Department of Microbiology and Immunology, University of Maryland, School of Medicine, 655 W. Baltimore St, Baltimore, MD 21201, USA
Correspondence
Harry L. T. Mobley
hmobley{at}umaryland.edu
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ABSTRACT |
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INTRODUCTION |
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The P. mirabilis urease gene cluster, ureDABCEFG, is induced in the presence of urea and UreR, the transcriptional regulator of the gene cluster (D'Orazio & Collins, 1993, 1995
; D'Orazio et al., 1996
; Island & Mobley, 1995
; Nicholson et al., 1993
). P. mirabilis UreR, a dimer of 33·4 kDa polypeptides is an AraC-like transcriptional regulator that activates transcription from pureD and pureR in a urea-inducible manner (D'Orazio et al., 1996
; Island & Mobley, 1995
). Thomas & Collins (1999)
have shown, using electrophoretic mobility shift assay, that there are two binding sites for UreR in the ureR-ureD intergenic region, resulting in a major band shift at low concentrations of UreR (occupation of a single binding site) and an additional minor band shift at higher concentrations of UreR (occupation of two sites). The UreR-binding sites upstream of both the ureR and ureD promoters have been mapped and have the consensus sequence T(A/G)(T/C)(A/T)(T/G)(C/T)T(A/T)(T/A)ATTG (Thomas & Collins, 1999
). Our laboratory has shown that UreR requires dimerization to bind its target DNA sequence in the ureR-ureD intergenic region and to activate transcription (Poore et al., 2001
). Collins and colleagues demonstrated that UreR binds urea (Gendlina et al., 2002
) and has a higher affinity for both DNA-binding sites in the presence of urea (Thomas & Collins, 1999
).
We previously implicated H-NS in the regulation of the urease gene cluster. H-NS, a 15·6 kDa histone-like nucleoid-structuring protein and a known transcriptional repressor, is involved in the negative regulation of the gene cluster (Coker et al., 2000). The ureR-ureD intergenic region contains poly(A) tracts that appear to be the target of H-NS (Coker et al., 2000
). Deletion of the poly(A) tracts in the intergenic region resulted in a derepression of urease expression (Coker et al., 2000
). Poly(A) tracts that are in phase on the same face of the DNA lead to DNA bending which can negatively or positively influence gene expression (Koo et al., 1986
; Lucht et al., 1994
; Owen-Hughes et al., 1992
). H-NS preferentially recognizes and binds to intrinsically curved DNA sequences (Yamada et al., 1991
) and can affect the DNA structure by compacting DNA (Spassky et al., 1984
), constraining DNA supercoils (Tupper et al., 1994
) and introducing DNA bending (Spurio et al., 1997
). There is no apparent sequence specificity for H-NS binding to DNA. However, because H-NS is involved in the regulation of the urease gene cluster and has been shown to bind poly(A) tracts that contain intrinsic curvature, we predicted that the poly(A) tracts found within the ureR-ureD intergenic region also contain intrinsic curvature which H-NS recognizes and binds.
Temperature and osmolarity affect the way in which H-NS regulates virulence gene expression in several species. For example, Shigella flexneri invasion genes, encoded on the large pINV plasmid, are repressed by chromosomally encoded H-NS under conditions of low osmolarity and temperatures less than 30 °C (Maurelli & Sansonetti, 1988; Porter & Dorman, 1994
). H-NS has also been shown to be a thermo-osmotic regulator of the enteroinvasive Escherichia coli pINV-encoded virulence genes, inhibiting expression of the virulence genes in medium or low osmolarity or at 30 °C (Dorman et al., 1990
; Hromockyj et al., 1992
; Maurelli & Sansonetti, 1988
; Porter & Dorman, 1994
). Several factors are involved in H-NS repression of the genes encoding cholera toxin and toxin-coregulated pilus in Vibrio cholerae, including temperature, osmolarity and pH (Gardel & Mekalanos, 1994
; Miller & Mekalanos, 1988
). H-NS is a global regulator clearly involved in the regulation of expression of many genes, including virulence genes.
