INTRODUCTION

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Salmonella enterica serovar Typhimurium is a gram-negative bacterium that causes various host-specific diseases. To do so, the pathogen must overcome barriers and manipulate host cells at specific sites along the course of infection. Following ingestion, the bacteria withstand the stomach's acid environment and subsequently colonize the small intestine. In calves and humans, S. enterica serovar Typhimurium induces cytokine production and neutrophil migration across the intestinal epithelium to elicit inflammatory diarrhea (69). In mice, however, the bacteria spread to systemic sites by traversing the intestinal epithelium to reach Peyer's patches, lymphatics, and the bloodstream. Before reaching the Peyer's patches, bacteria can also be intercepted by CD18⁺ phagocytes, which shuttle the bacteria directly to the liver and spleen (67). During systemic infection, the pathogen evades the host's immune response by residing within macrophages, causing a typhoid-like disease (19).

To execute such activities, S. enterica serovar Typhimurium produces virulence factors, including those encoded on the 40-kb Salmonella pathogenicity island 1 (SPI1) at centisome 63 (53). SPI1 genes encode several effectors and a type III secretory apparatus that translocates the effectors directly into the cytosol of intestinal epithelial cells (13, 64). There, the effectors interact with host cell proteins to rearrange the actin cytoskeleton and induce morphological changes that ultimately cause these normally nonphagocytic cells to take up the bacteria in a process called invasion (11, 21, 22, 31, 35). In addition to their roles in invasion, SPI1 invasion genes are important for intestinal colonization (55), destruction of M cells in Peyer's patches (39, 57), activation of cytokine secretion (69), and induction of neutrophil migration (24, 42, 49). Furthermore, effectors secreted by the SPI1 secretion apparatus activate proinflammatory and cytotoxic signal transduction pathways in host cells (11, 34, 35, 42, 54). Thus, the SPI1 type III secretion apparatus and its effectors may function in several ways to promote Salmonella virulence.

Virulence genes are thought to be regulated in the host such that they are expressed only at those sites where their products are needed (47). Unregulated production of virulence factors at inappropriate sites may inhibit the bacteria's ability to cause disease (32). SPI1 invasion genes are regulated in vitro by several transcription factors that may help limit SPI1 invasion gene expression to appropriate sites in vivo. HilA, an OmpR/ToxR family member encoded on SPI1, controls the expression of genes on SPI1, SPI4, SPI5, and SopE (1, 6, 12, 16). HilA directly binds to and activates promoters of SPI1 operons encoding the type III secretory apparatus, several secreted effectors, and InvF, an AraC-like transcriptional regulator (46). InvF promotes expression of HilA-activated effector genes on SPI1 by directly inducing their transcription from a second, HilA-independent promoter (12). InvF also appears to directly induce expression of effector genes outside of SPI1, including sigD(sopB) and sopE (12, 16). Because HilA directly modulates invF expression, InvF-dependent transcription of effector genes is regulated indirectly by HilA. Thus, HilA directly and/or indirectly activates the expression of genes encoding the SPI1 type III secretion apparatus and its secreted effectors, thereby playing a central role in the regulatory hierarchy controlling invasion-related gene expression.

Many two-component regulatory systems have been implicated in modulating hilA expression in vitro. One example is PhoP/PhoQ (7). PhoQ is a membrane sensor that phosphorylates its cognate transcriptional regulator PhoP only when extracellular cation levels are low (68). PhoP~P binds to and activates promoters of pags (PhoP-activated genes), including genes required for bacterial survival in macrophages (26, 27, 52). A point mutation in phoQ called pho-24 renders the sensor hyperactive, resulting in net phosphorylation of PhoP and activation of pag expression even when extracellular cation levels are high (28). The pho-24 mutation also greatly reduces hilA expression, suggesting that PhoP~P represses hilA (7). Interestingly, a disruption in the newly identified pag gene increases hilA expression, indicating that pag represses hilA (18). This suggests that the repression of hilA by PhoP/PhoQ may be mediated by pag. However, it has not yet been determined whether a disruption in pag can overcome the repression of hilA by a pho-24 mutation.

