Paediatric Molecular Infectious Diseases Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
Correspondence
Wendy A. Sweetman
wendy.sweetman{at}paediatrics.ox.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Haemophilus influenzae is a Gram-negative, obligate commensal of the upper human respiratory tract with the potential to cause diseases such as meningitis, septicaemia, epiglottitis, pneumonia and otitis media. H. influenzae has microsatellite regions associated with the promoter or 5' coding region of several contingency loci that have roles in virulence and disease. A dinucleotide (TA) repeat tract is present in the promoter region of the pilin locus (van Ham et al., 1993), heptanucleotide repeats are associated with the HMW adhesin genes (Dawid et al., 1999
), whilst tetranucleotide repeat sequences are present in loci whose functions include iron acquisition, biosynthesis of LPS epitopes and a putative type III restrictionmodification (mod) gene (Hood et al., 1996
).
Several studies have sought to determine factors that affect the frequency of replication slippage in microsatellite sequences. Cis factors (number of repeats) and trans factors (Pol I, RNase H, mismatch repair mechanisms) have been shown to have an effect on phase variation frequencies in H. influenzae (De Bolle et al., 2000; Bayliss et al., 2002
, 2004
, 2005
). Interestingly, Morel et al. (1998)
showed that, in Escherichia coli, induction of the SOS response greatly influenced the replication slippage rates of a dinucleotide repeat tract, increasing slippage rates by 8- to 22-fold, depending on the repeat sequence.
The SOS response was first described in E. coli and is a temporary, co-ordinated increase in transcription of a network of genes, the products of which are involved in DNA repair, replication restart and cell-division control. More than 40 genes have now been shown to be induced during the SOS response in E. coli (Fernández de Henestrosa et al., 2000; Courcelle et al., 2001
; Khil & Camerini-Otero, 2002
). Two proteins, RecA, the positive regulator, and LexA, the repressor, control induction of the SOS response. Single-stranded (ss) DNA accumulates at stalled replication forks following DNA damage (Sassanfar & Roberts, 1990
) and is bound by RecA, forming a nucleoprotein filament that then stimulates LexA to undergo self-cleavage (Little, 1993
). Under non-inducing conditions, LexA binds within the promoter regions of genes to a site referred to as the SOS box (Little & Mount, 1982
; Walker, 1984
).
Recently, Erill et al. (2003) looked in silico for the presence of SOS boxes in the published genome sequences of several gammaproteobacterial species. Only three genes (lexA, recA and recN) were found to have an SOS box present in the promoter region in all the organisms analysed, indicating that, although the SOS box sequence and RecA/LexA controlling mechanism are conserved within this subclass, the number of genes included in the LexA regulon varies considerably such that individual species may differ in the way in which they survive and repair DNA damage.
Zulty & Barcak (1993) showed that the recA gene of H. influenzae was induced approximately 3-fold in response to SOS-activating signals, but no detailed studies of the LexA regulon of H. influenzae have been performed. Some differences between the SOS regulons of H. influenzae and E. coli are apparent. For example, the SOS repair mechanisms of E. coli are associated with increased mutational frequencies due to the induction of three trans-lesion synthesis (TLS) polymerases, Pol II, IV and V, respectively encoded by the polB, dinB and umuDC genes (Napolitano et al., 2000
; Wagner et al., 2002
). Notani & Setlow (1980)
noted that prophage induction could be stimulated in H. influenzae by exposure to UV light but, in contrast to E. coli, with no associated increase in mutation rates. Annotation of the entire H. influenzae genome sequence (Fleischmann et al., 1995
) reveals the apparent absence of any TLS polymerases.
The aim of this work was to determine whether or not induction of the SOS response influences phase variation rates in H. influenzae. In order to achieve this aim, the published H. influenzae Rd KW20 genome sequence (Fleischmann et al., 1995) was first screened for genes that have the E. coli minimal consensus SOS-box sequence CTG(N10)CAG in their promoter region in order to identify genes with the potential to be LexA regulated. Secondly, a non-inducible LexA mutant (lexANI) was constructed and shown to have increased sensitivity to both irradiation with a UV light source and treatment with mitomycin C (mitC). Thirdly, mRNA levels of candidate SOS genes in wild-type (WT) RM118 and lexANI mutant strains of H. influenzae were analysed following exposure to UV and mitC, confirming the presence of a LexA-dependent SOS response in WT H. influenzae strains and disabling of the SOS response in the engineered mutants. Finally, we investigated whether induction of the SOS response in H. influenzae had an effect on phase variation events, mediated by slippage at either dinucleotide (AT) or tetranucleotide (AGTC) repeat tracts.