In this study, we investigated the regulation of the P. mirabilis gene cluster by UreR and H-NS. Purified H-NS-Myc-His bound to the ureR-ureD intergenic region; competition experiments between H-NS and UreR elucidated the ability of these proteins to compete with each other for the intergenic region DNA. Structural characteristics of the intergenic region DNA and the effect of H-NS on DNA bending were also shown. Finally, because H-NS regulates other virulence factors in a temperature-dependent manner, we studied the differential regulation of ureR and urease gene expression by H-NS and UreR at 25 and 37 °C in the absence and presence of urea.
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METHODS |
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Bacterial strains and growth conditions.
Bacterial strains used in this study are listed in Table 1. Bacteria were cultured either in LuriaBertani (LB) broth (l-1: 10 g Tryptone, 5 g yeast extract and 0·5 g NaCl) at 37 °C with aeration in a shaking incubator (200 r.p.m.) or on LB plates containing 1·5 % agar at 37 °C. Plates were supplemented with the antibiotics chloramphenicol (10 µg ml-1), tetracycline (7·5 µg ml-1), ampicillin (100 µg ml-1) or kanamycin (50 µg ml-1) as necessary.
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Sequencing.
Both strands of each plasmid construct were sequenced across each junction and throughout the insert. Plasmids were sequenced by dideoxy chain termination at the Biopolymer Core Facility at the University of Maryland, Baltimore, using an Applied Biosystems model 373A automated DNA sequencer using the Big Dye Terminator Cycle Sequencing Kit.
Purification of UreR-Myc-His and H-NS-Myc-His.
Purification of both proteins followed protocol described previously for UreR-Myc-His (Poore et al., 2001).
Gel shift analysis for H-NS binding.
Purified H-NS-Myc-His (0350 nM) was incubated with target DNA (2·6 nM) in binding buffer [10 mM Tris/HCl (pH 8·0) 10 mM KCl, 100 mM NaCl, 0·5 mM EDTA, 0·5 mM DTT, 5 % (v/v) glycerol, 10 µg BSA ml-1] for 30 min at 25 °C. Binding reactions were separated on 5 % non-denaturing polyacrylamide gels. Transfer of the DNA to the membrane and development of the membrane were performed as described previously (Poore et al., 2001).
Competition experiments between UreR and H-NS for the ureR-ureD intergenic region using gel shift analysis.
For the competition studies, the first protein was incubated with target DNA (2 nM) in binding buffer (as above) for 15 min at the temperature indicated. The second of the two proteins was added to the binding reaction and incubated for an additional 15 min at the indicated temperature. Binding reactions were separated on 5 % non-denaturing polyacrylamide gels. Transfer of the DNA to membrane and development of the membrane were performed as described previously (Poore et al., 2001).
Circular permutation analysis of the ureR-ureD intergenic region.
The permutation vector, pBend3 carrying 322 bp of the ureR-ureD intergenic region (pCP106), was cut singly with one of the following enzymes: MluI, BglII, NheI, XhoI, PvuII, SmaI, RsaI and BamHI. Linear fragments were separated on a 5 % non-denaturing polyacrylamide gel at 10 °C at 10 V cm-1 and stained with 1 µg ethidium bromide ml-1. The bend angle () was calculated using the equation (µM/µE)=cos(
/2), where µM is the relative migration of the fragment where the bend centre is in the centre and µE is the relative migration of the fragment where the bend centre is at the end of the fragment (Thompson & Landy, 1988
). All fragments were assayed in three or more independent experiments.
Circular ligation assay.