The PhoR/PhoB two-component signal transduction system may also regulate hilA expression. The PhoR sensor kinase phosphorylates PhoB when extracellular P_i levels are low (70). PhoB~P then binds to and activates promoters in the Pho regulon. However, when extracellular P_i concentrations are high, PhoR dephosphorylates PhoB~P, thereby preventing the transcriptional regulator from binding promoters and modulating gene expression. For its phosphatase activity, PhoR requires the presence of the Pst high-affinity P_i uptake system. Disruptions in the pstSCAB-phoU operon, which encodes the Pst system, result in an accumulation of PhoB~P and activation of the Pho regulon even when extracellular P_i levels are high. Such mutations also reduce hilA expression in a phoB-dependent manner, suggesting that PhoB~P represses hilA (48).

Mutant analyses also imply that BarA/SirA and EnvZ/OmpR regulate hilA. BarA and SirA are homologs of the Pseudomonas two-component regulatory factors LemA and GacA, respectively. BarA is believed to activate the transcriptional regulator SirA in response to an unknown environmental signal. Disruptions in sirA and barA repress hilA and invasion gene expression, suggesting that this putative two-component system activates hilA expression (1, 4, 38). Similarly, the EnvZ/OmpR two-component system may activate hilA expression since disruptions in envZ and ompR repress hilA (48). The sensor EnvZ modulates the activity of its cognate transcriptional regulator OmpR in response to changes in osmolarity (60).

In addition to two-component regulatory systems, small nucleoid binding proteins H-NS, HU, and Fis may modulate hilA expression. Recent genetic evidence suggests that H-NS can repress hilA under certain conditions while HU and Fis help activate hilA expression (L. M. Schechter and C. A. Lee, unpublished results, and reference 72). H-NS is a homodimeric protein that preferentially binds to and condenses curved DNA, causing local as well as global transcriptional effects (5). HU is composed of two similar but nonidentical subunits encoded by hupA and hupB. It binds DNA nonspecifically with respect to sequence and influences expression of a number of genes (59). In contrast, Fis is a site-specific DNA binding protein that induces sharp bends (58) and can behave both as an activator and a repressor of gene expression (20). It is unknown whether these proteins modulate hilA expression directly or indirectly.

hilA expression also appears to be modulated in vitro by several other factors whose roles in transcriptional regulation are not fully understood. One such factor, called CsrB, is an RNA that sequesters CsrA, which is a protein that selectively destabilizes specific mRNAs (43, 74). A disruption in csrB or expression of csrA from a plasmid represses hilA, suggesting that one of CsrA's target mRNAs encodes an activator of hilA expression (4). Loss of csrA also decreases hilA expression, suggesting that CsrA affects a repressor of hilA as well (3). A disruption in ams, which encodes RNase E, increases hilA expression, suggesting that RNase E inhibits hilA expression (18). Like CsrA, RNase E may target an RNA that induces hilA expression. Another potential inhibitor of hilA expression is a protein of unknown function called HilE. A disruption in hilE increases hilA expression, suggesting that HilE somehow represses hilA (18).

FliZ, whose function is also not well understood, appears to induce hilA. An enhancer of class II flagellar gene expression (40), FliZ is encoded in an operon with fliA (36), which encodes the alternative sigma factor required for class III flagellar gene expression (56). The fliAZY operon requires the master flagellar gene regulators, FlhD and FlhC, for expression (44). Disruptions in flhDC and fliA that reduce fliZ expression also repress hilA, and the effects of these mutations on hilA expression are complemented by a plasmid expressing fliZ from an inducible promoter (48). Furthermore, controlled expression of fliZ results in high-level expression of hilA, suggesting that FliZ somehow induces hilA expression.

