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Journal of Bacteriology, August 2006, p. 6034-6038, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00340-06

The Mycobacterium-Specific Gene Rv2719c Is DNA Damage Inducible Independently of RecA

Patricia C. Brooks, Lisa F. Dawson, Lucinda Rand, and Elaine O. Davis^*

Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Received 9 March 2006/ Accepted 31 May 2006

	ABSTRACT

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The mycobacterium-specific gene Rv2719c was found to be expressed primarily from a promoter that was clearly DNA damage inducible independently of RecA. Upstream of the transcriptional start site for this promoter, sequence motifs resembling those observed previously at the RecA-independent, DNA damage-inducible recA promoter were identified, and the –10 motif was demonstrated by mutational analysis in transcriptional fusion constructs to be important for expression of Rv2719c.

	TEXT

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The expression of numerous genes is increased following DNA damage in bacteria including Mycobacterium tuberculosis. In Escherichia coli this is primarily controlled by the repressor protein LexA (9, 16) and is dependent on RecA (23). Although this system has been identified and shown to be functional in M. tuberculosis (4, 8, 17), the majority of DNA damage-inducible genes in this organism are controlled by an alternative mechanism(s) that has yet to be characterized but is independent of RecA (19).

The M. tuberculosis gene Rv2719c is a mycobacterium-specific gene of unknown function located adjacent to, and divergently transcribed from, the lexA gene (6). This gene has been shown to be cotranscribed with the two downstream genes Rv2718c and Rv2717c, and microarray analysis indicated that expression of all three of these genes is increased following DNA damage (19).

Previously we had examined the role of three SOS boxes located in the upstream region of Rv2719c in the DNA damage-mediated induction of its expression by using lacZ transcriptional fusions (6). This analysis indicated that regulation was mediated by LexA interacting with these SOS boxes, since altering the sequence of all three SOS boxes to prevent LexA binding resulted in high-level, constitutive expression. Regulation by LexA is dependent on the presence of functional RecA in M. tuberculosis (5) as in E. coli (23). Surprisingly, however, the microarray study revealed that expression of Rv2719c remained DNA damage inducible in a recA deletion strain of M. tuberculosis (19). This was also the case for the cotranscribed genes Rv2718c and Rv2717c; in each case the induction ratio for the recA deletion strain was approximately 64% that observed for the wild type.

Here we resolve these apparently contradictory results and clarify the control of expression of the Rv2719c operon.

E. coli strains DH5 and XL1-Blue were grown under standard conditions (21). M. tuberculosis strain 1424 and its recA deletion derivative were grown and induced by exposure to mitomycin C as described previously (5), except that the uninduced control for quantitative reverse transcription-PCR (RT-PCR) was harvested at time zero. Cell lysates were prepared and used for protein and ß-galactosidase assays as described elsewhere (4). Statistical significance was determined using a two-tailed t test for samples of unequal variance.

Plasmid DNA was prepared using SNAP miniprep kits (Invitrogen) according to the manufacturer's instructions. For other DNA manipulations, standard protocols were followed (21). The reporter plasmids used are listed in Table S1 in the supplemental material. Base changes in predicted –10 motifs were introduced by site-directed mutagenesis as described in the QuikChange site-directed mutagenesis kit (Stratagene) using primers listed in Table S2 of the supplemental material. For each construct, the sequences of the inserts and the junctions to the vector were confirmed. Clones were introduced into M. tuberculosis by electroporation (13) and verified by PCR and sequencing as described elsewhere (4).

RNA was isolated as described elsewhere (4). RNA concentrations were determined spectrophotometrically at 260 nm or by using a Bioanalyser (Agilent Technologies) in the case of samples for RNase protection assays. Real-time quantitative RT-PCR was performed as described previously (19) using the primers given in Table S2 of the supplemental material.

Primer extension analysis was carried out with Thermoscript RNase H reverse transcriptase (Invitrogen) at 55°C for 1 h according to the manufacturer's instructions using 60 µg of total RNA annealed to the ³²P-labeled primer, before separation alongside sequencing reactions performed on pEJ544 with the same primer using a Sequenase kit (USB/Amersham) on an 8% polyacrylamide-urea gel. The primers used were XbaRv2719c for analyzing chromosomal Rv2719c and LACR for analyzing the reporter construct pEJ544 (see Table S2 in the supplemental material).

