMR mutations are known to increase the mutation rates of such tracts in other organisms, it was of interest to see whether PV rates mediated by dinucleotide repeat tracts were affected by MMR mutations in Hi. A mod–lacZ fusion carrying 20 5′AT dinucleotide repeat units was constructed and transferred into strains Rd, RdΔmutS and RdΔpolI. PV rates in RdGΔZ–AT20R strains (Table IV) were within the range of rates observed for the RdGΔZ strains with 23 and 17 5′AGTC repeats (Table III; De Bolle et al., 2000), suggesting that dinucleotide and tetranucleotide repeat tracts, containing similar numbers of repeat units, phase vary at similar rates in Hi. Rates for RdΔmutS dinucleotide mod/lacZ reporter strains were significantly higher than those of control strains for both directions of switching (Table IV; P <0.0001, calculated as for the tetranucleotide repeat tracts). Rates for the RdΔpolI mutants were not significantly different from the controls (Table IV). Thus, dinucleotide repeat tracts were destabilized by loss of MMR but not polI. The types of mutational events producing variants were analysed as described above. Interestingly, for the ON-to-OFF direction of switching, the Rd and RdΔpolI dinucleotide mod/lacZ reporter strains exhibited ∼2-fold more two, rather than one, repeat unit changes, whilst in equivalent ΔmutS strains, all the changes were by a single repeat unit (Figure 2). One possible explanation is that dinucleotide repeat tracts form 4 bp single-stranded (ss) DNA loops (i.e. two unpaired repeat units) that cannot be corrected by MMR. These results also indicate that a large number of mutational events occur in dinucleotide repeat tracts, which are corrected by MMR. Dinucleotide repeat tracts may, therefore, be more prone to mutation than tetranucleotide repeat tracts. Figure 2.Types of alterations occurring in dinucleotide repeat tracts in Hi strains carrying mutations in mutS or polI. Alterations were analysed and are presented as described for Figure 1. Relevant genotypes of and total number of variants examined for (in parentheses) each strain are indicated below each column. (A) ON-to-OFF switching. The parental colonies contained 20 5′AT repeats. (B) OFF-to-ON switching in which either the +1 (columns 1–3) or −1 (column 4) reading frame produces a Mod–LacZ fusion protein. Parental colonies contained 22 5′AT (wt), 19 5′AT (ΔmutS or ΔpolI, column 3) or 18 5′AT (ΔpolI, column 4) repeats. Download figure Download PowerPoint Table 4. Influence of mutations in DNA metabolism genes on the rates of PV of dinucleotide repeat tracts in Hi Relevant genotypea Direction of switching ON-to-OFF OFF-t
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