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Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
Correspondence
Andrew J. Darwin
darwia01{at}med.nyu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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54) and the enhancer binding protein PspF. PspA, PspB and PspC, encoded within the pspA operon, also regulate expression by participating in a putative signal transduction pathway that probably serves to modulate PspF activity. All of this suggests that appropriate expression of the pspA operon is critical. Previous genetic analysis of the Y. enterocolitica pspA operon suggested that an additional level of complexity might be mediated by PspF/RpoN-independent expression of some psp genes. Here, an rpoN null mutation and interposon analysis were used to confirm that PspF/RpoN-independent gene expression does originate within the psp locus. Molecular genetic approaches were used to systematically analyse the two large non-coding regions within the psp locus. Primer extension, control region deletion and site-directed mutagenesis experiments led to the identification of RpoN-independent promoters both upstream and downstream of pspA. The precise location of the PspF/RpoN-dependent promoter upstream of pspA was also determined. The discovery of these RpoN-independent promoters reveals yet another level of transcriptional complexity for the Y. enterocolitica pspA operon that may function to allow low-level constitutive expression of psp genes and/or additional regulation under some conditions.
Present address: Albert Einstein College of Medicine, Graduate Program in the Biomedical Sciences, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It was originally identified in Escherichia coli K-12 (Brissette et al., 1990), where it has been studied extensively. Expression of the E. coli psp regulon (pspABCDE and pspG) occurs in response to several stresses, including heat shock, osmotic shock, ethanol treatment, exposure to proton ionophores, and the overexpression of some cell envelope proteins, most notably secretins (for reviews see Darwin, 2005; Model et al., 1997).
The homologous psp regulon of Yersinia enterocolitica (pspABCDycjXF and pspG) is predicted to have similar inducing signals. Recently, specific induction of Y. enterocolitica pspA operon expression was observed following overexpression of secretins and some cytoplasmic membrane proteins, and upon disruption of the F0F1-ATPase (Maxson & Darwin, 2004). The Psp system of Y. enterocolitica is also essential for virulence in a mouse model of infection (Darwin & Miller, 1999, 2001). This is probably because Psp is essential during production of the Ysc type III secretion system (Darwin & Miller, 2001). Apparently, the Y. enterocolitica Psp system must respond to a stress caused by mislocalization of the YscC secretin component of the type III secretion system (Darwin & Miller, 2001). Consequently, null mutations of some psp genes cause severe growth defects when yscC is overexpressed (Darwin & Miller, 2001; Green & Darwin, 2004; Maxson & Darwin, 2006).
Regulation of E. coli psp gene expression has been well studied, and much of the current understanding is probably applicable to the homologous Y. enterocolitica psp regulon. The E. coli pspA and pspG control regions each contain an RpoN (54)-dependent promoter, as well as binding sites for integration host factor (IHF) and PspF, a member of the enhancer binding protein family (Jovanovic et al., 1996; Lloyd et al., 2004; Weiner et al., 1991, 1995). Induction of the psp regulon is completely dependent on PspF. Regulation is also mediated by several of the other Psp proteins. The peripheral cytoplasmic membrane protein PspA acts as a negative regulator, by directly interacting with PspF and inhibiting its activity (Dworkin et al., 2000; Elderkin et al., 2002, 2005). The integral cytoplasmic membrane proteins PspB and PspC act as positive regulators, presumably by interacting with PspA (Adams et al., 2003) and possibly inhibiting its ability to interfere with PspF.
Previous analysis of Y. enterocolitica pspA expression, together with examination of the DNA sequence of its control region, indicated that it appeared to have a PspF/RpoN-dependent promoter (Darwin & Miller, 2001; Green & Darwin, 2004). Here, we confirm the presence of this promoter and determine its exact location. In addition, earlier genetic experiments raised the possibility of PspF- and, therefore, RpoN-independent expression of at least some of the genes within the pspA operon (Darwin & Miller, 2001). In this study, we strengthened this hypothesis by constructing an rpoN null mutation and using interposon analysis of 
(pspABC'–lacZY) operon fusion expression. We then identified RpoN-independent transcription initiation sites both upstream and immediately downstream of pspA. These promoters, which are predicted to be 70-dependent, offer a plausible explanation for the predicted low-level PspF/RpoN-independent expression of some Y. enterocolitica psp genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. For routine plasmid manipulations the host strain was E. coli DH5
. Plasmids with an R6K ori were maintained in E. coli CC118 
pir, and conjugated into Y. enterocolitica from either E. coli S17-1 pir or E. coli SM10 pir. In most cases E. coli strains were grown at 37 °C, and Y. enterocolitica strains were grown at 26 °C or 37 °C as noted. All strains were grown in Luria–Bertani (LB) broth and on LB agar (Miller, 1972). Antibiotics were used as before (Maxson & Darwin, 2004).