RNase protection assays were performed using 150 µg RNA and 7.5 x 10⁵ cpm of the purified ³²P-labeled probe, prepared from BamHI-linearized pEJ639 (see Table S1 in the supplemental material) using a MAXIscript in vitro transcription kit (Ambion) and NucAway columns (Ambion) in accordance with the manufacturer's instructions, and digestion with an RNase A-T₁ mix at a ratio of 1:75 was performed as described for the RPAIII RNase protection assay kit (Ambion). The protected RNA was resolved on a 5% denaturing polyacrylamide gel.

DNA damage induction of Rv2719c is independent of RecA. To establish whether or not the expression of Rv2719c remained DNA damage inducible in the recA deletion strain of M. tuberculosis as suggested by microarray analysis, two further assays comparing induction following DNA damage in the wild-type and recA mutant strains were performed. In the first of these, expression levels were measured by quantitative real-time RT-PCR using TaqMan probes, with normalization to levels of sigA, the expression of which has been shown previously not to alter following DNA damage (1). In the second approach, expression levels were assessed using the lacZ reporter construct (pEJ544) used previously (6). Both analyses revealed that Rv2719c was induced to the same extent in the recA mutant as in the wild type (Fig. 1), there being no significant difference between the induction ratios in the two strains (P > 0.05). The apparent difference in the induction ratios obtained by the two assays was not statistically significant for either strain (P > 0.05).

View larger version (11K): [in this window] [in a new window]   FIG. 1. Rv2719c remains DNA damage inducible in a recA deletion strain of M. tuberculosis. The extent of induction of Rv2719c was determined in wild-type (black bars) and recA (white bars) strains of M. tuberculosis using two methods. In the first, labeled Taqman, RNA was isolated from uninduced and induced (with 0.2 µg/ml mitomycin C) cultures of each strain and the amount of Rv2719c RNA in each sample was measured by quantitative real-time RT-PCR and normalized to the amount of sigA RNA. In the second, labeled lacZ, cell extracts from uninduced and induced (with 0.2 µg/ml mitomycin C) cultures of each strain carrying the reporter clone pEJ544 were assayed for ß-galactosidase activity, which was normalized to total protein concentration. Values are means from triplicate (Taqman) or duplicate (lacZ) assays from at least three independent inductions. Error bars, standard deviations.

The initial approach taken was to map the end-points of the mRNA directly by primer extension. Preliminary experiments using avian myeloblastosis virus reverse transcriptase under standard conditions at 42°C resulted in a ladder of bands (data not shown), suggesting that secondary structure in this region of the RNA was causing premature termination of the reverse transcriptase. However, by using a more thermotolerant reverse transcriptase and increasing the temperature of the reaction to 55°C, this problem was largely eliminated. RNA isolated from both the wild-type and recA strains was analyzed using a primer within the coding sequence of Rv2719c. In each case, a strong primer extension product, which was only barely detectable in the uninduced sample, was obtained from the induced sample (data not shown). This transcriptional start site was located 97 bp upstream of the coding region, coincident with the more upstream possibility identified by RACE analysis. In addition, there was an indication from the wild-type samples that a second transcriptional start site, corresponding to that previously identified by RACE 36 bp upstream of the initiation codon, might exist.

To clarify whether this represents a genuine transcriptional start site, we analyzed expression using an RNase protection assay, which should be less affected by local secondary structures. A protected fragment was observed that was strongly inducible in both the wild-type and recA strains (Fig. 2a) and that had the expected size (150 bases) to correspond with the clear primer extension product mentioned above. However, no protected band specific to the samples containing RNA was identified that was equivalent to the potential smaller primer extension product.

