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Department of Applied Microbiology, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland
Correspondence
Katarzyna Brzostek
kbrzostek{at}biol.uw.edu.pl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). During infection, the bacteria are able to penetrate the intestinal epithelium through M cells and subsequently grow and multiply in the lymphoid tissue, i.e. Peyer's patches. The first critical step in Y. enterocolitica infection is tight adhesion followed by active uptake. Both attachment to eukaryotic cells and subsequent invasion are mediated by virulence factors including invasin and adhesins Ail, Myf and YadA (Iriarte et al., 1993; Miller et al., 1989; Pepe & Miller, 1993; Schulze-Koops et al., 1992; Skurnik et al., 1994). It has been demonstrated that invasin plays a major role in the ‘zipper mechanism’ by which Y. enterocolitica invades intestinal epithelial cells (Isberg & Van Nhieu, 1995). Complete virulence of Y. enterocolitica depends on the activity of the type III secretion system (encoded by the 70 kb virulence plasmid pYV), which secretes the Yop proteins, the main virulence markers responsible for resistance to non-specific host defences (Cornelis et al., 1998).
Intensive genetic and physiological studies of invasin in Y. enterocolitica, and in the other enteric pathogen of this genus, Yersinia pseudotuberculosis, revealed the complicated nature of its expression. Invasin is encoded by the inv gene, which is regulated in response to growth phase and a variety of environmental parameters such as temperature, osmolarity and pH (Nagel et al., 2001; Pepe et al., 1994). Expression of this protein is maximal during the late exponential growth phase at 25 °C but is inhibited when bacteria are grown at 37 °C at neutral pH. In addition, stressful conditions such as high concentrations of salts and low pH decrease the expression of invasin at 25 °C. So far, at least three proteins, RovA, H-NS and YmoA, have been identified as factors regulating inv expression in Yersinia cells (Ellison et al., 2004). The transcriptional activator RovA positively regulates the expression of inv in both Y. enterocolitica and Y. pseudotuberculosis (Nagel et al., 2001; Revell & Miller, 2000; Tran et al., 2005). RovA is a member of the large MarR/SlyA family of transcriptional regulators identified in Enterobacteriaceae, which control a diverse range of physiological processes in pathogenic bacteria (Ellison & Miller, 2006a). These factors activate the expression of virulence genes that play crucial roles in survival, stress adaptation and pathogenesis. The RovA-dependent inv gene in Y. pseudotuberculosis is subject to silencing by the small chromatin-associated H-NS protein (Heroven et al., 2004). H-NS modulates the expression of a large number of genes in enterobacteria, most encoding factors having a negative effect on transcription. RovA and H-NS bind directly and specifically to a similar region of the inv promoter of Y. pseudotuberculosis, competing for overlapping binding sites. The same function for these two protein factors in Y. enterocolitica has recently been reported by Ellison & Miller (2006b). These authors showed that the inv gene of Y. enterocolitica is negatively regulated by YmoA, a histone-like protein, which together with H-NS forms a repression complex on the inv promoter. In addition, it has been demonstrated that temperature-dependent expression of inv, i.e. reduced inv expression at 37 °C compared with that at 26 °C, is a consequence of differences in the level of RovA, probably acting as a derepressor of the H-NS/YmoA complex. The higher levels of RovA present at 26 °C effectively compete with H-NS/YmoA for binding to the inv promoter, resulting in increased inv transcription at this temperature.
In spite of significant progress in our understanding of the molecular mechanisms of inv regulation in response to changes in growth temperature, the influence of the osmolarity of the environment on inv expression is still unclear.
The EnvZ-OmpR regulatory system of Escherichia coli is a paradigm of a two-component signal transduction system in which EnvZ is the membrane-bound osmosensor kinase and OmpR is the response regulator. This system governs the expression of the ompC and ompF genes, encoding the two major outer-membrane porins (Egger et al., 1997; Russo & Silhavy, 1991). Upon sensing a signal (increased osmolarity), EnvZ phosphorylates itself at histidine 243 and then transfers the phosphate to aspartate 55 of OmpR. Phosphorylation of OmpR induces conformational changes in the C-terminal domain of the protein, increasing its binding affinity for the ompC and ompF promoters. Interaction of bound OmpR with the RNA polymerase 
-subunit activates transcription. In addition to its role in porin osmoregulation, OmpR is involved in the regulation of flagellar gene expression, fatty acid transport, curli fibre formation and cell division as a dual, i.e. negative or positive, regulator (Higashitani et al., 1993; Jubelin et al., 2005; Shin & Park, 1995; Yamamoto et al., 2000). Finally, it has been found that the OmpR protein influences the virulence properties of Salmonella, regulating the acid induction of the SsrAB two-component signal transduction system located within pathogenicity island 2 (Lee et al., 2000). The pleiotropic effects of OmpR suggest that this protein acts as a global regulator.
It has recently been shown that the ompR gene is present in both Y. enterocolitica O : 8 and O : 9 serotypes, the so-called American and European serotypes, respectively, and that there is a correlation between the functioning of the OmpR protein and pathogenicity (Brzostek & Raczkowska, 2003; Dorrell et al., 1998).
Our previous study revealed that the OmpR protein of Y. enterocolitica O : 9 exhibits 95 % amino acid identity to E. coli OmpR and that it can participate in the control of some physiological properties. We also demonstrated that OmpR is involved in the adaptation of this bacterium to multiple stresses as well as in survival and replication within macrophages (Brzostek & Raczkowska, 2003).