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red recombinase gene replacement system (Datsenko & Wanner, 2000), adapted for use in Y. enterocolitica (Maxson & Darwin, 2004), was used to construct an rpoN in-frame deletion mutation. Briefly, a 
rpoN : : kan mutation was constructed using Red recombinase-mediated allelic exchange. The kanamycin-resistance gene was then removed by FLP recombinase-mediated excision and the in-frame deletion mutation was confirmed by colony PCR and Southern hybridization analysis (data not shown). The rpoN null mutant was grown in the presence of 5 mM alanyl-glutamine to alleviate a minor growth defect in LB medium. The growth and regulatory phenotypes of rpoN deletion mutants could be fully complemented by a plasmid encoding RpoN (data not shown). Single-copy lacZ operon fusions were constructed, integrated into the ara locus and confirmed by colony PCR analysis as described previously (Maxson & Darwin, 2005). When necessary, strains were cured of the virulence plasmid as described by Darwin & Miller (2001).
Interposon analysis of (pspABC'–lacZY).
The pspF'pspABC' insert fragment of pAJD457 (Table 1
) has a unique HindIII site within pspA, and a unique HpaI site within pspB. These sites were used as the insertion point for the 
-Sp cassette from plasmid pHP45 (Fellay et al., 1987). The -Sp cassette was inserted into the HpaI site of pspB as a blunt-ended SmaI fragment. To be consistent, the -Sp cassette was also inserted into the HindIII site of pspA as a SmaI fragment. Therefore, pAJD457 was digested with HindIII and treated with DNA polymerase I large (Klenow) fragment and dNTPs, prior to insertion of the -Sp cassette. For consistency, for both the pspA and pspB insertions, clones were chosen with the -Sp in the same orientation. Following insertion of the -Sp cassette, the pspF'pspA : : -Sp pspBpspC' and pspF'pspApspB : : -Sp pspC' fragments were cloned into pAJD905 (Table 1). Each operon fusion was integrated into the ara locus of Y. enterocolitica strains and confirmed by colony PCR analysis as described previously (Maxson & Darwin, 2005).
-Galactosidase assays.
The effect of YscC production on the expression of (pspABC'–lacZ) and (pspA–lacZ) operon fusions was determined as described previously (Darwin & Miller, 2001). Briefly, saturated cultures were diluted into 5 ml LB broth containing appropriate antibiotics, in 18 mm diameter test tubes, so that the OD600 was approximately 0·04. Cultures were grown on a roller drum at 37 °C for 2 h. Then 0·2 mM IPTG (final concentration) was added to induce yscC expression. Growth at 37 °C continued for a further 2 h prior to harvest for enzyme assays.
To monitor expression of the (pspB–lacZ) operon fusion, saturated cultures were diluted into 5 ml LB broth, in 18 mm diameter test tubes, so that the OD600 was approximately 0·06. The cultures were grown on a roller drum at 26 °C for 4 h, prior to harvest for enzyme assays.
-Galactosidase enzyme activity was determined at room temperature (approx. 22 °C) in permeabilized cells as described by Maloy et al. (1996). Activities are expressed in arbitrary units, which were determined according to the formula of Miller (1972). Individual cultures were assayed in duplicate, and values were averaged from at least three independent cultures. Data were rounded to two significant figures.
RNA isolation and primer extension analysis.