The fragment used in the circularization assay was PCR-amplified using the Roche PCR DIG Probe Synthesis Kit. For PCR amplification of the 168 bp fragment, a 1 : 30 final dilution of labelled DIG-11-dUTPs to unlabelled dTTPs was used which led to a final incorporation of 3·5 labelled DIG-11-dUTPs per DNA molecule. The fragment was PCR-amplified with Pfu polymerase and separated by agarose gel electrophoresis. Fragments were purified using the Qiaquick Gel Extraction Kit and digested with XbaI cutting at the ends of both DNA fragments. Ligation reactions were done in the presence of ligase (4 U per 20 µl reaction) and different concentrations of purified H-NS-Myc-His (01·6 µM). Ligase and protein were removed from the DNA in the ligation reactions by phenol/chloroform extraction followed by ethanol precipitation of the DNA. The gel shift control reaction contained the same components as the ligation reactions, minus the ligase, and was not subject to the extraction/precipitation steps. The precipitated ligation reactions and gel shift control were separated on a 5 % non-denaturing polyacrylamide gel. Transfer and development of the membrane was performed as described previously (Poore et al., 2001
).
Construction of hns null mutant in E. coli MC1061.
A P1 phage lysate was prepared from ATM121 [E. coli MC4100(hns : : Tn10)] and used to transduce E. coli MC1061 to Tcr by standard procedures (Hromockyj et al., 1992). An hns disruption was verified in Tcr transductants by PCR analysis using primers that amplify hns and the hns knockout isolate [MC1061(hns : : Tn10)]. MC1061 and MC1061 (hns : : Tn10) were transformed with pMID1010 encoding the P. mirabilis urease gene cluster by CaCl2 transformation (Sambrook et al., 1989
) for the measurement of urease activity using the urease phenol-hypochlorite assay. MC1061 and MC1061(hns : : Tn10) were transformed with pCC002 and pLC9801 by CaCl2 transformation (Sambrook et al., 1989
) for the
-galactosidase expression assay.
Urease extract preparations and protein determinations.
E. coli MC1061 and MC1061(hns : : Tn10), both transformed with pMID1010, and P. mirabilis HI4320 were cultured in LB containing the appropriate antibiotic and 1 µM NiCl2 overnight at 37 °C with aeration. Overnight cultures were used to inoculate fresh LB medium (1 : 100) containing antibiotics and 1 µM NiCl2. Cultures were grown to an OD600 of 0·40·6 and then induced in the absence or presence of 100 mM urea for 2 h. Extracts were prepared as described by McGee et al. (1999).
Phenol-hypochlorite urease assay.
Urease activity was determined as described by McGee et al. (1999).
-Galactosidase expression assays.
Fresh medium was inoculated with a single colony from LB agar plates containing the appropriate antibiotics and cultured at 37 °C overnight. Overnight cultures (5 ml) were used to inoculate fresh medium (1 : 100). Cultures (5 ml) were monitored until they reached an OD600 of 0·40·6, at which time urea was added and cultures were incubated for an additional 2 h. Cultures were then placed on ice and the OD600 was measured. Chloroform (100 µl) and 0·1 % SDS (50 µl) were added and the cultures were vortexed. The suspension of permeabilized cells (50 µl) was added to 950 µl of Z buffer (Platt et al., 1972) and 200 µl O-nitrophenyl-
-D-galactopyranoside (4 mg ml-1 in dH2O). Timed reactions were stopped with 500 µl 1 M Na2CO3. OD420 and OD550 measurements were recorded and Miller units were calculated (Platt et al., 1972
). All constructs were assayed in three or more independent experiments.
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RESULTS |
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Competition of H-NS with UreR for the ureR-ureD intergenic region
Competition of H-NS for the DNA (609 bp fragment) occupied by UreR was also studied using gel mobility shift assays. UreR binding to the target DNA retarded the mobility of the fragment into the gel (Fig. 3c, lanes 4, 5, 9 and 13). Higher concentrations of H-NS resulted in elimination of the UreR-shifted bands (Fig. 3c
, lanes 6 and 11). Interestingly, bands corresponding to either an H-NS-shifted or UreR-shifted complex were not evident in these lanes, possibly suggesting an interaction between UreR and H-NS. However, when 0·4 µM H-NS was added to 3·2 µM UreR (Fig. 3c
, lane 14), a supershifted band appeared, which differed from the complete loss of a shifted band seen for lower concentrations of UreR (Fig. 3c
, lanes 6 and 11). This supershift could also be the result of an interaction between UreR and H-NS during the displacement of one protein for the other. For each concentration of UreR assayed, 0·81·6 µM H-NS was sufficient to displace UreR from the target DNA (Fig. 3c
, lanes 8, 12 and 15), with the resulting H-NS-DNA complex migrating the same distance into the gel as the shift seen in the lane containing only H-NS and DNA (Fig. 3c
, lane 4). These data indicate that H-NS has the ability to displace UreR from the intergenic region in the absence of urea.
