1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
2 John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK
Correspondence
Andrew W. B. Johnston
a.johnston{at}uea.ac.uk
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ABSTRACT |
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Both authors contributed equally to this work.
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INTRODUCTION |
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In Gram-negative bacteria, the most widely used (and most studied) Fe-responsive gene regulator is Fur (ferric uptake regulator). In model -Proteobacteria (e.g. Escherichia coli or Pseudomonas), in the presence of Fe2+, this dimeric protein (Kolade et al., 2002
; Pohl et al., 2003
) binds to sequences, known as fur boxes, and prevents transcription of the target promoters. Fur mutants in E. coli also have reduced expression of several transcripts (McHugh et al., 2003
). In some cases, this is via Fur-mediated repression of a small RNA molecule, RyhB, which then represses transcription of genes whose expression appeared to be induced by Fur (Masse & Gottesman, 2002
).
Other Fe-responsive regulators include those of the Dtx class, which are important in Gram-positive bacteria and cyanobacteria (see Andrews et al., 2003). Although differing in their primary sequences, the structures of the Dtx and the Fur families share broad structural similarities (see Pohl et al., 2003
).
A third class of wide-ranging, Fe-responsive transcriptional regulators, termed RirA, was recently described for Rhizobium leguminosarum, the symbiotic N2-fixing -proteobacterium that induces root nodules on peas, beans and clover (Todd et al., 2002
), a process that is very demanding for iron (see Johnston et al., 2001
). Mutations in rirA cause deregulated expression of several genes whose transcription is normally repressed in cells grown in Fe-replete medium. These include the vbs and fhu genes, which are involved, respectively, in the synthesis and uptake of the siderophore vicibactin (VB) (Carter et al., 2002
; Stevens et al., 1999
; Yeoman et al., 2000
); hmuPSTUV, which specifies a haem ABC transporter (Wexler et al., 2001
); tonB (Wexler et al., 2001
); and rpoI, which encodes an ECF
factor that specifically recognizes promoters of the vbsADL and vbsGSO VB biosynthetic genes (Yeoman et al., 1999
, 2003
). Transcription of rirA itself is enhanced in Fe-replete conditions and is auto-regulated (Todd et al., 2002
).
There is no detectable sequence similarity between RirA and members of the Fur or Dtx families, suggesting that RirA represents a novel class of Fe-responsive regulator. There are close homologues (>70 % amino acid identity) of RirA, but these are confined (so far) to Sinorhizobium, Mesorhizobium, Agrobacterium and the animal pathogen Brucella, all of which are near relatives of Rhizobium. However, RirA has 30 % amino acid identity to the large (181 members) Rrf2 family, which occurs in all eubacteria, with many genera (including Rhizobium) having more than one paralogue. Rrf2 itself regulates cytochrome biosynthesis in Desulfovibrio (Keon et al., 1997
). The only other member to have been studied in detail, IscR of E. coli, regulates the adjacent iscRSUA, operon, which is involved in the synthesis of FeS clusters. IscR is itself an FeS-cluster protein, repressing iscRSUA transcription, but is only fully effective if charged with this co-factor (Schwartz et al., 2001
). IscR does not repress the iscRSUA operon in Fe-depleted or oxygen-stressed E. coli cells, indicating that this regulator responds to both of these environmental signals (Outten et al., 2004
).
It is possible that a subset of the Rrf2 superfamily evolved into a specialized Fe-sensing regulator in Rhizobium and its very close relatives, with RirA mediating many functions that in E. coli are undertaken by Fur. Consistent with this, Rhizobium has a gene whose product resembles a classical Fur, but mutations in the fur-like gene do not affect Fe-responsive regulation of the fhu, vbs, rpoI, hmu and tonB genes alluded to above (Wexler et al., 2003). Indeed, this Fur-like protein is actually a manganese-sensing regulator, termed Mur, which represses the operon that specifies a Mn2+ ABC transporter, SitABCD, in Mn2+-replete cells (Diaz-Mireles et al., 2004
; Chao et al., 2004
).
Here we try to elucidate how RirA regulates Fe-responsive genes in R. leguminosarum and identify potential RirA-binding sites near promoters of some of its target genes.