Total RNA was isolated from Y. enterocolitica rpoN+ or rpoN strains containing the multicopy psp locus plasmid pAJD113 and the tacp-yscC expression plasmid pAJD126. Cultures were grown as described above for the -galactosidase assay experiments. RNA was isolated with the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. End-labelling of oligonucleotides and primer extension reactions were done with the Primer Extension System–AMV Reverse Transcriptase (Promega). For analysis of the region upstream from pspA the primer 5'-CTGTGGATCTTCAGCTTTATCCAG-3' corresponds to pspA codons 20 to 26 in the template strand. For the region downstream from pspA the primer 5'-CGTGCATCGTCAGTTAACTGCGAT-3' corresponds to pspB codons 45 to 52 in the template strand. The primers were labelled at the 5' end with [
-32P]ATP and used in extension reactions containing 5 µg RNA. To generate size markers the same primers were used in DNA sequencing reactions of the pAJD113 template using the fmol DNA Cycle Sequencing System (Promega). Samples were resolved by denaturing 8 % polyacrylamide-urea electrophoresis and visualized by autoradiography.
Promoter deletion analysis.
Truncated control region fragments were generated by PCR using a common downstream primer and upstream primers that annealed at various distances upstream. The fragments were cloned into the lacZ operon fusion plasmid pAJD905 and the DNA sequences were confirmed. The operon fusions were integrated into the ara locus and confirmed by colony PCR analysis as described previously (Maxson & Darwin, 2005).
Site-directed mutagenesis.
The Altered Sites II in vitro Mutagenesis System (Promega) was used. The starting point for these constructions was plasmid pAJD457, which has a pspF'pspABC' insert (Table 1). For mutagenesis of the region upstream of pspA, a 0·7 kb pspF'–pspA' KpnI–HindIII fragment of pAJD457 was cloned into pALTER-1. For mutagenesis of the region downstream from pspA, a 0·56 kb 'pspApspB' HindIII–HpaI fragment was cloned into pSL1180 (Table 1), and then transferred to pALTER-1 as a HindIII–KpnI fragment. The oligonucleotides for mutagenesis were designed according to the manufacturer's instructions, and incorporated mismatches as indicated in Fig. 2. After mutagenesis, the DNA sequence of the insert fragment was determined to confirm the desired mutations, and to ensure that no spurious mutations had been introduced. Mutagenic fragments were then cloned into the lacZ operon fusion plasmid pAJD905 and the operon fusions were integrated into the ara locus and confirmed by colony PCR analysis exactly as described previously (Maxson & Darwin, 2005). Colony PCR and DNA sequence analysis was used to verify the presence of each site-directed mutation on the chromosome.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). pspABCDycjXF operon expression depends on the enhancer binding protein PspF, which is predicted to activate an RpoN-dependent promoter upstream of pspA (Green & Darwin, 2004). However, a pspF null mutation causes a less severe YscC-induced growth defect than a pspC null mutation, which led to a prediction of some PspF-independent expression of pspC (Darwin & Miller, 2001). Consistent with this, we have found that an rpoN null mutant also has a less severe YscC-induced growth defect than a pspC null mutant and that the YscC-related growth phenotypes of pspF and rpoN null strains are indistinguishable (data not shown). A likely location for an RpoN/PspF-independent promoter driving some pspC expression was thought to be the 161 bp non-coding region separating pspA and pspB (Darwin & Miller, 2001). We used insertional mutagenesis with the interposon (a strong transcriptional terminator cassette; Fellay et al., 1987) to begin to address this hypothesis.
Merodiploid strains were constructed with a (pspABC'–lacZ) operon fusion integrated into the ara locus (Maxson & Darwin, 2005) and an intact pspF–pspABCDycjXF locus. Therefore, polar effects of -Sp interposon insertions on lacZ expression were determined in strains that retained intact copies of all psp genes. Derivatives were constructed with an -Sp transcriptional terminator cassette inserted into the pspA or pspB genes of the (pspABC'–lacZ) fusion and -galactosidase activites were determined in the presence or absence of YscC overexpression (Fig. 1). The hypothesis predicts that the -Sp insertion in pspA should abolish lacZ expression from the RpoN-dependent pspA promoter. The -Sp insertion in pspB should abolish lacZ expression from both the RpoN-dependent pspA promoter, and the putative promoter in the pspA–pspB intergenic region.