Other factors promoting hilA expression may include FadD and SPI2 gene products, since disruptions in fadD and certain SPI2 genes repress hilA expression (14, 48). fadD encodes acyl coenzyme A synthetase, which is required for the uptake and degradation of long-chain fatty acids (15). SPI2 genes encode another type III secretion system required for S. enterica serovar Typhimurium's ability to survive in macrophages (33). The mechanisms whereby these factors influence hilA expression remain cryptic.

Several environmental conditions, including oxygen, osmolarity, and pH, also regulate hilA expression in vitro, but the sensors and transcription factors responsible for this environmental regulation have not yet been identified (7). Previous studies indicate that phoP is not required for regulation of hilA by any of these conditions (7). pmrA, an Fe³⁺-responsive regulator (73) whose expression and activity are influenced by PhoP/PhoQ and pH (29, 65), is not required for pH regulation of hilA expression (unpublished observations). Furthermore, arcA and oxrA, two transcriptional regulators known to modulate expression of many genes in response to changes in redox states (8), are not required for oxygen-mediated repression of hilA (unpublished observations). However, it is unknown whether the environmental regulation of hilA expression is mediated by two-component signal transduction systems such as PhoR/PhoB, BarA/SirA, or EnvZ/OmpR, which are known to influence hilA expression.

It is also unclear how all of these environmental and regulatory inputs are integrated to modulate hilA expression. Recent evidence indicates that sequences 332 to 39 upstream of the hilA promoter are required for repression of hilA by low osmolarity, high oxygen, the pho-24 mutation, or a disruption in sirA (63). This suggests that the upstream repressing sequences (URS) define a common regulatory node where all of these conditions and mutations converge to reduce hilA expression. It was proposed that an unknown repressor binds to the URS under repressing conditions to reduce hilA expression (63). Two AraC-like transcriptional regulators encoded on SPI1, HilC (also called SirC [61] or SprA [17]) and HilD, also bind to the URS (Schechter and Lee, unpublished results) and appear to derepress hilA expression (63). Thus, the environmental and regulatory inputs may affect hilA expression by altering the expression or activity of HilC, HilD, or the unknown repressor.

Previously, we provided evidence that FadD, FliZ, PhoB, SirA, and EnvZ regulate hilA expression by independent pathways (48). In this paper, we demonstrate that these pathways require the URS to regulate hilA expression. This finding suggests that these regulatory pathways ultimately modulate the repression-derepression mechanism at this site to alter expression of hilA. Our results suggest that the EnvZ/OmpR regulatory pathway modulates hilA expression primarily by altering the expression and/or activity of hilC. In contrast, our results favor models in which all other regulatory factors and environmental conditions tested affect hilA expression by transcriptional and/or posttranscriptional modulation of hilD or by altering the expression or activity of the repressor.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Unless otherwise specified, bacterial cultures were grown at 37°C in Luria-Bertani (LB) medium comprised of 0.5% Bacto yeast extract, 1% Bacto tryptone, and 1% NaCl. For "no salt" growth conditions, NaCl was omitted from the medium. When appropriate, the medium was supplemented with 10 µg of chloramphenicol per ml, 100 µg of ampicillin per ml, 50 µg of kanamycin per ml, or 10 µg of tetracycline per ml. To induce genes under P_BAD control, the medium was supplemented with 0.02% arabinose before bacterial inoculation. All strains containing plasmids that express hilC or hilD from P_BAD also carry a deletion in the chromosomal araBAD operon and therefore cannot metabolize arabinose. -Galactosidase assays were performed on bacterial cultures grown under low-oxygen or high-oxygen conditions as previously described (7, 41), and activities were quantified by the Miller method (51).

DNA methods. Restriction enzymes were obtained from New England Biolabs. PCR was done using Ex Taq polymerase from Takara Shuzo Co. Plasmid DNA was isolated using Qiagen columns, and chromosomal DNA was purified using the Easy-DNA kit from Invitrogen. Enzymes and kits were used according to the manufacturers' directions.