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METHODS |
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To map transcript starts (Sawers & Böck, 1989), cells were grown in Fe-replete or Fe-depleted media and total RNA was isolated. Using primers located within the structural, promoter-proximal gene, primer extensions were done, together with manual sequencing of the corresponding cloned segment of DNA to identify transcription initiation sites. The sequences of the primers used for these experiments were as follows: vbsC (5'-GATCGGCAGAGCTCCGCCTTG-3'); fhuA1 (5'-CTTGTGAACACACGCAACTTTCCC-3'); fhuA2 (5'-GGCTATCTCTATAAATTCGCG-3'); fhuC (5'-CGGCGACAAAAGTTTCAGCCGTTC-3').
Site-directed mutagenesis (SDM).
The 10 bp deletion in IRORi was generated using the ExSite kit (Stratagene) and primers that hybridized on either side of the designated deletion. The template DNA was plasmid pBIO1327, which contains a 400 bp DNA fragment spanning the rpoIvbsC intergenic space, cloned in pUC18. Mutant plasmids were verified by DNA sequence analysis and the mutated insert was recloned into the wide host-range transcriptional fusion vector pMP220 in the appropriate orientation. Site-directed substitution mutations were generated using the QuikChange XL kit (Stratagene) to mutate the wild-type plasmid pBIO1328, which contains the same insert as in pBIO1327, but cloned in pMP220, forming a rpoIlacZ fusion in pMP220. Mutagenic primers, 25 bp in length (synthesized by MWG-Biotech) and containing a single base pair substitution compared to wild-type (Table 2), were used in a PCR-based mutagenic reaction, and the products were transformed into E. coli DH10B-T1R (Invitrogen), selecting tetracycline resistance. Mutations were verified by DNA sequencing. To mutate the IROVc, the vbsClacZ fusion plasmid pBIO1306, which contains 780 bp, spanning the rpoIvbsC intergenic space, cloned in pMP220 was used as a template. Using a QuikChange XL kit (Stratagene), 8 bp of IROVc were removed and replaced with a NotI site (5'-GCGGCCGC-3') 16 bp downstream from the vbsC transcriptional start site. Mutations were verified by sequencing. The resultant mutant plasmid was termed pBIO1466. The mutant rpoIlacZ fusion plasmid pBIO1467, which lacks IROVc, was derived from pBIO1466, from which the insert was cloned into pMP220, but in the opposite orientation.
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RESULTS |
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In the 163 bp intergenic region between rpoI and vbsC of R. leguminosarum strain J251, we noted two similar (11 of 17 bp identical) sequences, termed iron-responsive operators (IROs), with opposing orientation (see Fig. 1). Similar sequences occur near other Fe-regulated promoters (see below). The conserved sequence 5' of rpoI, which we term IRORi, is centred 40 bp 5' of the previously identified (Yeoman et al., 2003
) transcription initiation site of rpoI. The other, termed IROVc, is immediately 5' of the rpoI transcript start site (Fig. 1
).
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Having established its importance in the control of rpoI expression, we examined IRORi in more detail. Mutant derivatives of pBIO1328, which contains the intact rpoI promoter and regulatory region, were constructed by site-directed mutagenesis (SDM) in which individual bases of IRORi were substituted. The resulting mutant plasmids were each mobilized into wild-type R. leguminosarum and the transconjugants assayed for -galactosidase activity after growth in high-Fe and low-Fe media. As shown in Table 2
, most of these substitutions had little or no effect on rpoIlacZ expression in either medium, but those at the 3' end of the motif abolished Fe-responsive repression of the fusion. However, these mutations at the 3' end of the IRORi also reduced rpoI expression, even in Fe medium, indicating they affect promoter activity and the response to Fe availability. This is consistent with their location near the 35 region relative to the rpoI transcriptional start. Two other adjacent mutations, in pBIO1473 and pBIO1427 (Table 2
), elevate rpoIlacZ expression, especially in low-Fe medium. Although upstream from the 35 region, these two mutations may enhance rpoI promoter activity, either directly through interactions with RNA polymerase or, perhaps, by indirect interaction with another, unknown, factor.