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(pspABC'–lacZ) expression was increased by approximately 50 % when YscC was produced (Fig. 1). As expected, the pspA : : -Sp insertion reduced (pspABC'–lacZ) expression and abolished YscC-dependent induction. The pspB : : -Sp insertion reduced expression to a greater extent. These data support the prediction that some lacZ expression originates from downstream of the -Sp insertion site within pspA.
When the operon fusions were analysed in an rpoN null mutant the interpretation became more complex. An rpoN null mutation abolished YscC-dependent induction of (pspABC'–lacZ) expression, which is consistent with the predicted RpoN-dependence of the pspA promoter. However, the effect of the rpoN mutation on (pspABC'–lacZ) was much less than that of the pspA : : -Sp insertion in the rpoN+ strain (Fig. 1). Furthermore, even in the rpoN null strain the pspA : : -Sp insertion reduced (pspABC'–lacZ) expression by 50 %. Taken together, all of these data suggest the following. First, RpoN-dependent expression originates only from upstream of the -Sp insertion site within pspA. Second, significant RpoN-independent expression originates both upstream and downstream of the -Sp insertion site within pspA. Therefore, we next analysed the non-coding regions upstream and downstream of pspA (Fig. 2) for the presence of RpoN-dependent and RpoN-independent transcription initiation sites.
Identification of RpoN-dependent and RpoN-independent 5' mRNA ends expressed from the pspA control region
Interposon analysis had suggested that both RpoN-dependent and RpoN-independent transcription initiation might originate upstream from pspA (Fig. 1). Therefore, we used primer extension analysis to identify 5' mRNA ends originating from the pspF–pspA intergenic region of the intact psp locus. RNA was isolated from both rpoN+ and rpoN strains and analysed with a primer complementary to the 5' end of pspA (see Methods). The most abundant mRNA molecule isolated from the rpoN+ strain had a 5' end at the expected position downstream of the predicted RpoN-binding site (P1 in Fig. 3). This confirms the location of the RpoN-dependent promoter. As expected, this mRNA was undetectable in the rpoN null mutant (Fig. 3). We also identified two additional 5' mRNA ends separated by 2 nucleotides, which mapped a short distance upstream of P1 (P2 in Fig. 3), indicating that they did not arise from processing of the RpoN-dependent transcript. Furthermore, these mRNAs were detected in both rpoN+ and rpoN null strains. Sequences similar to the –10 element of a 70-dependent promoter were identified upstream (Figs 2 and 3). All of these results were also confirmed by 5' RACE analysis of mRNA initiated from a different template [a multicopy (pspA–lacZ) fusion plasmid; data not shown].
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). Next we undertook a 5' deletion analysis in order to identify how much upstream DNA was required for RpoN-dependent and RpoN-independent expression of a (pspA–lacZ) operon fusion made with the isolated pspA control region. A set of single-copy (pspA–lacZ) operon fusion strains was constructed with different 5' deletions of the pspA control region fragment (Fig. 2). Strains were grown under both non-Psp-inducing (–YscC) and Psp-inducing (+YscC) conditions and -galactosidase activities were determined (Table 2). YscC overproduction does not induce (pspA–lacZ) expression as much as some other proteins (Maxson & Darwin, 2004, 2005, 2006). However, it does cause a severe growth defect in some psp null mutants (Darwin & Miller, 2001; Green & Darwin, 2004; Maxson & Darwin, 2006) and so its effect on pspA operon expression is probably physiologically significant.
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-galactosidase activities of deletion constructs 252, 132 and 71 were indistinguishable from that of the original construct (278) under uninduced conditions (–YscC). However, the YscC-induced activities of constructs 132 and 71 were slightly reduced and completely abolished, respectively (Table 2). This shows that DNA upstream of position –71 is needed for regulated expression of (pspA–lacZ). PspF is required for YscC-dependent induction. In E. coli the PspF-binding site overlaps the pspF translation initiation codon (Jovanovic et al., 1999). Sequence alignment suggests that the same is probably true in Y. enterocolitica (Fig. 2 and data not shown). Therefore, the PspF-binding site is likely to have been compromised in construct 132 and completely removed in construct 71 (Fig. 2). All (pspA–lacZ) expression was completely abolished in deletion constructs 39 and 12 (Table 2). The remaining 8–9 Miller units can be attributed to endogenous Y. enterocolitica -galactosidase activity and basal expression from an empty (–lacZ) operon fusion (Maxson & Darwin, 2005 and data not shown). From this it can be concluded that the region upstream of position –39 is required for all basal (pspA–lacZ) expression.