In the presence of 100 mM urea, however, H-NS was unable to bind the target DNA (Fig. 3d, lanes 14). UreR-binding to the DNA in the presence of urea resulted in two shifted bands, similar to those seen in Fig. 3(b)
. The addition of 1·6 µM H-NS appears to have an effect on UreR binding to the target DNA as shown by the disappearance of the UreR-shifted complexes (Fig. 3d
, lanes 8 and 12).
Circular permutation analysis of the ureR-ureD intergenic region
Because H-NS preferentially binds intrinsically curved DNA (Yamada et al., 1991), we hypothesized that the ureR-ureD intergenic region contains specific sequences that mediate intrinsic curvature to which H-NS binds. Circular permutation analysis enables the estimation of a bend angle and the location of the bend with a curved sequence in a DNA fragment. As described by Wu & Crothers (1984)
, the electrophoretic mobility of a curved DNA fragment is decreased as the bending locus is moved towards the centre of the fragment. Differences in mobility are greatest when the fragments are separated at 10 °C (Mizuno, 1987
). Thus, to determine the position in a sequence that contained the static curvature, circular permutation analysis was performed on a DNA fragment from the ureR-ureD intergenic region. This assay enabled the movement of the putative bend centre from the centre of the DNA fragment to the end using unique restriction sites within the pBend3 permutation vector (Fig. 4
a). A portion of the ureR-ureD intergenic region (322 bp; Fig. 1
) was cloned into the XbaI and SalI sites in pBend3 producing pCP106. After digesting pCP106 individually with eight different enzymes (MluI, BglII, NheI, XhoI, PvuII, SmaI, RsaI and BamHI), the fragments were separated on a 5 % non-denaturing polyacrylamide gel at 10 °C. The relative migration of each fragment was calculated and plotted (Fig. 4
). The net bend angle of the entire intergenic region was calculated to be 46° (see Methods). The mapped location of the bend at the -60 position in relation to the ureD transcriptional start falls just outside the poly(A) tract (* in Fig. 1
). This experimental calculation is supported by a prediction made by the bend.it server at http://www.icgeb.trieste.it/dna, which predicted the curved sequence (# in Fig. 1
) to be located approximately 15 bp from the mapped site (Munteanu et al., 1998
). Furthermore, the analysis of two additional DNA fragments (410 bp insert size and 401 bp insert size), each containing the poly(A) tracts at a different location within the insert, place the bend centre within 10 and 11 bp, respectively, on either side of the predicted -60 bend site (data not shown).
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A thermoregulatory function of H-NS was also evident in the case of urease expression. Urease activity was significantly lower for MC1061 (H-NS+ strain) grown at 25 °C than at 37 °C (P<0·0001), both in the absence of urea (Fig. 6b). The urease activities of the H-NS-deficient strain cultured in the absence of urea at 25 and 37 °C were not significantly different. Because there was a difference in the urease activity in the cultures grown at 25 vs 37 °C in the presence of H-NS and no difference in the urease activity in the cultures grown at the two different temperatures in the absence of H-NS, this indicated that H-NS represses urease expression in a temperature-dependent manner in the absence of urea. In the presence of urea, a 4·5-fold increase was seen in urease activity for the wild-type strain cultured at 37 vs 25 °C. The H-NS-deficient strain, in the presence of urea, showed only a 2·7-fold increase in urease activity when cultures were grown at 37 vs 25 °C. In the presence of urea, MC1061 (H-NS+ strain) showed a 1·7-fold higher activation than the H-NS-deficient strain when cultures were assayed at 37 vs 25 °C, indicating that H-NS plays a role in the temperature-dependent repression of the urease gene cluster in the presence of urea.