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METHODS |
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Screening of the H. influenzae genome sequence for LexA-binding boxes.
Each occurrence of the minimal E. coli SOS-box consensus sequence, CTG(N10)CAG, in the genome sequence of H. influenzae Rd KW20 was determined from a search of the published sequence (Fleischmann et al., 1995) using a search algorithm within an Artemis database. Each SOS box was checked to determine its position relative to the nearest open reading frames (ORFs) in order to assign it as being located in a promoter (here defined as being within 200 bp upstream of an initiation codon), coding or non-coding region.
Generation of LexANI mutants and reporter strains.
The lexA gene, together with approximately 800 bp of 5' DNA and 950 bp of 3' DNA, was amplified from strain RM118 genomic DNA as two fragments using the pairs of primers lex1EcoRI/lex5HindIII and lex6HindIII/lex7BamHI, respectively (Table 1; Fig. 1a
). The HindIII sites generated by primers lex5HindIII and lex6HindIII were restricted with HindIII and the two fragments were ligated together to generate the full-length gene construct with an engineered HindIII site immediately downstream of the stop codon of the lexA coding region. This full-length gene construct was then restricted with the appropriate enzymes and cloned into the EcoRI and BamHI sites of plasmid pUC19. A HindIII fragment containing a tetracycline-resistance cassette (derived from plasmid pHVT1; Danner & Pifer, 1982
) was then inserted into the engineered HindIII site generating plasmid pLexAWTtet.
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pLexAG91Dtet was linearized by digestion with SalI and used to transform strain RM118 by the method of Herriott et al. (1970). Transformants were selected on BHI tetracycline plates and allelic replacement of the WT lexA gene with the mutated lexANI allele was confirmed by PCR, Southern analyses and DNA sequencing (data not shown). Two independent lexANI mutants, G91D-1 and G91D-2, were selected for use in these studies. Reporter plasmids containing the coding region of the H. influenzae mod that is 5' of the repeat site, fused, in-frame, to a repeat region consisting of AGTC or AT repeats fused, in turn, to a lacZ gene have been described elsewhere (De Bolle et al., 2000
; Bayliss et al., 2002
). Slippage at the repeat tracts in these constructs results in gain or loss of expression from the lacZ gene. The lexANI strains G91D-1 and G91D-2 were further transformed with reporter plasmids that contained either 38 AGTC or 20 AT repeats in order to create reporter strains for phase variation experiments that also carried the lexANI mutation. The plasmids were linearized by digestion with SalI prior to transformation. Transformants G91D-1AGTC38, G91D-1AT20, G91D-2AGTC38 and G91D-2AT20 were selected for use in phase variation experiments.
Determination of UV sensitivity.
Overnight bacterial cultures were diluted 10-fold in BHI broth and grown to mid-exponential phase (OD650 0·20·4). Cells were diluted 1 : 105 in BHI and 2550 µl aliquots were spread onto BHI plates. Following recovery at 37 °C for 1 h, plates were exposed, with lids off, to a model UV6-54 mineralight lamp (UV Products Inc.) 254 nm UV light source at a distance of 16 cm for 15 s (calibration using a Blak-Ray model J-22S short-wave UV meter showed this to be equivalent to a dose of 4 J m2 s1). The number of colonies that survived the exposure to UV light was counted after overnight incubation at 37 °C and percentage survival was calculated relative to the number of colonies obtained from unexposed samples of the same cultures.
mitC-sensitivity experiments.
Overnight cultures were diluted 10-fold to a final volume of 20 ml BHI broth, incubated for 2 h at 37 °C (estimated to be mid-exponential phase) and split into four 5 ml aliquots. A 2 mg mitC ml1 stock in DMSO was diluted to a working concentration of 0·02 mg ml1 in DMSO and added to the split cultures to give final concentrations of 0, 0·1, 0·05 and 0·01 µg ml1. Cultures were then incubated at 37 °C for 30 min, serial dilutions were prepared and aliquots of 104 and 105 dilutions were plated onto BHI plates. The number of colonies that survived treatment was counted after overnight incubation at 37 °C and percentage survival was calculated relative to the number of colonies in the untreated sample.