The major goal of these experiments was to characterize RpoN-independent (pspA–lacZ) expression. To more clearly identify the region required specifically for RpoN-independent expression the deletion constructs were analysed in an rpoN null strain (Table 2). The results confirmed that the region upstream of position –39 is required for RpoN-independent expression of (pspA–lacZ). This region includes almost the entire DNA region upstream from where the RpoN-independent 5' mRNA ends were mapped (Figs 2 and 3). Furthermore, the 39 construct removes part of a putative –10 element (Fig. 2). Together, all of these data suggest that one or both of the 5' mRNA ends represents an RpoN-independent transcription initiation site. Finally, in the rpoN null strain only, we noticed that (pspA–lacZ) expression was elevated in the 71 construct (Table 2). We do not yet understand the explanation for this, and do not draw any conclusions from it. It may be an artifact resulting from the relative positioning of vector sequences upstream of the control region end-point in this particular deletion construct.
Site-directed mutagenesis of the pspA control region
To complete the analysis of the pspA control region, putative promoter elements were targeted by site-directed mutagenesis. Mutations were introduced into the original 278 (pspA–lacZ) construct and -galactosidase activities were determined (Table 2). First, the highly conserved –24 and –12 dinucleotides of the predicted RpoN-binding site were disrupted (Fig. 2). In an rpoN+ strain this mutation abolished YscC-dependent induction and reduced basal expression by almost 50 % (Table 2). This effect was indistinguishable from that of an rpoN null mutation on expression of the original (pspA–lacZ) construct with the wild-type control region sequence. In an rpoN null strain the –24/–12 mutation had no effect on (pspA–lacZ) expression (Table 2). Therefore, as expected the mutation only affected RpoN-dependent expression.
The region immediately upstream of the putative RpoN-independent transcription initiation sites has several overlapping sequences that might represent –10 elements (CATTATATTTT; Fig. 2), although there is no recognizable –35 element. To test the functionality of the putative –10 elements, and to distinguish between them, we made three different trinucleotide substitutions. Two of these (CATTAT to GCGTAT) and (TATATT to CCAATT) did not reduce RpoN-independent expression of (pspA–lacZ) (data not shown). However, a TATTTT to TTGGTT mutation (Fig. 2) essentially abolished (pspA–lacZ) expression in an rpoN null strain and also reduced expression in an rpoN+ strain (Table 2). Therefore, we tentatively assigned the TATTTT motif as a –10 element. The TATATT to CCAATT mutation, which also disrupts the first position of this –10 element, did not affect expression, possibly because it leaves the most highly conserved positions intact (Harley & Reynolds, 1987; Hawley & McClure, 1983). It also remains possible that multiple sequences serve as –10 elements. Nevertheless, when taken together the primer extension, 5' deletion and site-directed mutagenesis data strongly suggest the presence of at least one RpoN-independent transcription initiation site upstream of pspA.
Identification of 5' mRNA ends expressed from the pspA–pspB intergenic region
Interposon analysis had also suggested that some RpoN-independent transcription initiation might originate downstream from pspA (Fig. 1). Therefore, we also used primer extension analysis to identify 5' mRNA ends originating from the pspA–pspB intergenic region of the intact psp locus. RNA was analysed by primer extension analysis with an oligonucleotide complementary to the 5' end of pspB (see Methods). Surprisingly, the 5' ends of mRNA molecules mapped to a single C residue that was just within the pspB gene (Fig. 4). We are confident that the pspB translation initiation codon has been correctly identified. It has a Shine–Dalgarno (SD) motif, there are no alternative downstream ATG or GTG codons with SD motifs, and the predicted PspB amino terminus is conserved between different species (data not shown). We also confirmed the primer extension result by 5' RACE analysis of mRNA initiated from a different template [a multicopy (pspB–lacZ) fusion plasmid; data not shown]. This 5' mRNA end may be generated from a 70-dependent promoter because putative –35 and –10 elements were identified upstream (Fig. 4).