Cloned rirA confers Fe-responsive repression of rpoI and vbsC in P. denitrificans
Carter et al. (2002) showed that the cloned vbsGSO and vbsADL genes were not expressed in the
-proteobacterium P. denitrificans unless the cloned R. leguminosarum
factor gene rpoI was also present in this heterologous host. Thus, the promoters of vbsGSO and vbsADL are not recognized by a native P. denitrificans
factor, but rpoI itself is expressed in this bacterium. We therefore directly examined rpoI expression in P. denitrificans and, for comparison, the divergently transcribed vbsC gene. Plasmids pBIO1328 and pBIO1306 (rpoIlacZ and vbsClacZ fusions, respectively), were each mobilized into P. denitrificans, and the transconjugants were grown in medium with high and low concentrations of Fe. Both fusions were expressed in this host, but their expression was unaffected by the Fe status of the medium (Table 3
), showing that P. denitrificans cannot mediate Fe-responsive regulation of rpoI or vbsC.
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To show that an IRO box is needed for this repression, the mutant rpoIlacZ fusion plasmid pBIO1335, which has a deletion in IRORi, was transferred to P. denitrificans. As with its intact parent, pBIO1328, there was constitutive, high-level expression of rpoIlacZ in P. denitrificans harbouring pBIO1335. Significantly, introduction of the cloned rirA gene no longer conferred Fe-responsive repression to this mutated rpoIlacZ fusion (Table 3). For reasons that are not clear, the presence of rirA further enhanced the level of expression from pBIO1335.
With pBIO1466, the mutated vbsClacZ fusion plasmid that lacks an intact IROVc, a similar pattern was found. Whereas the expression of -galactosidase from the parental plasmid pBIO1306 was repressed by introducing cloned rirA into P. denitrificans, when IROVc was removed, the inhibitory effect of RirA in this heterologous host was abolished (Table 3
).
Taken together, these experiments, in which P. denitrificans was used as a null background, provide strong circumstantial evidence that RirA protein acts directly on the IRO motifs of vbsC and rpoI, resulting in a very significant drop in the levels of transcription of both genes. In the case of the rpoIlacZ fusion, this repression is much greater in the high-Fe than in the low-Fe medium just as it is in R. leguminosarum itself. However, with the vbsC fusion, RirA is almost equally repressive in both media. The reasons for this difference are unknown, but may suggest the presence of other regulatory factors that are needed for the precise regulation of some, but not all, RirA-responsive promoters. It might also be, though, that the presence of extra copies of the cloned rirA can confer repression of vbsC, even in the absence of Fe.
Different arrangements of fhuA genes in different field isolates of R. leguminosarum
In R. leguminosarum field-isolated J251, we had shown that the uptake of VB is mediated by the Fhu transport proteins, the outer-membrane receptor being FhuA, the periplasmic binding protein FhuD, the inner-membrane transporter FhuB and the ATPase FhuC (Stevens et al., 1999; Yeoman et al., 2000
). Analysis of the genome of the sequenced R. leguminosarum field isolate 3841 showed that it has two fhuA genes, with fhuA1 being on the symbiotic plasmid, pRL10JI,
145 kb from the nod genes (http://www.sanger.ac.uk/Projects/R_leguminosarum/) and the other copy, fhuA2 being in the fhuA2FrpoIvbs gene cluster on a different native plasmid, pRL12JI. The finding of two apparently intact copies of fhuA in strain 3841 was surprising, since field isolate J251 had been shown to have one intact fhuA, in the same location relative to rpoI as that of fhuA2 in field isolate 3841. In the locus occupied by fhuA1 in strain 3841, field isolate J251 has a non-transcribed pseudogene,
fhuA, with many stop codons, which, like fhuA1 of 3841, is orientated divergently from fhuCDB. Despite this,
fhuA is 81 % identical over 460 bp at its 3' end to fhuA1 of strain 3841. Thus, J251 probably had an intact fhuA1, but its function was lost by accumulated mutations (Yeoman et al., 2000
). In strain 3841, both fhuA1 and fhuA2 are functional: when individually cloned, each can confer VB uptake ability to a fhuA mutant of strain J251 (results not shown).