The urease activity for the H-NS-deficient strain cultured at 25 °C was not statistically different in the absence and presence of urea, 1·86 versus 2·14 µmol min-1 mg-1, respectively (P>0·1), indicating that derepression and a loss of UreR/urea inducibility was occurring in the H-NS-deficient strain. These data demonstrated that H-NS is involved in the repression of the P. mirabilis urease gene cluster in the E. coli MC1061 model.
Effect of culture temperature and urea on -galactosidase expression from pureR in E. coli MC1061 and MC1061(hns : : Tn10) transformed with pCC002 and pLC9801
The target of H-NS repression of the urease gene cluster is unclear. To clarify this, H-NS modulation of the ureR promoter was studied by transforming E. coli MC1061 and MC1061(hns : : Tn10) with both pCC002 (constitutively expresses UreR) and pLC9801 (ureR-lacZ reporter construct). MC1061 had higher activity when cultured at 37 °C (420 and 844 units in the absence and presence of urea, respectively) than at 25 °C (240 and 648 units in the absence and presence of urea, respectively). At 25 and 37 °C, MC1061 is inducible with 100 mM urea (Fig. 6c). However, in MC1061(hns : : Tn10), ureR transcription was completely derepressed in the absence of urea at either temperature. MC1061(hns : : Tn10) (± urea) did, however, have significantly more
-galactosidase activity at 37 °C (2590 and 2835 Miller units, in the absence and presence of urea, respectively) than at 25 °C (1183 and 1147 Miller units, in the absence and presence of urea, respectively) (P<0·0001), indicating that temperature is responsible for the increased transcription of ureR at 37 °C in the H-NS-deficient strain.
A significant increase in -galactosidase activity was apparent in the H-NS-deficient strain in comparison to the wild-type strain under all conditions (P<0·0001 at 25 °C in the absence of urea, P=0·001 at 25 °C in the presence of urea, P<0·0001 at 37 °C in the absence of urea and P<0·0001 at 37 °C in the presence of urea). H-NS clearly represses transcription from pureR under all conditions assayed.
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DISCUSSION |
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This differential regulation of urease by H-NS and UreR is biologically relevant. P. mirabilis urease, a recognized virulence factor, is expressed by this pathogen in the urinary tract (Johnson et al., 1993; Jones et al., 1990
; Li et al., 2002
). At 37 °C (the temperature in the host) and in the presence of high concentrations (
400 mM) of urea in urine, H-NS repression is alleviated and UreR activates transcription of the urease gene cluster. Outside the host at 25 °C or lower in the absence of urea, H-NS represses urease genes and UreR binds only weakly to the intergenic region. Urease is not likely to be advantageous under these circumstances and is not required for growth. Indeed, unnecessary synthesis of the enzyme in the absence of substrate is energetically inefficient.
H-NS has been genetically implicated by our group in the negative regulation of the urease gene cluster (Coker et al., 2000). In the present study, however, binding of purified H-NS-Myc-His to the 609 bp ureR-ureD intergenic region was directly demonstrated by gel shift analysis. The addition of >100 nM P. mirabilis H-NS protein to the target DNA resulted in retardation of the electrophoretic mobility of the bands, all of which migrated the same distance into the gel, indicating that the H-NS binding sites in the intergenic region were saturated at and above that protein concentration (Fig. 2
). Thus, we conclude that H-NS specifically binds the ureR-ureD intergenic region, supporting a regulatory role of H-NS in urease expression.