Plasmid construction. pSA4 was produced by cloning hilD downstream of P_BAD in pBAD33 (S. Akbar, unpublished results). pRL692 was constructed by inserting lacZ into the hilD open reading frame (ORF) on pSA4. lacZ was cut out of pCS3 (71) using BamHI and BglII. This fragment was inserted into pSA4's unique BglII site 398 bp downstream of hilD's translational start site, creating a hilD::lacZ transcriptional fusion under P_BAD control. To ensure that lacZ was in the proper orientation for a transcriptional hilD::lacZ fusion, candidates were tested for arabinose induction of lacZ expression and their plasmids were tested by restriction digests.

Bacterial strain construction. Marked mutations were transduced into different strain backgrounds by using P22. Plasmids were passed through the rm⁺ LT2 strain LB5000 (10, 62) before being electroporated into SL1344 derivatives.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Multiple regulatory pathways act at a common node to modulate hilA expression. Because the URS is required for regulation of hilA expression by oxygen, osmolarity, PhoP/PhoQ, and SirA/BarA (63), we tested whether this cis element is also required for regulation of hilA expression by FadD, FliZ, PhoB, and EnvZ. For these experiments, we used two pRW50 reporter plasmids, pLS50 and pLS79, to measure the activity of the hilA promoter. In these plasmids, portions of the hilA promoter and the 5' untranslated region of hilA are cloned upstream of a promoterless lacZ gene. pLS50 contains 332 to +416 of hilA, and pLS79 contains 39 to +416 of hilA.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Disruptions in fadD, fliA, pstS, and envZ require region 332 to 39 upstream of the hilA promoter to reduce hilA expression. The following mutations were used in this experiment: fadD1::Tn5, fliA51::Tn5, pstS55::Tn5, and envZ182::cam. -Galactosidase assays were performed on cultures grown in high-salt LB medium (1% NaCl) under oxygen-limiting conditions (7). Averages were calculated using four or more values from at least two different experiments. a, standard deviation of 633; b, standard deviation of 263. WT, wild type.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Disruptions in fadD, fliA, pstS, envZ, and sirA do not abolish regulation of hilA080::Tn5lacZY expression by oxygen and osmolarity. The following mutations were used in this experiment: fadD1::Tn5, fliA51::Tn5, pstS55::Tn5, envZ182::cam, and sirA2::kan. -Galactosidase assays were performed on cultures grown as indicated. Cultures exposed to activating conditions were grown in high-salt LB medium (1% NaCl) under oxygen-limiting conditions. No-salt cultures were grown in LB medium lacking NaCl under oxygen-limiting conditions. High-oxygen cultures were grown in high-salt LB medium (1% NaCl) under high-oxygen conditions to OD600s of approximately 0.2 to 0.3 (7, 41). Averages were calculated using six or more values from at least three different experiments. Error bars represent standard deviations. WT, wild type.

Regulation of mutants by oxygen and osmolarity. The apparent convergence of multiple regulatory pathways at a common site upstream of the hilA promoter could be explained in part if regulation of hilA expression by oxygen or osmolarity is mediated by one of these regulatory factors. For example, if the EnvZ/OmpR two-component system is responsible for osmoregulation of hilA expression, this would explain why both low osmolarity and a disruption in envZ require the URS to reduce hilA expression.