RT-PCR analysis of H. influenzae SOS genes.
Induction of the SOS response, prior to RNA extraction and RT-PCR, was achieved by exposure to a UV light source or treatment with mitC as follows. For UV exposure, overnight bacterial cultures were diluted 10-fold in 20 ml BHI broth, incubated at 37 °C and harvested at mid- to late-exponential phase (OD650 0·40·8) by centrifugation at 1800 g for 10 min in a Sorvall RT7 centrifuge. The cell pellet was resuspended in 12 vols ice-cold PBS (10 µM phosphate buffer, 27 µM KCl, 137 µM NaCl)/10 % (v/v) BHI and 4 ml aliquots of this suspension were put into 8·5 cm Petri dishes and placed on a gently rotating platform beneath the UV light source. The dishes were exposed to UV light (4 J m2 s1), with lids off, for 4 s. Cells were allowed to recover at 37 °C for 20, 30 or 40 min before cells were again recovered by centrifugation as above. Cell pellets were resuspended in 100 µl TE (10 mM Tris/HCl, 1 mM EDTA, pH 7·9) and RNA was extracted using the Promega SV total RNA isolation kit. For mitC treatment, overnight bacterial cultures were diluted and regrown in the same way as for UV exposure. The 20 ml culture was split into two and mitC added to one half to a final concentration of 0·1 µg ml1. Cultures were then incubated at 37 °C for a further 90 min. Aliquots (0·25 ml) from each culture were then taken, pelleted at 13 000 g for 3 min and resuspended in 100 µl TE. RNA was extracted as for UV exposure.
For RT-PCR analysis, 500 ng isolated RNA was reverse-transcribed with 1 µl random primers (500 µg ml1; Promega) and 200 U Moloney murine leukaemia virus reverse transcriptase (Invitrogen) in a 25 µl total reaction volume. The equivalent of 1 µl undiluted cDNA was used in PCRs. PCR amplifications were performed in 1x PCR buffer (Invitrogen) containing 200 µM each of dATP, dCTP, dGTP and dTTP (Invitrogen), 1 µl of a 20 µM dilution of each of the target SOS gene primers and 1 µl of a 6 µM dilution of each of the control gene (frdB) primers (primer sequences are listed in Table 1). Cycling conditions were 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min for 30 cycles. Amplifications were performed on untranscribed RNA samples to monitor for DNA contamination. If DNA was found to be present, RNA samples were treated with DNase I (Promega) and rescreened for DNA contamination prior to PCR amplification.
PCR products were separated on 2 % agarose gels. Gels were post-stained in 0·5 µg ethidium bromide ml1 and band intensities were measured using Imagemaster1D image analysis software. Induction ratios were calculated from gel-band intensity measurements in the following manner. For each gel lane, the target gene band intensity was first divided by the frdB gene band intensity to obtain an expression ratio. Induction ratios were then calculated as the expression ratio of UV-exposed or mitC-treated cells divided by the expression ratio obtained from control, unexposed cells.
Northern blot analysis.
Overnight bacterial cultures were diluted 10-fold in 6080 ml BHI and regrown to mid-exponential phase as before. Each culture was split into two and one half was treated with mitC (0·2 µg ml1) for 1 h. RNA was extracted using an adaptation of the hot acid phenol method (Miller, 1972). In brief, cultures were pelleted at 6000 g at 4 °C for 5 min, washed once in cold PBS/azide (120 mM NaCl, 2·7 mM KCl, 10 mM phosphate buffer, 20 mM NaN3, pH 7·0) and then resuspended in 10 ml lysis buffer (20 mM sodium acetate, 0·5 % w/v SDS, 1 mM EDTA, pH 5·4). Twenty millilitres of phenol equilibrated in lysis buffer and pre-heated to 65 °C was added and mixed thoroughly. The extracts were then centrifuged at 6000 g at 20 °C for 10 min and the upper, aqueous phase was removed to a second clean, sterile tube. One millilitre of 1 M KCl was added with 2 vols ice-cold ethanol. Precipitates were allowed to form at 20 °C for 112 h and then pelleted at 10 000 g at 4 °C for 15 min. Pellets were drained and resuspended in 250 µl water treated with 0·1 % diethyl pyrocarbonate.