View larger version (46K): [in this window] [in a new window]   FIG. 2. (a) Identification of transcription start sites for Rv2719c within genomic DNA by RNase protection. One hundred fifty micrograms of RNA isolated from uninduced and induced (with 0.2 µg/ml mitomycin C) cultures of wild-type and recA strains of M. tuberculosis, or a no-RNA control, was analyzed. The inducible transcription start site is indicated by T and the undigested probe by P. Lane 1, RNA from the uninduced wild-type strain; lane 2, RNA from the induced wild-type strain; lane 3, RNA from the uninduced recA strain; lane 4, RNA from the induced recA strain; lane 5, no-RNA control; lane 6, undigested probe control; lane M, markers. (b) Identification of transcription start sites for Rv2719c within reporter clones by primer extension analysis. Sixty micrograms of RNA isolated from uninduced and induced (with 0.2 µg/ml mitomycin C) cultures of wild-type M. tuberculosis carrying pEJ544 or pEJ544-B234, or a no-RNA control, was analyzed by extension of primer LACR (see Materials and Methods), which binds within the coding sequence of the lacZ reporter gene. The extension products were run alongside sequencing reactions performed on plasmid pEJ544 with the same primer. The inducible transcription start site is indicated by T and the band that could represent either an additional start site or a premature termination product by T?. Lane 1, RNA from uninduced pEJ544; lane 2, RNA from induced pEJ544; lane 3, RNA from uninduced pEJ544-B234; lane 4, RNA from induced pEJ544-B234; lane 5, no-RNA control.

Transcriptional start sites in the original reporter constructs. To confirm that transcription initiation in the reporter constructs used previously corresponded to that at the native locus of M. tuberculosis, a similar primer extension analysis was conducted but using a primer within the reporter gene so that RNA expressed from the integrated plasmid could be detected without interference from the natural chromosomal copy of the gene itself. When RNA isolated from M. tuberculosis carrying pEJ544 was analyzed in this way, a strongly inducible primer extension product was indeed observed and aligned to the same base on the DNA sequence as seen above (Fig. 2b). A faint band corresponding to the smaller band was also observed, but some stuttering indicated that this may again result from secondary structure in the region preventing complete extension.

A further primer extension analysis was performed using RNA isolated from M. tuberculosis carrying pEJ544-B234, which had exhibited constitutive expression (6), and the primer within the reporter gene. Again, the same primer extension product was obtained (Fig. 2b), and expression was strongly inducible. However, in this case there was a stronger signal for the smaller band; thus, it is possible that the changes introduced in the SOS boxes could have resulted in the enhancement of a very weak promoter in this region or in the creation of an artifactual promoter.

Promoter element identification using transcriptional fusions. Upstream of the inducible transcription start site, there are motifs similar to those originally identified at the recA P1 promoter (12), which is also DNA damage inducible independently of RecA (5), and subsequently found upstream of other M. tuberculosis DNA damage-inducible genes (10). Although the smaller primer extension product appeared most likely to be an artifact from the above analyses, inspection of the DNA sequence upstream of this potential start site identified motifs resembling the E. coli ⁷⁰ promoter elements (Fig. 3).

View larger version (29K): [in this window] [in a new window]   FIG. 3. Sequence of the DNA upstream of Rv2719c contained in pEJ544. The inducible transcription start site identified in this study is shown by a bent arrow and labeled T, while the location of the potential additional start site is indicated by a dotted bent arrow labeled T?, corresponding to the labeling in Fig. 2. The motifs showing similarity to M. tuberculosis recA P1 promoter elements or to E. coli 70 promoter elements are underlined and labeled. The SOS boxes are boxed with dotted lines, while the predicted translation initiation codon is double underlined. The small arrows above the sequence indicate the limits of the fragment cloned in pEJ630, and those below the sequence indicate the extent of the insert in pEJ629 (see Fig. 4).

View larger version (14K): [in this window] [in a new window]   FIG. 4. Effect of mutation of the predicted –10 promoter elements of Rv2719c on expression of the lacZ reporter gene in transcriptional fusion constructs. (a) Schematic indicating the relative locations of the fragments cloned and the presence of mutations in the –10 regions (indicated by stars) relative to the locations of the transcription start sites shown by the bent arrows. In each case three consecutive bases were altered as described in the text. (b) Expression of pEJ544 and its derivatives in wild-type (wt) and recA strains of M. tuberculosis. ind, induced; unind, uninduced. (c) Expression directed by small DNA fragments containing the promoters individually as indicated in panel a along with the pEJ414 vector in wild-type and recA strains of M. tuberculosis. ß-Galactosidase activity determined with (hatched and solid bars) or without (open and striped bars) exposure to 0.2 µg/ml mitomycin C for 24 h is shown for wild-type (open and hatched bars) and recA (striped and solid bars) M. tuberculosis. nt, not tested (pEJ631 and pEJ632 were not tested in the recA strain). Values are means from duplicate assays of at least three independent cultures in each case. Error bars, standard deviations. Note the difference in scales for the two graphs.