The ability of UreR to displace H-NS from the intergenic region is obviously critical in the transcriptional activation of the urease genes by UreR. Our data support the work of Thomas & Collins (1999) in that two UreR-induced shifts were observed in the presence of urea, signifying UreR-binding to one or both of the UreR binding sites on the target DNA. UreR was able to displace H-NS from the DNA in the absence of urea (Fig. 3a
). Interestingly, under conditions where maximal induction of urease expression is achieved (e.g. 100 mM urea), H-NS was unable to bind to the target DNA (Fig. 3b
). Although we were unable to directly show that UreR could displace H-NS from its binding site in the presence of urea, binding reactions containing higher concentrations of H-NS did not present conditions suitable for UreR binding to the DNA (Fig. 3b
, lane 15). It is important to note that the concentrations of proteins and urea used in the competition experiments may not necessarily reflect the actual concentrations in the cytosol of the bacterium. In addition, competition assays required the development of a buffer that allowed binding of both UreR and H-NS. This buffer was different from the buffers used for H-NS or UreR alone. Thus some differences can be noted on gel shift experiments conducted for one protein alone (e.g. Fig. 1
) as opposed to both proteins (e.g. Fig. 3
). Nevertheless, the gel shift studies provide insight into how UreR and H-NS may behave in the presence of each other and the target DNA.
We also hypothesized that the displacement of UreR by H-NS is essential for reintroducing H-NS repression of urease gene transcription when the enzyme is no longer required (e.g. at 25 °C in the absence of urea, indicative of when the bacterium leaves the host). Gel shift competition experiments showed that H-NS can displace UreR from the intergenic region in the absence of urea (Fig. 3c). An interaction between H-NS and UreR may be required for the removal of UreR by H-NS, which is supported by the loss of UreR-shifted bands without the formation of H-NS-shifted bands upon the addition of H-NS (Fig. 3c
). Formation of a supershifted band (Fig. 3c
, lane 14), which migrates more slowly through the gel than the shifted bands for either UreR or H-NS, also supports the idea of an interaction between H-NS and UreR.
In the presence of urea (i.e. inducing conditions for urease expression), higher concentrations of H-NS were required to remove, but not necessarily replace, UreR from the DNA as shown by the loss of the UreR-shifted bands (Fig. 3d). H-NS binding to the DNA under these conditions was not determined. Concentrations of H-NS used in the absence of urea (Fig. 3c
) that allowed for H-NS-binding to the DNA were not sufficient for H-NS-binding in the presence of urea (Fig. 3d
).
H-NS preferentially binds the ureR-ureD intergenic DNA sequence at an AT-rich region with intrinsic curvature. Circular permutation analysis demonstrated that the area of curvature, with a bend angle of 46°, was localized to the -60 position (±10 bp) in relation to the ureD transcriptional start site. Circular permutation analysis of larger fragments yields an overall estimate of intrinsic curvature for the entire fragment, without elucidating small, structural intricacies throughout the fragment. However, three individual fragments and the prediction from the bending server place the bend centre within a 20 bp area in the ureR-ureD intergenic region.
Further evidence for intrinsic curvature of the intergenic region and additional H-NS-induced bending was provided by the ligase-mediated circularization assay. A portion of the ureR-ureD intergenic region (168 bp) containing both the UreR mapped binding sites and the curved poly(A) tract was incubated in the presence of ligase and/or H-NS. The 168 bp dsDNA fragment was unable to be ligated in the presence of ligase alone; however, adding H-NS to the ligation reaction resulted in the formation of circular monomers, which was achieved by the ligation and closure of one 168 bp DNA molecule (Fig. 5). Indeed, Spurio et al. (1997)
showed that when short uncurved fragments (155 bp) were incubated in the presence of H-NS and ligase, circular monomers formed. Thus, we conclude that H-NS binds to the intrinsically curved ureR-ureD intergenic region bending this fragment to a greater degree.