RNA (1020 µg; 510 µl) was run on 1·2 % agarose, 3·7 M formaldehyde gels in 1x MOPS buffer (0·4 M MOPS/NaOH, 0·1 M sodium acetate, 10 mM EDTA, pH 7·2) and then transferred to nylon filters. Filters were placed in 25 ml pre-hybridization solution (5x SSPE, 50 % v/v formamide, 5x Denhardt's solution, 1 % w/v SDS, 100 µg heat-denatured salmon sperm DNA ml1) at 42 °C for several hours. DNA probes were synthesized from the appropriate gel-purified PCR product using the Megaprime DNA-labelling system and [-32P]dCTP (Amersham). PCR products were amplified from RM118 genomic DNA using primer pairs listed in Table 1
and the amplification conditions stated above. Hybridization was performed at 42 °C overnight in 10 ml hybridization solution (5x SSPE, 50 % v/v formamide, 5 % w/v dextran sulfate, 5x Denhardt's solution, 1 % w/v SDS). Filters were washed in 2x SSC at 20 °C for 15 min and then in 2x SSC, 0·1 % SDS (w/v) at 65 °C for 30 min before exposure to X-ray film.
Phase variation experiments.
The method for estimating phase variation frequencies using the modrepeatlacZ reporter gene constructs has been described previously (De Bolle et al., 2000; Bayliss et al., 2002
). Adaptations of the published method were as follows: to determine the effects of UV induction, strains to be investigated were plated onto BHI plates containing 40 µg X-Gal ml1 and incubated overnight at 37 °C. A single blue (lacZ-expressing) colony was picked, diluted and plated onto BHI/X-Gal plates. These plates were exposed to a UV light source (4 J m2 s1) for 4 s. Exposure occurred either immediately after or several hours after plating. To determine the effects of mitC induction, the single blue colony that was selected after overnight incubation was diluted and plated onto plates containing 0·01 µg mitC ml1and incubated overnight at 37 °C. The concentration of mitC that was used was determined experimentally to give an observable reduction in colony size but still allow sufficient growth of the colonies for estimates of phase variation to be made. Induction of the SOS response by the mitC treatment was confirmed from RT-PCR analysis of RNA extracts prepared from cells harvested from mitC plates. Harvested cells were resuspended in 100 µl TE and RNA was extracted from them as described above.
Eight colonies were picked from either the UV-exposed or mitC-containing plates after overnight incubation and analysed as described previously (De Bolle et al., 2000; Bayliss et al., 2002
) to allow estimation of phase variation frequencies. In brief, serial dilutions were made from selected colonies and aliquots of the 102105 dilutions were plated. The number of c.f.u. present in the 104 and 105 dilutions was used to estimate total cell number in each of the selected colonies, whilst the total number of revertants was estimated from duplicate platings of the higher dilutions. The frequency of revertants for each of the eight colonies was calculated from the mean number of revertants divided by the total number of cells. The median value and range of frequencies from the eight colonies were recorded and used to calculate mutation rates according to the method of Drake (1991)
and 95 % confidence intervals according to Kokoska et al. (1998)
. The changes in repeat number that resulted in the revertant phenotype were analysed as described previously (Bayliss et al., 2002
). In brief, revertant colonies were picked and the repeat regions were PCR-amplified using fluorescent primers. PCR products were separated and sized by electrophoresis of the fragments using an ABI Prism 377 automatic sequencer and the ABI GeneScan 3.1 program (Perkin-Elmer).