Effects of environmental conditions and regulatory mutations on hilC and hilD expression. Although the regulatory pathways modulating hilA expression appear to act independently of each other (48), their convergence at the URS suggests that they all ultimately influence a common mechanism that directly regulates hilA expression. HilC and HilD are thought to bind directly at this site to derepress the hilA promoter under activating conditions (Schechter and Lee, unpublished results). Thus, the environmental conditions and regulatory mutations that modulate hilA expression may do so by regulating expression of either hilC or hilD. HilC and HilD are encoded at different locations on SPI1 and are transcribed separately. Therefore, we examined the effects of these conditions and mutations on expression of hilC9::Tn5lacZY and hilD696::lacZ chromosomal fusions.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Regulation of hilC and hilD expression by environmental conditions and regulatory mutations. -Galactosidase activity for each fusion is expressed as a percentage of its activity in a wild-type (WT) background under activating conditions. Error bars represent the standard deviation of normalized values. (A) No-salt and high-oxygen conditions reduce expression of hilC9::Tn5lacZY and hilD696::lacZ. -Galactosidase assays were performed on cultures grown as described for Fig. 2. Average percentages were calculated by using 10 or more values from at least three different experiments. (B) Effects of mutations on hilC9::Tn5lacZY and hilD696::lacZ expression. The mutations used in these experiments were as follows: fadD1::Tn5, fliA51::Tn5, pstS55::Tn5, sirA2::kan, envZ182::cam, hilD1::kan, and hilC1::kan. -Galactosidase assays were performed on cultures grown in high-salt LB medium (1% NaCl) under oxygen-limiting conditions. Average percentages were calculated by using four or more values from at least two different experiments. Typical -galactosidase activities (Miller units) for fusions in a wild-type background under activating environmental conditions were as follows: hilC9::Tn5lacZY, 1,081 units; hilD696::lacZ, 1,456 units.

Roles of hilC and hilD in regulation of hilA expression by environmental conditions and regulatory mutations. The modulation of hilC and hilD expression by oxygen and osmolarity might account for the regulation of hilA expression under these conditions. Furthermore, although the repression of hilC by fadD, fliA, pstS, envZ, and sirA mutations is mild, it is still possible that the modest effects of these mutations on hilC expression may help to cause the repression of hilA in these mutants. Similarly, although the repression of hilD in sirA and pstS mutants is very modest, it is still possible that this mild reduction in hilD expression helps mediate the repression of hilA in these mutants. Alternatively, these conditions and mutations may modulate hilC or hilD posttranscriptionally, thereby affecting hilA expression.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Roles of hilC and hilD in regulation of hilA expression by environmental conditions and regulatory mutations. (A) Regulation of iagB87::lacZY expression by oxygen and osmolarity is hilC independent and hilD dependent. -Galactosidase assays were performed on cultures grown as described for Fig. 2. Averages were calculated using four or more values from at least two different experiments. (B) The effects of fadD, fliA, pstS, and sirA disruptions on iagB87::lacZY expression are hilC independent. The following mutations were used in this experiment: fadD1::Tn5, fliA51::Tn5, pstS55::Tn5, sirA2::kan, ompR1009::Tn101617, and hilC1::cam. -Galactosidase assays were performed on cultures grown in high-salt LB medium (1% NaCl) under oxygen-limiting conditions. Averages were calculated using three or more values from at least two different experiments. (C) The effects of disruptions in envZ, pst, and fliA on iagB87::lacZY expression are hilD dependent. The following mutations were used in this experiment: envZ182::cam, pst-4::Tn10, fliA36::Tn5B50, and hilD1::kan. -Galactosidase assays were performed on cultures grown in high-salt LB medium (1% NaCl) under oxygen-limiting conditions. Averages were calculated using six or more values from at least two different experiments. Error bars represent standard deviations. WT, wild type.