Expression from pEJ630 was highly inducible in both the wild-type and recA strains of M. tuberculosis (Fig. 4), even though it did not include any SOS boxes. When the same base changes that had been examined in the full-length construct were introduced into the –10 region in pEJ632, expression was reduced to background levels, similar to those seen with vector pEJ414, in both uninduced and induced cultures (Fig. 4). These observations confirm the presence of a promoter in this region that is DNA damage inducible independently of RecA and LexA, and they indicate that the mutation in its –10 region eliminates promoter function. Thus, the residual promoter activity observed with pEJ597 is most likely due to the presence of an additional promoter within the 315-bp fragment contained in the series of constructs derived from pEJ544.

The clone containing the proximal region, pEJ629, exhibited low-level but significant (P < 0.01) promoter activity in both uninduced and induced cultures of both strains (Fig. 4); expression did not increase following DNA damage despite the inclusion of the SOS boxes. Introduction of the same base changes in the –10 region that had been examined in the full-length construct did not alter the level of expression significantly (P > 0.05) (Fig. 4, pEJ631). Taken together, these observations support the presence of a weak constitutive promoter for Rv2719c in this region but suggest that the promoter elements have not yet been identified. Comparison of the data in Fig. 4b and 4c suggests that the activity of this promoter is enhanced in the presence of additional DNA from upstream of the gene, as has been found previously for the M. tuberculosis recA promoters (12). Nevertheless, the contribution made by such a promoter to the expression of Rv2719c appears to be small under DNA-damaging conditions.

The expression of Rv2719c remains inducible in the recA mutant strain of M. tuberculosis to an extent very similar to that seen in the wild-type strain, indicating that its induction is independent of RecA and therefore of the repressor LexA. An inducible promoter corresponding to a transcription start site 97 bp upstream of the coding region was identified; it contained elements resembling the promoter motifs found at the DNA damage-inducible P1 promoter of recA, which is also independent of RecA and LexA (5), suggesting that these two promoters are regulated by a common mechanism. Similar motifs have been identified upstream of a number of other DNA damage-inducible genes in M. tuberculosis (10), indicating the likely extent of this regulon, although the components responsible for its control have yet to be identified.

This promoter is located upstream of the previously identified binding sites for LexA (6), implying that RNA polymerase initiating from this promoter can proceed past these binding sites under conditions in which LexA cleavage is prevented. The process of transcription across sites bound by LexA may be facilitated by the fact that these sites contain multiple mismatches with the LexA consensus sequence (6), presumably resulting in reduced affinity of LexA for these sites and hence sufficient "off" time during equilibrium binding for RNA polymerase to pass. Transcription of some LexA-regulated genes has been reported to be induced under conditions that do not cause cleavage of LexA during the stringent response in E. coli (15).

Although LexA has been demonstrated to bind to the two SOS boxes nearest to the gene by gel shift analyses (1, 6), binding to the most distal site was not detected. Curiously, though, the effect of changing the sequence of a single SOS box on the expression of a downstream reporter gene was greatest when this most distal site was the one affected (6). Thus, it may be that the changes introduced enhanced the activity of the naturally weak promoter located in this region such that it masked the effect of induction of the regulated promoter.

DNA damage induction independent of RecA is relatively rare but has been described previously (11, 14, 20). Nevertheless, it has recently become apparent that gene expression can be induced following DNA damage independently of LexA in a number of bacterial species (2, 3). In some cases, the alternative mechanisms involved are beginning to be unraveled: Lactococcus lactis HdiR has a more complex mechanism of action, requiring degradation by Clp protease in addition to RecA-dependent self-cleavage for optimal DNA damage induction (22), while in Clostridium perfringens enhanced expression of a bacteriocin gene following DNA damage was shown to be dependent on a novel sigma factor termed UviA (7). Determining the mechanisms of the RecA-independent response to DNA damage in mycobacteria will be of great interest.

. . .

	ACKNOWLEDGMENTS

 
This work was supported by the Medical Research Council.

	FOOTNOTES

 
* Corresponding author. Mailing address: Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. Phone: 020 8959 3666. Fax: 020 8913 8528. E-mail: edavis{at}nimr.mrc.ac.uk.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: TB Unit, 10 Biopolis Road, #05-01 Chromos, Singapore 138670.

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This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brooks, P. C. Articles by Davis, E. O. Search for Related Content PubMed PubMed Citation Articles by Brooks, P. C. Articles by Davis, E. O.