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RESULTS |
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Engineering H. influenzae lexANI mutants
Four amino acid residues are essential for the self-cleavage reaction of LexA in E. coli. These are Ala84 and Gly85 at the cleavage site and Ser119 and Lys156 at the active site (Lin & Little, 1988). Mutation of any of these amino acids can prevent cleavage of LexA and result in mutants showing a non-inducible SOS phenotype (lexANI). The published Rd KW20 lexA sequence shows 67 % identity to the E. coli lexA sequence. The Rd KW20 lexA sequence differs from the E. coli sequence in that the conserved Ala and Gly residues that form the cleavage site are at positions 90 and 91, respectively, of the H. influenzae LexA protein. Sequencing of the lexA gene from strain RM118 used in these studies showed that there was a further polymorphism in this strain relative to the published strain KW20 sequence that would result in the substitution of Arg for Ala at position 89 (the E. coli sequence also has Arg at the equivalent position 83). Primers GD1F and GD1R (Table 1
) were designed to accommodate this polymorphism and were used in site-directed mutagenesis reactions to generate a mutation in lexA that would result in Gly91 at the cleavage site being replaced by Asp (Fig. 1b
). This mutation has previously been described in E. coli, resulting in a non-inducible LexA phenotype (lexANI) (Lin & Little, 1988
). Following site-directed mutagenesis and transformation, two independent lexANI mutants, G91D-1 and G91D-2, in which the WT lexA allele had been replaced by the mutated allele, were selected for use in studies of the H. influenzae SOS response. The allelic replacement was confirmed by PCR, Southern blots and sequencing (data not shown).
Sensitivity of H. influenzae lexANI mutants to DNA damage
Fig. 2 shows that the H. influenzae lexANI mutants were more sensitive than WT cells to both UV exposure and mitC treatment but were less sensitive to these DNA-damaging treatments than recA mutants. The recA mutant was more sensitive (13·5 % survival) than the WT strain RM118 (88·3 % survival) following only 1 s exposure to UV light, whilst the lexANI mutants showed increased sensitivity only after >3 s of exposure, at which time the survival rates were similar to recA mutants (37, 0·5, 0·44 and 0·7 % survival for strains RM118, G91D-1, G91D-2 and recA, respectively). The lexANI and recA mutants were more sensitive to mitC than WT, as shown by both a decrease in the number of surviving colonies and a reduction in the size of the colonies following overnight growth. The difference in survival rates between the WT and lexANI or recA mutants is less marked following mitC treatment (4·2 % survival for WT and 0·52, 0·81 and 0·62 % survival for strains G91D-1, G91D-2 and recA, respectively, using 0·05 µg mitC ml1).
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Northern blots were performed with RNA extracted from WT strain RM118 and the lexANI strains, grown in the presence or absence of mitC, and were probed to analyse expression patterns of recA and ruvA. Fig. 3(a) shows that message levels for recA and ruvA were increased following treatment with mitC in strain RM118 but not the lexANI strains, indicating that transcription of these genes is regulated in a LexA-dependent manner (the results from only one of the independent mutants is shown; the second mutant gave the same result). Analysis of other transcripts by this method, however, was not successful, and RT-PCR was adopted as the preferred method of analysis.
Typical gel profiles obtained from the RT-PCRs are shown in Fig. 3(b). For each putative SOS gene analysed, RT-PCRs were performed on mRNA extracted from WT strain RM118 either unexposed or exposed to UV light or mitC and compared to RT-PCRs performed on RNA extracted from either the recA strain or one of the lexANI strains that were unexposed or exposed to the DNA-damaging treatments. Products from these four RT-PCRs were loaded in adjacent lanes on agarose gels (see Fig. 3b
for examples). The housekeeping gene frdB (fumarate reductase B) was amplified in each PCR (upper band in each track) as an internal control for the RT-PCR efficiency. Amplification of the frdB gene gave consistent amounts of product following growth under all the conditions used in these studies (see Figs 3b and 4c
) and preliminary microarray data have also shown that the transcript levels of this gene are not changed following SOS induction (data not shown). Target gene bands of increased intensity in the post-exposure lanes relative to the unexposed lanes indicate an increase in transcription or stability of transcripts of the target gene in response to DNA damage, as is evident in Fig. 3(b)
for lexA, recN, impA, recA and recX of strain RM118.
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Fig. 3(c) shows the results of the semi-quantitative analysis of the RT-PCRs. The results obtained from the two isogenic lexANI mutants pre- and post-UV exposure have been combined, as have the results of the effect of mitC treatment on RM118 and the lexANI mutant G91D-1 following exposure in culture or on plates. The efficiency of the RT-PCRs showed some variation both within and between experiments and thus incomplete datasets were obtained for some genes. The values plotted are the means of all successful analyses for each gene in each strain and the number of successful analyses is indicated below the axis in Fig. 3(c)
.