Controlled expression of hilC or hilD abolishes regulation of hilA expression by environmental conditions and regulatory mutations. Another way to investigate whether the regulation of hilC or hilD expression plays any role in the regulation of hilA expression by environmental conditions and regulatory factors is to induce hilC or hilD expression under repressing conditions. If repression by a particular regulatory pathway is specifically overcome by expressing either hilC or hilD, it might suggest that this regulatory pathway affects hilA expression primarily by controlling hilC or hilD expression. Because certain environmental conditions and regulatory mutations affect hilC or hilD expression and appear to regulate hilA expression in a hilC- or hilD-dependent manner, we expected that controlled expression of hilC or hilD would abolish the effects of some conditions and mutations on hilA expression but not others.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Repression of hilA080::Tn5lacZY expression by low osmolarity is suppressed by arabinose-induced expression of hilC or hilD. -Galactosidase assays were performed on cultures grown as described for Fig. 2. Media were supplemented with 0.02% arabinose prior to inoculation as indicated. Averages were calculated using six or more values from at least three different experiments. Error bars represent standard deviations.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Repression of hilA080::Tn5lacZY expression by high-oxygen conditions is overcome by arabinose-induced expression of hilC or hilD. -Galactosidase assays were performed on cultures grown in high-salt LB medium (1% NaCl) with or without 0.02% arabinose under extended high-oxygen conditions as described in Materials and Methods. (A) After an initial lag, arabinose induction of hilC or hilD results in high-level expression of hilA080::Tn5lacZY under high-oxygen conditions. philC is pLS119, and philD is pSA4. Each curve is comprised of data points from at least four different experiments. (B) The delay in philD-mediated derepression of hilA expression is due to a lag in PBAD induction. pRL692 is philD (pSA4) with lacZ cloned into the hilD ORF. Each curve is comprised of data points from three different experiments.

View larger version (34K): [in this window] [in a new window]   FIG. 7.   Repression of hilA080::Tn5lacZY expression by disruptions in fadD, fliA, pstS, sirA, envZ, and hilD is overcome by arabinose-induced expression of hilC or hilD. The mutations used in these experiments were as follows: fadD1::Tn5, fliA51::Tn5, pstS55::Tn5, sirA2::kan, envZ182::cam, and hilD1::kan. philC is pLS119, and philD is pSA4. -Galactosidase assays were performed on cultures grown under oxygen-limiting conditions in high-salt LB medium (1% NaCl) with or without 0.02% arabinose as indicated. Averages were calculated using four or more values from at least two different experiments. wt, wild type.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Previously, Schechter et al. demonstrated that hilA expression is repressed in the absence of hilD (63). Because the URS is required for repression, an unidentified repressor is thought to bind to this region, thereby inhibiting hilA expression. When present, HilC or HilD binds to this same site and promotes hilA expression (63; Schechter and Lee, unpublished results). However, when this site is removed, the hilA promoter is no longer repressed even when hilD and hilC are absent. These results suggest a model in which HilC and HilD, unlike most other AraC-like transcriptional regulators (23), are not activators but instead behave as derepressors of hilA expression (Fig. 8). In this model, the repressor is always present but cannot inhibit hilA expression under activating conditions because HilD (and, to a lesser extent, HilC) displaces it from the URS. However, when hilD is absent, the repressor can bind to this site to inhibit hilA expression even under activating environmental conditions.

View larger version (23K): [in this window] [in a new window]   FIG. 8.   Model for regulation of hilA expression. hilA expression may be regulated by modulating the expression or activity of hilC or hilD. R is an unknown repressor. Under activating conditions, HilC and HilD bind to region 332 to 39 upstream of the hilA promoter, displacing the repressor from this site. This allows derepression of hilA expression. High oxygen, low osmolarity, and PhoB~P repress expression of hilC and hilD and may modulate hilD posttranscriptionally. SirA, OmpR, FadD, FliZ, and HilD promote hilC expression, and OmpR may also modulate hilC posttranscriptionally. SirA, OmpR, FadD, and FliZ may also modulate hilD posttranscriptionally to regulate hilA expression.

Previous results as well as data from this study demonstrate that the URS is also required for modulation of hilA expression by oxygen, osmolarity, PhoP/PhoQ, FadD, FliZ, PhoB, SirA, and EnvZ (63). None of the regulatory factors affecting hilA expression appears to be responsible for regulating the expression of hilA in response to oxygen or osmolarity. Also, previous results indicate that many of these regulatory factors act independently of each other to modulate hilA expression (48). Thus, multiple independent regulatory pathways converge at the URS, suggesting that they may all modulate the repression-derepression mechanism to regulate hilA expression. Such modulation might include altering the expression or activity of HilD, HilC, or the unknown repressor.