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(pspB–lacZ) operon fusion strain. The fragment used for this fusion encompassed the 3' end of pspA (position –270 in Fig. 2) to 63 bp within the 5' end of pspB. The single-copy (pspB–lacZ) fusion strain produced 180 Miller units of -galactosidase activity when grown to mid-exponential phase at 26 °C in LB broth (Table 3). This compares to only 5–10 Miller units due to endogenous Y. enterocolitica -galactosidase activity and basal expression of an empty (–lacZ) operon fusion (Maxson & Darwin, 2005 and data not shown). Therefore, these results support the hypothesis that the 'pspA–pspB' fragment contains an active promoter. (pspB–lacZ) expression was not affected by rpoN and pspF null mutations, or by yscC overexpression (data not shown).
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(pspB–lacZ) expression. For this we constructed a set of single-copy (pspB–lacZ) operon fusion strains with different 5' deletions of the 'pspA–pspB' fragment (Fig. 2). The strains were grown to mid-exponential phase at 26 °C in LB broth and -galactosidase activities were determined (Table 3). Deletion constructs 177, 115 and 55 expressed -galactosidase activities similar to that of the original construct (270). This suggests that DNA upstream of position –55 is not required for (pspB–lacZ) expression, making it unlikely as a target for a regulatory protein under the growth conditions we used. However, the -galactosidase activity of deletion construct 17 was reduced by 60 % in comparison to the full-length fragment (270). This may be because this deletion removed a putative –35 element (Fig. 2).
Finally, if the putative promoter within pspB is authentic then disruption of the predicted –10 element should reduce expression. Therefore, we tested whether (pspB–lacZ) expression was affected by a trinucleotide substitution in the –10 region of the full-length 270 construct (Fig. 2). This mutation reduced (pspB–lacZ) expression by over 80 % (Table 3), which further corroborates the identification of a putative 70-dependent promoter.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The phenotypes of an rpoN null mutant further supported this hypothesis (Fig. 1 and data not shown). The pspF–pspABCDycjXF locus contains two large non-coding regions that we decided to focus on for this study (Fig. 2): the region containing promoters for the divergently transcribed pspF and pspA genes (187 bp) and the pspA–pspB intergenic region (161 bp). RpoN-independent promoters were identified both upstream and downstream of pspA. The expected location of the PspF/RpoN-dependent pspA promoter was also confirmed.
Three 5' mRNA ends were identified upstream of pspA (Fig. 3). The most abundant was expressed from the RpoN/PspF-dependent promoter and served to confirm its anticipated location. This RpoN-dependent promoter is apparently highly conserved upstream of pspA in different bacteria (Green & Darwin, 2004). Two RpoN-independent 5' mRNA ends were also identified, separated by only 2 nucleotides, and mapping approximately 30 bp upstream of the RpoN-dependent transcription initiation site. These two mRNA ends might represent two different promoters or alternative transcription initiation sites for the same promoter. Another possibility is that there is a single initiation site and the shorter transcript arose from a processing event. Multiple putative –10 elements were found upstream, but only one mutation reduced RpoN-independent (pspA–lacZ) expression (Table 2 and data not shown). Although a –35 element could not be identified we are confident that at least one of the 5' mRNA ends is indicative of a promoter. First, RpoN-independent (pspA–lacZ) expression was abolished by deletion of upstream DNA (Table 2). Second, RpoN-independent (pspA–lacZ) expression was also abolished by the –10 mutation (Table 2). RpoN-independent pspA transcription initiation sites were not detected in E. coli by ribonuclease protection assays (Weiner et al., 1991). It is possible that the sensitivity was insufficient. E. coli pspA primer extension experiments were also only reported to have identified the RpoN-dependent 5' mRNA end, but the data were not shown (Weiner et al., 1991). Either RpoN-independent expression upstream of pspA is unique to Y. enterocolitica, or it also occurs in E. coli but was below the limit of detection in the published experiments.