Several studies have shown the thermoregulatory nature of H-NS regulation of virulence gene expression. At temperatures less than 32 °C, expression of virulence genes is negatively regulated; derepression occurs at the permissive temperature of 37 °C (Colonna et al., 1995; Dorman et al., 1990
; Falconi et al., 1998
; Gardel & Mekalanos, 1994
; Hromockyj et al., 1992
; Maurelli & Sansonetti, 1988
; Nye et al., 2000
; Porter & Dorman, 1994
; Prosseda et al., 1998
). These findings are consistent with the bacterium expressing a critical virulence factor when present within the mammalian host (37 °C) and downregulating expression under growth conditions outside the host. Because urease is critical for P. mirabilis infection and H-NS binds to the ureR-ureD intergenic region, we hypothesized that H-NS represses urease gene expression in a temperature-dependent manner. H-NS plays a role in the negative regulation of urease expression and transcription from pureR (Coker et al., 2000
). UreR initiates transcription in a urea-inducible manner from both pureD and pureR. P. mirabilis HI4320 cultured in the presence of urea at 37 °C had
twofold more urease activity than bacteria cultured at 25 °C in the presence of urea (P=0·016) (Fig. 6a
). Expression of the urease gene cluster appears to be thermoregulated, with maximal activity occurring at a culture temperature of 37 °C. These data are consistent with upregulation of virulence factor expression under permissive temperatures found in the host.
The role of H-NS repression of the urease gene cluster was also studied by insertionally inactivating the hns gene in E. coli MC1061 (Fig. 6b). The absence of H-NS in MC1061(hns : : Tn10) resulted in statistically significant increases in urease activity compared to the wild-type MC1061 strain under all conditions examined; a derepression of urease expression was observed in the H-NS-deficient strains. Interestingly, the H-NS-deficient strain was still inducible with 100 mM urea at 37 °C.
H-NS also mediates temperature-dependent repression of urease expression. The higher urease activity in MC1061 (H-NS+) at 37 vs 25 °C, both in the presence and absence of urea, reflects regulation from two different promoters, pureR and pureD. Therefore, because we see two different mechanisms of H-NS repression, temperature-dependent and temperature-independent repression, we hypothesized that a different form of repression occurs at each of the promoters. The ureR promoter appears to be under the control of H-NS temperature-independent repression, leaving the ureD promoter under the control of H-NS temperature-dependent regulation. We were, however, unable to directly determine whether pureD was regulated by H-NS in a temperature-dependent manner. Thus, it remains unclear how temperature exhibits its effects. It is possible, however, that an increase in temperature induces a conformational change in the DNA topology that creates a preferable binding site for H-NS or UreR.
While the repression of urease expression by H-NS was established, the target promoters, either pureD and/or pureR, of this repression were previously not known. H-NS-mediated repression of the ureR promoter was studied using E. coli MC1061 and MC1061(hns : : Tn10) containing a ureR-lacZ reporter construct and a construct expressing ureR constitutively. A small, but significantly different, temperature effect was observed with the activity of MC1061 cultures incubated at 25 versus 37 °C; activity was higher at the higher temperature (Fig. 6c). Moreover, the ureR promoter in MC1061 was urea-inducible at both temperatures. The ureR promoter in E. coli MC1061(hns : : Tn10), however, is dramatically affected by temperature, as shown by the significant increase in activity from 25 to 37 °C. H-NS represses pureR as shown by the derepression in MC1061(hns : : Tn10) compared to the wild-type strain (H-NS+) under all conditions. This H-NS-mediated repression appears to be temperature-independent. Derepression of the ureR promoter results in constitutive expression at both temperatures due to the loss of H-NS. Three promoters lie upstream of the ureR promoter; one of the promoters shows strong homology to the
70 promoter consensus sequence. We hypothesize that the loss of H-NS affects transcription by permitting constitutive expression from the
70 promoter, which would otherwise be silenced by H-NS binding.
This study has shown the preferential binding of UreR and H-NS to the P. mirabilis ureR-ureD intergenic region, which contains sufficient intrinsic curvature for H-NS recognition. H-NS then facilitates further bending of the DNA. Because both UreR and H-NS are able to displace one another from the intergenic region DNA under certain conditions, these proteins differentially regulate both UreR and urease expression. Expression of the urease gene cluster is urea-inducible, thermoregulated and is ultimately controlled by both H-NS and UreR.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 July 2003;
revised 18 September 2003;
accepted 18 September 2003.
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