An induction ratio greater than 1·0 indicates that transcript levels are increased following exposure to DNA-damaging treatments (induction ratios were calculated from gel-band intensity measurements as described in Methods). The induction ratios of lexA, recN, recX and recA are all greater than 1·0 following either UV exposure or mitC treatment of strain RM118. The induction ratios of these genes were not increased in the same way in the recA or lexANI mutants following UV exposure or mitC treatment, indicating that induction of these genes is LexA dependent.
Gel profiles for impA (Fig. 3b) showed it to be very tightly regulated by LexA. No message was detected in RNA samples from unexposed strain RM118 but a strong product band was obtained from RT-PCR following exposure. recA and lexANI strains showed no detectable message in either pre- or post-exposure samples. The lack of detectable transcript in the mutant and unexposed WT samples meant that an induction ratio could not be determined.
Analysis of the recJ transcript showed an increase in transcription of this gene only in strain G91D-1 following mitC induction. The reasons for this result are not clear, but we note that RecJ is an ssDNA-specific exonuclease and it may be that recJ is upregulated in the G91D-1 mutant because it permits survival of mitC but not UV damage through a process that is achieved in WT cells by another SOS-regulated gene product. The remaining genes, himD, recF and HI1222, did not show an increase in transcription after induction using this analysis method.
Transcription of recX was further investigated to determine whether it was co-transcribed with recA, as it is in E. coli (Pagès et al., 2003). Fig. 4
(b) shows representative results of RT-PCR amplifications performed on RNA extracts isolated from strain RM118 and the lexANI mutant G91D-1 before and after treatment with mitC in culture. A strong product was produced by amplification with primers spanning recA and recX, indicating the presence of a recAX readthrough transcript. The mean band intensities obtained following mitC treatment (Fig. 4c
) show that the recA, recX and recAX transcripts were all more abundant in strain RM118 but not in the lexANI strain. The frdB control gene was amplified separately in these experiments and Fig. 4(c)
demonstrates that the transcript level of this gene was constant between strains and induction conditions (see also Fig. 3b
). It is not possible from these experiments to draw conclusions about the relative levels of recA or recX transcription relative to the co-transcript, as the lower intensities obtained for the recAX transcript may be due to a reduced amplification efficiency of the longer product.
Phase variation experiments
The lexANI strains G91D-1 and G91D-2 were transformed with plasmids containing either 38AGTC or 20AT repeats in a modrepeatlacZ reporter construct, as described in Methods, to generate strains G91D-1AGTC38, G91D-1AT20, G91D-2AGTC38 and G91D-2AT20, in which the modrepeatlacZ construct replaces the WT mod allele. Phase variation rates were first measured for the reporter constructs in the lexANI background under normal culture conditions, i.e. overnight growth on BHI agar at 37 °C. The results, given in Table 3, show that there was no significant difference in the phase variation frequencies measured for the reporter construct in the lexANI background compared with those for the reporter construct in the RM118 strain, indicating that the lexANI mutation has no detectable effect on the repeat-mediated phase variation frequency of the reporter construct under conditions where the SOS response is not induced.
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However, although there was no significant difference in phase variation rates of the reporter constructs observed between RM118 and lexANI strains following either UV or mitC induction of the SOS response, a significant (approx. 2-fold) increase in phase variation rates was observed between mitC-treated and untreated cells for the 20AT reporter construct in RM118 (P=0·0298), the 38AGTC reporter construct in RM118 (P=0·0156) and the 38AGTC reporter construct in the lexANI strains (P=0·0002). The difference in the phase variation rate of the 20AT reporter construct in the lexANI strains was just outside the level of significance (P=0·0501) (P values obtained from MannWhitney non-parametric rank sum tests). The mechanism by which mitC treatment could give rise to an increase in phase variation frequency is not clear, but may be due to the constant DNA damage resulting from the prolonged exposure leading to repeated interruptions of DNA replication and hence more opportunities for the occurrence of slippage events.
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DISCUSSION |
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Only 12 of the 31 E. coli SOS genes reported by Fernández de Henestrosa et al. (2000) have homologues in the H. influenzae genome. Those that are absent include the TLS polymerases (Pols II, IV and V) that are responsible for increased mutation rates associated with induction of the SOS response in E. coli (Napolitano et al., 2000
; Wagner et al., 2002
).