IRO-like sequences and the promoters of fhu genes involved in VB uptake
The reason for describing the differences in the organization of fhuA genes in these two strains is that when we examined the R. leguminosarum strain 3841 genome for IRO-like sequences (based on IRORi), the closest match (14 of 17 identical, Figs 1a and 4) was located 5' of fhuA2. A near-identical sequence was also seen in the corresponding region 5' of fhuA2 of field isolate J251. We also examined the DNA 5' of the other two fhu operons, fhuA1 and fhuCDB, in strain 3841 for IRO-like sequences. As seen in Figs 1(b) and 4
, there was a region 88 bp 5' of fhuA1 with limited similarity to IRORi, this being most pronounced at the 3' end of the motif, around the 35 bp region. In contrast, we found no IRO-like motifs near the transcript start of the fhuCDB operon, which had been located by primer extension experiments, even though fhuCDB expression is regulated in response to Fe availability, this being mediated by RirA (Todd et al., 2002
; unpublished observations).
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fhuA1.
A transcriptional fhuA1lacZ fusion plasmid, pBIO1508, which contains DNA extending 60 bp 5' of the fhuA1 transcriptional start, exhibited normal Fe-responsive expression in wild-type R. leguminosarum (Fig. 4). In the RirA mutant J397 (Todd et al., 2002
), expression of this fhuA1lacZ fusion was at a high level, even in high-Fe medium (data not shown), formally demonstrating that RirA mediates Fe-responsive regulation of fhuA1. When the slightly smaller fusion plasmid pBIO1510, which lacks IROFa1, was mobilized to wild-type R. leguminosarum, the expression of
-galactosidase was essentially the same in +Fe and Fe media (Fig. 4
). Thus, removal of IROFa1 abolishes Fe-responsive regulation of fhuA1.
These two fhuA1lacZ fusion plasmids were also mobilized into P. denitrificans. In this background, both of them expressed -galactosidase constitutively. Thus, as with rpoI and vbsC, this heterologous species can transcribe the introduced R. leguminosarum gene but cannot effect Fe-responsive regulation of its expression. When pBIO1451 (rirA cloned in pOT2), was introduced into P. denitrificans that harboured pBIO1508, there was significant reduction in the level of
-galactosidase activity. This was more pronounced in the high-Fe than in the low-Fe medium, but in both regimes there was still a significant level of expression, showing that the repression was not as effective as that of the analogous experiments using the rpoIlacZ fusion plasmid in P. denitrificans (see above). Importantly, though, the presence in P. denitrificans of the cloned rirA gene had no effect on the expression from pBIO1510, which lacks an intact IROFa1 (Fig. 4
).
The IROFa1 motif is therefore likely to be a cis-acting regulatory element that interacts with RirA, despite its considerable sequence divergence compared to IRORi and IROVc.
fhuA2.
As mentioned above, the best match to IRORi in the genome of R. leguminosarum is to a motif (IROFa2) 5' of the fhuA2F promoter, but further upstream of the fhuA2F promoter than were the other IRO sequences relative to their promoters. Nevertheless, this IROFa2 motif appears to be important in the Fe-responsive regulation of fhuA2F, as seen by the behaviour of two fhuA2lacZ fusion plasmids in wild-type R. leguminosarum grown in high-Fe or in low-Fe medium. One of these, pBIO1512, which contains the intact IROFa2 and extends 87 bp 5' of the fhuA2 transcriptional start site, exhibited Fe-responsive repression of -galactosidase in R. leguminosarum grown in Fe-replete medium (Fig. 4
). However, the smaller plasmid pBIO1514, whose 5' end is within IROFa2, shows no Fe-dependent regulation, its expression being essentially the same in the +Fe and Fe media.