The close proximity of RpoN-dependent and RpoN-independent promoters upstream of pspA (Figs 2 and 3) raises the question of whether they can be occupied (active) simultaneously. Mutation of the putative –10 element of the RpoN-independent promoter reduced (pspA–lacZ) expression in both rpoN+ and rpoN strains (Table 2). This suggests that this promoter does contribute to (pspA–lacZ) expression when the RpoN-dependent promoter is active. However, it is also possible that this mutation independently compromises both promoters. Alternatively, only one promoter may be occupied at any one time and the total -galactosidase activity measured reflects the average of the population of cells. It might be interesting to investigate this question in future experiments, particularly with an in vitro transcription system.
The literature contains numerous examples of bacterial genes with multiple upstream promoters, including the E. coli rpoE operon, which, like the pspA operon, is involved in extracytoplasmic stress response (Raina et al., 1995). In many cases the precise physiological function of the multiple promoters is unknown and this is certainly true for RpoN-independent expression of the Y. enterocolitica pspA operon. RpoN-independent expression from upstream of pspA might ensure low-level constitutive expression of some psp genes or regulation under unknown conditions. Constitutive expression might indicate that low levels of some Psp proteins are critical under non-inducing conditions, perhaps to facilitate a fast response to inducing stress conditions.
Another RpoN-independent promoter was identified downstream from pspA. Unexpectedly, the promoter overlaps the 5' end of pspB (Fig. 2). There are many examples of promoters within bacterial genes, but the location of this one at the extreme 5' end of pspB was surprising. Nevertheless, we are confident of the veracity of this promoter. The 5' mRNA end was mapped from different templates by both primer extension analysis (Fig. 2) and 5' RACE (data not shown). Putative –35 and –10 elements were identified at the appropriate locations and their disruption by deletion (–35) or mutation (–10) significantly reduced (pspB–lacZ) expression (Table 3). Expression of (pspB–lacZ) was not completely abolished by these disruptions. However, it is not unprecedented for control region mutations in –35 and –10 elements to leave some promoter function intact. The location of this promoter indicates that it can only control expression of pspC and the downstream genes. Its activity was unaffected by pspF and rpoN null mutations, by YscC overproduction or by heat or osmotic shock (Fig. 1 and data not shown). Therefore, the promoter may drive low-level constitutive expression of pspC and downstream genes. Alternatively, it could be responsible for regulated expression under as-yet-unidentified conditions.
Examination of the genome sequences from closely related Enterobacteriaceae suggests that the relatively large size of the pspA–pspB intergenic region is a signature of Yersinia species. It occurs in Y. enterocolitica, Y. pseudotuberculosis and Y. pestis (data not shown). Furthermore, the –10 element of the promoter is perfectly conserved in all three of these species. In contrast, the pspA–pspB intergenic region is approximately 100 bp shorter in both E. coli and Salmonella enterica serovar Typhimurium. Therefore, the RpoN-independent promoter and any upstream control elements identified here may not be present in all species. However, it is possible that different internal promoters are present in the psp loci of other species. For example, Northern hybridization analysis of the E. coli pspABCDE operon detected a constitutively expressed transcript encoding only pspBCDE, although its origin was not determined (Brissette et al., 1991).
Appropriate expression of the Psp response system is probably critical. Indeed, pspA operon expression is highly regulated in both E. coli and Y. enterocolitica. This regulation is mediated entirely by a conserved RpoN/PspF-dependent promoter upstream of pspA. In addition, the PspA, PspB and PspC proteins all modulate PspF activity via a putative signal transduction pathway, which further increases the complexity of pspA operon regulation. This study has revealed yet another level of transcriptional complexity within the Y. enterocolitica pspA operon. RpoN-independent promoters are present both upstream and downstream of pspA. These promoters may only be responsible for a relatively small amount of the total level of psp gene expression in comparison to the highly inducible PspF/RpoN-independent promoter (e.g. see Maxson & Darwin, 2004). Nevertheless, a goal for future experiments will be to investigate the role of these promoters and whether there are any conditions under which their activity is altered.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 November 2005;
revised 12 January 2006;
accepted 13 January 2006.
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