We next created a mutation in the lexA gene of H. influenzae that gave a non-SOS-inducible phenotype (lexANI) and showed that strains with the mutation were more sensitive than WT cells to DNA-damaging agents but not as sensitive as recA mutants. These results demonstrate that the SOS response is compromised in the lexANI and recA mutants and suggest that the H. influenzae lexA gene mediates survival following DNA damage in a process that is mechanistically similar to that mediated by E. coli lexA. Transcriptional analysis of nine of the candidate H. influenzae SOS genes identified from the genome analysis showed increased message levels for lexA, recA, recN, ruvA, recX and impA following induction of the SOS response. These induced genes include the core response genes (lexA, recA and recN) identified in the work of Erill et al. (2003). However, although the sensitivity of the semi-quantitative RT-PCR analysis method was sufficient to determine that these six genes are upregulated as part of the SOS response, it did not permit accurate comparisons of the relative induction levels of these genes or a definitive demonstration that other genes analysed (himD, recF, recJ) are not induced. A comprehensive investigation using full genome microarrays will clarify whether these three genes are upregulated by H. influenzae in response to DNA damage and whether this response includes some of the additional members of the potential H. influenzae LexA regulon identified herein but not analysed (Table 2
) or indeed other genes regulated by non-consensus SOS boxes or by alternative induction mechanisms.
We have shown evidence that the H. influenzae recX is co-transcribed with recA. In E. coli, only 510 % of the recA transcripts are recArecX co-transcripts, because the majority of transcripts terminate at a palindromic repeat sequence that is located between these two genes (Pagès et al., 2003). Whilst we have not accurately determined the relative levels of the recA and recX transcripts, it is interesting to note that there are two inverted copies of the H. influenzae uptake sequence 19 bp 3' of the H. influenzae recA stop codon, which could form a stemloop and thereby terminate transcription between recA and recX (Zulty & Barcak, 1993
). Additionally, it was recently shown that the E. coli RecX competes with DinI (an E. coli SOS-regulated protein that is absent from the H. influenzae genome) to modulate RecA activity; RecX being a negative regulator and DinI positive (Lusetti et al., 2004
). It will be of interest to investigate the role of recX further in H. influenzae, given the extreme sensitivity of the H. influenzae recA mutant (Fig. 2
) and the absence of the SOS-regulated dinI gene from the H. influenzae genome (Table 2
).
The results of our phase variation studies showed that, in contrast to the findings of Morel et al. (1998), induction of the SOS response in H. influenzae did not affect phase variation frequencies mediated by either an AT dinucleotide or AGTC tetranucleotide repeat tract in the mod locus. Phase variation frequencies at a second locus, lic2A, which contains CAAT tetranucleotide repeats fused to the lacZ reporter gene, were also measured for strain RM118 with and without mitC treatment (data not shown). A similar result to that obtained for the AGTC repeats following mitC treatment was observed, confirming that the tetranucleotide repeat tracts in H. influenzae are not destabilized by the SOS response. One interpretation of our findings is that the destabilization of dinucleotide repeats observed by Morel et al. (1998)
was due to expression of one or more of the E. coli SOS genes that are absent in H. influenzae; particularly strong candidates are the TLS polymerases. Each of the three TLS polymerases in E. coli can cause 1 or 2 bp frameshifts, dependent upon the nature of the lesion and the combination of polymerases present (reviewed by Tippin et al., 2004
).
In our studies, treatment with mitC caused an increase in phase variation rates at both dinucleotide and tetranucleotide repeats, independently of the SOS response. The reasons for the small increase in phase variation rates observed with mitC are unclear, but we have suggested that they may be due to repeated interruptions of DNA replication resulting from prolonged exposure to mitC allowing more opportunities for the occurrence of slippage events. Alternatively, DNA-repair pathways that are not LexA controlled may be responsible for this observed increase.
In summary, we have shown that H. influenzae has a LexA-dependent SOS response to DNA damage that aids survival following DNA assault and have demonstrated that at least five genes involved in DNA repair and recombination (lexA, recA, recX, recN and ruvA) are expressed as part of this response. Induction of the H. influenzae SOS response did not affect slippage rates at an AT dinucleotide or tetranucleotide repeat tract. The observed stability of H. influenzae repeats during an SOS response may be due to the absence of TLS polymerases in the H. influenzae genome.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 February 2005;
revised 19 May 2005;
accepted 25 May 2005.
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