HilD. Mutations in fadD, fliA, envZ, and hilC have no effect on hilD expression. This suggests that these mutations do not repress hilA by modulating hilD expression. However, the finding that the fliA, envZ, and hilC mutations no longer affect hilA expression in a hilD mutant suggests that they may be modulating hilA expression by posttranscriptional effects on hilD. The fadD mutation may also affect hilA expression by modulating hilD posttranscriptionally. Alternatively, regulation by all of these mutations may be mediated by modulation of the repressor (see below).

HilC. Interpreting the impact of controlled hilD expression on the modulation of hilA expression was made even more complicated by the fact that hilC, when expressed to high levels, can substitute functionally for hilD. Schechter et al. found that high-level expression of hilC could derepress the hilA promoter even in the absence of hilD (63). Furthermore, we found that controlled expression of hilC overcomes repression of chromosomal hilA expression by high oxygen levels, low osmolarity, and all mutations tested (Fig. 5, 6, and 7). Arabinose-induced expression of hilC overcomes repressing conditions and mutations even when hilD is disrupted (data not shown), suggesting that HilC is directly derepressing hilA expression. In support of this hypothesis, HilC binds to the URS in vitro (Schechter and Lee, unpublished results). Thus hilC, which is also a member of the AraC family, can behave as a derepressor of hilA expression when produced at high levels.

Parallels between regulation of hilC and hilA expression. Rakeman et al. observed that hilC expression is reduced by a disruption in sirA (61). Our results confirm this observation, though our sirA mutation has a milder effect on hilC expression than that reported by Rakeman et al. In addition, we found that hilC expression is repressed by high-oxygen conditions, low osmolarity, and all regulatory mutations tested. Thus, although hilC is not required for the regulation of hilA expression by any of these conditions or mutations, hilC expression is regulated in a manner that parallels the regulation of hilA expression. hilC expression is unaffected by a disruption in hilA (61), but it is mildly reduced by a disruption in hilD. This suggests that the same repression-derepression mechanism that regulates hilA expression may also affect hilC expression. If so, future studies on the regulation of hilC expression by all of these environmental conditions and regulatory mutations may provide clues about how hilA expression is regulated. Interestingly, preliminary results indicate that in a hilD mutant, hilC expression is even further repressed by low-osmolarity conditions, suggesting that osmoregulation of hilC expression is hilD independent (data not shown). The same may be true for osmoregulation of hilA expression.

Repression. Although this study focused on the roles of hilC and hilD in regulating hilA expression, this regulation could also be mediated by affecting the expression or activity of a repressor. The finding that arabinose-induced expression of hilD overcomes the repression of hilA by all environmental conditions and regulatory mutations would seem to argue against this possibility. However, controlled expression of hilD from the P_BAD promoter may result in such high levels of HilD that the repressor is completely outcompeted for binding at the URS. If repression by a particular condition or mutation is mediated by increasing the expression or activity of the repressor, this effect could be counteracted by flooding the system with so much HilD that the repressor is unable to bind to the URS at all. In such a situation, the abundance and activity of the repressor would become irrelevant, such that environmental conditions and regulatory mutations acting through the repressor would no longer affect hilA expression. Similarly, if hilD is absolutely required for expression of hilA, increased expression or activity of the repressor might have no effect in a hilD mutant. This might explain why hilA expression is not strongly affected by environmental conditions or regulatory mutations in the absence of hilD. Thus, our data do not rule out models in which environmental conditions and regulatory mutations modulate hilA expression by altering the expression or activity of a repressor.

Future directions. Our results have excluded hilC from playing a major role in the in vitro regulation of hilA expression by all environmental conditions and regulatory factors tested (except EnvZ/OmpR). However, regulation of hilC expression by these conditions and regulatory factors parallels the regulation of hilA expression. This implies that studies on the regulation of hilC expression may provide more clues about how hilA is regulated. In fact, something equivalent to the URS may be present upstream of the hilC promoter, and future studies must explore this possibility.