In contrast to other lac fusions described here, when pBIO1512 was transferred to P. denitrificans, its expression was affected by the Fe availability of the medium, the levels of -galactosidase being approximately twofold lower in the high-Fe than in the low-Fe medium. With the smaller fusion plasmid pBIO1514, which lacks an intact IROFa2, no such Fe-responsive regulation was seen. This suggests that the region 5' of fhuA2 contains a sequence that is recognized by an (unknown) Fe-responsive regulator that is native to P. denitrificans. Introducing the cloned rirA into P. denitrificans harbouring the fhuA2lacZ fusion pBIO1512 had little further effect, the ratio of expression in high-Fe and low-Fe media being similar (
0·3) to that when the P. denitrificans did not contain rirA.
This difference in behaviour of the fhuA2 regulatory region in the heterologous host was not due to the use in P. denitrificans of a promoter different to that used in R. leguminosarum. From primer extension experiments, using RNA from P. denitrificans harbouring the cloned fhuA2 gene, it was found that the fhuA2 transcriptional start was identical in the two bacteria, and that the levels of expression were significantly greater in cells that had been grown in Fe-depleted medium (not shown).
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DISCUSSION |
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Here, a combination of deletion analyses and expression studies of various Fe-responsive Rhizobium genes in Paracoccus has provided strong circumstantial evidence that RirA likely does recognize IRO motifs. Although this work revealed some similarities in the ways in which the IROs identified here affect Fe-responsive gene expression, there are also some differences, pointing to differences in the exact way in which RirA mediates its regulatory effects on individual target genes. In what follows, it should be kept in mind that although we know that expression of vbsC, rpoI and the fhu genes does not involve the RpoI factor, it has not been established whether transcription of one or more of these genes is mediated by the housekeeping
70 or by one (or more) of the other
factors that Rhizobium possesses. It cannot be discounted, therefore, that the different responses of the genes examined here may be due to their being transcribed by different
factors.
Here, we have used an operational definition of a consensus of a 17-mer as the IRO sequence. However, since in some cases (rpoI and fhuA1) the 3' end of the IRO motif overlaps the 35 region of the promoter, we have not formally distinguished bases that may be conserved because they constitute part of the RNA polymerase recognition sequence from those that may be specifically involved in interacting with RirA. Indeed, some nucleotides may be involved in both these functions.
The most detailed studies were done on rpoI and the divergently transcribed vbsC. Removal (in the case of rpoI) or replacement (for vbsC) led to their enhanced expression, especially in medium that was Fe-supplemented. Thus, the importance of these sequences in Fe-responsive regulation is unequivocal. The locations of the IRORi and IROVc motifs relative to the transcription starts of the corresponding genes were different, but in both cases would be in accord with transcriptional repression of rpoI and vbsC. Further, SDM of individual bases in the 5' regions of the IRORi motif had little or no effect on Fe-responsive regulation of rpoI. These sets of observations suggest that there may be considerable flexibility in the sequences required for the interaction with RirA. This was further borne out by the examination of the IRO-like sequences in the promoter regions of fhuA1 and fhuA2. In the former, the IROFa1 motif overlaps the 35 region of the fhuA1 transcript, just as does IRORi relative to the rpoI promoter. However, the DNA sequences of IROFa1 and IRORi are not strikingly similar, especially towards their 5' ends. The reverse is true for the IROFa2 motif, which strongly resembles the sequence of IRORi, but is significantly further upstream of the fhuA2F promoter than are the other IROs from their corresponding transcription start sites. Such limited sequence similarity between factor-binding sites has been observed previously for other transcriptional regulators. For example, with OxyR from E. coli, the nucleotide contacts required for protein recognition are separated by 10 bp intervals (Toledano et al., 1994) and different genes have different spacings between the OxyR-binding sites and the promoter.
All the fusions studied here were expressed in the heterologous -proteobacterium P. denitrificans, but with one exception (fhuA2F) they were not subject to Fe-responsive repression in this background. The behaviour of most of the lac fusions in this study indicates that P. denitrificans does not have a functional equivalent of RirA. This is not unexpected, since close homologues of RirA exist only in the very near relatives of the rhizobia and are not found even in other
-proteobacteria.
However, the Fe-responsive repression of the fhuA2FlacZ fusion indicates that, at least in some cases, the regions 5' of Fe-regulated R. leguminosarum genes can be recognized by regulators in this -proteobacterium. It is becoming increasingly clear that bacterial genomes are remarkably mosaic in form, with large regions having likely been acquired by lateral gene transfer (Lawrence & Hendrickson, 2003
). Possibly, therefore, fhuA2F was acquired from another bacterium relatively recently, and had retained some of the cis-acting regulatory sequences that were used in its previous host, in addition to having acquired the cognate IRO sequences appropriate for RirA-dependent regulation in R. leguminosarum. Thus, it might be potentially subject to one of two different mechanisms of Fe-responsive regulation, one appropriate for Paracoccus and the other for Rhizobium. We do not know what is/are the Fe-responsive regulator(s) in Paracoccus. Indeed, little or nothing is known of Fe-responsive gene regulation in any
-proteobacterium, even in such genetically well-characterized genera as Caulobacter and Rhodobacter.
However, the lack of Fe-responsive regulation of most of the RirA-regulated operons studied here in P. denitrificans presented an effective means of studying the effects of RirA on its target genes in a heterologous null background. There was a striking repression of fhuA1lacZ, vbsClacZ and rpoIlacZ fusion expression when the cloned rirA was also introduced into P. denitrificans, and this required the corresponding cognate IRO motif to be present. Although the introduced rirA gene may affect the expression of another regulator, native to P. denitrificans, which in turn regulates the fusion plasmid, we feel that this is inherently unlikely. It was noticeable that in P. denitrificans the cloned rirA gene markedly reduced expression of rpoI, fhuC and fhuA1, even in cells that had been grown in Fe-depleted medium. With rpoIlacZ and, to a lesser extent, with fhuA2lacZ, growth in high-Fe medium enhanced the repression, but this was not the case with the vbsClacZ fusion. Therefore, R. leguminosarum (but not P. denitrificans) may have some other factor that contributes to its ability to sense the availability of Fe, ensuring the appropriate level of Fe-dependent control in the native host.
This is the first report to examine the factors involved in RirA-mediated gene regulation in response to Fe availability. Although IRO motifs are clearly important to the ability of RirA to repress gene expression, the differences in the exact behaviour of the promoters that were studied here point to other unknown factors that influence the ability of RirA to affect the expression of specific transcriptional units. Indeed, one of the operons, fhuCDB, whose transcription quite clearly responds to Fe availability in an RirA-dependent way, does not even have a recognizable IRO motif near its promoter.
In addition to the genes involved in the synthesis and uptake of VB, Todd et al. (2002) showed that the Fe-dependent repression of other R. leguminosarum genes, namely haem-uptake (hmu) genes, ABC transporter (fbp) genes and tonB, also requires RirA. The transcriptional start sites of the hmuPSTUV and the orf1tonB operons have been identified (Wexler et al., 2001
); in both cases, a sequence similar to the 3' end of an IRO motif was located at the
35 position. More recently, proteomic studies have shown that >100 polypeptide gene products are differentially expressed in rirA mutant strains of R. leguminosarum, compared to the near-isogenic wild-type strains (J. D. Todd, unpublished observations). The transcript initiation sites for the corresponding genes have not been determined, but it does appear that at least some of these newly discovered RirA-responsive genes do not have a motif with similarity to the IRO motifs in their putative promoter regions.
Lynch et al. (2001) identified a more local form of Fe-responsive gene control in Sinorhizobium meliloti. In this species, rhrA specifies a positive regulator that activates expression of the rhb genes, which are involved in the synthesis of the siderophore rhizobactin 1021. It will be of interest to see if there is any interplay between the wide-ranging regulator RirA and the more specialised RhrA of S. meliloti.
We already know that RirA participates in one regulatory cascade, repressing the transcription of rpoI, whose factor product is, in turn, involved in the transcription of the vbsGSO and vbsADL genes that are required for siderophore biosynthesis (Carter et al., 2002
; Yeoman et al., 2003
). It will be interesting to discover how many of the large number of genes whose expression is affected by RirA involve a direct interaction between this regulator and their cognate promoter regions.
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ACKNOWLEDGEMENTS |
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Received 18 June 2004;
revised 30 August 2004;
accepted 31 August 2004.
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