The present study further examined the role of OmpR in Y. enterocolitica using experimental and in silico approaches and our results suggest that this protein functions as a negative regulator of invasin expression. We propose that OmpR, acting as a repressor of inv transcription, might disturb RovA- and H-NS/YmoA-dependent inv expression at 25 °C.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Y. enterocolitica O : 9 strains were routinely grown in LB medium (10 g tryptone, 5 g yeast extract and 5 g NaCl per litre) at 25 °C or 37 °C. The osmolarity of the growth medium was raised by adding NaCl to a final concentration of 385 mM. When necessary, media were supplemented with the following antibiotics: kanamycin (Km), 50 µg ml–1; chloramphenicol (Cm), 25 or 12.5 µg ml–1; nalidixic acid (Nal), 15 µg ml–1 and tetracycline (Tc), 12.5 µg ml–1.
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) with enzymes supplied by Promega or MBI Fermentas. DNA fragments were amplified by PCR using Taq DNA polymerase (Invitrogen) and oligonucleotide primers (described below). Individual PCR fragments were purified from the reaction using a PureLink PCR purification kit (Invitrogen) or following agarose gel electrophoresis with a PureLink Gel extraction kit (Invitrogen).
RT-PCR gene expression analysis.
Total RNA was isolated from 107 bacterial cells using TRI reagent (Sigma) according to the manufacturer's recommendations. Following treatment with RNase-free DNase (Sigma), RNA was reverse-transcribed with random hexamers and AMV reverse transcriptase (Sigma). Multiplex PCR was carried out for 28 cycles with cDNA derived from 5x104 cells using primer pairs RTInv1 (5'-GTCGCAATACCCTTAATATCG-3') and RTInv390 (5'-GGATCGGCGTATAAATAAG-3') for inv, and GapA1 (5'-ACGCTGACTACATGGCATAC-3') and GapA2 (5'-ACGTCAACGTTAACTTCGTTC-3') for gapA (encoding D-glyceraldehyde-3-phosphate dehydrogenase, used as an external control). The PCR products were 390 and 166 bp for inv and gapA, respectively. All amplified fragments were resolved by electrophoresis on 2 % agarose gels and the density of the inv product band was compared with the band corresponding to gapA. To confirm that there was no DNA contamination in the RT-PCR mixture, samples of RNA obtained before the RT reaction were used in separate reactions with the same primer pairs.
Construction of a chromosomal inv' : : lacZYA fusion in Y. enterocolitica.
To construct a chromosomal inv' : : lacZYA transcriptional fusion, the suicide vector pFUSE, an ori R6K derivative carrying genes of the lacZYA operon lacking their own promoter, was used (Baumler et al., 1996). The chromosomal fusion was created by plasmid insertion via homologous recombination.
The 607 bp intragenic fragment of the inv gene (starting at nucleotide position 1085 and terminating at position 1692) was amplified by PCR with primers InvA1 (5'-TGTCTAGAAGTCTCAATCTGCACTACAAC-3') and InvA2 (5'-TGCCCGGGGAGCCATCGGCAACAATATC-3'). The product was cloned into the pDrive cloning vector (Qiagen) to generate plasmid pDI2, then an XbaI/SmaI fragment of inv was subcloned into XbaI/SmaI-restricted pFUSE to produce suicide construct pFI4. Biparental conjugation was used to transfer plasmid pFI4 from E. coli S17-1
pir (which encodes the R6K replication protein pi and the transfer functions of RP4) to Y. enterocolitica strains Ye9 NalR (Ye9N) and AR4. Transconjugants were selected on LB plates supplemented with Nal and Cm, and in the case of Y. enterocolitica AR4, also with Km. The functionality of the invasin promoter driving lacZYA expression in the selected transconjugant strains (Ye10 and AR5) was confirmed by production of a blue colour following growth at 25 °C on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (20 µg ml–1). The presence of the inv' : : lacZYA fusion in chromosomal DNA isolated from these strains was confirmed by PCR with two oligonucleotide primer pairs. The first primer pair consisted of RTInv1 (5'-GTCGCAATACCCTTAATATCG-3'), designed to amplify a region of inv in the chromosome, and PlacZ (5'-AGTCTCAATCTGCACTACAA-3'), designed to amplify the lacZ sequence present in pFUSE. The second pair consisted of InvA3 (5'-AGTCTCAATCTGCACTACAAC-3') and PLacZ, designed to amplify the inv fragment included in pFI4 and the lacZ sequence, respectively.
β-Galactosidase assays.
Triplicate cultures of Y. enterocolitica strains containing the inv' : : lacZYA transcriptional fusion were grown overnight in LB broth or LB supplemented with NaCl at 25 °C and 37 °C. The activity of β-galactosidase was measured in vitro with ONPG as the substrate, according to a standard method (Miller, 1972).
Complementation of ompR mutation.
To complement the ompR mutation in Y. enterocolitica strains, the XhoI/PstI fragment from plasmid pQR1 carrying ompR with the RBS and 6His-tag region was subcloned into XhoI and PstI-digested mobilizable cloning vector pBBR1 MCS-3 (5.1 kb). The resulting construct, pBR3, was used to transform E. coli DH5. The pBR3 plasmid was mobilized into recipient Y. enterocolitica ompR strains by triparental mating using E. coli DH5 carrying the helper plasmid pRK2013, which provides tra and mob functions.
Triparental conjugation.
Overnight cultures of the recipient Y. enterocolitica strains, the E. coli DH5 strain carrying plasmid pBR3 as the mating donor and E. coli DH5 harbouring the helper plasmid (pRK2013) were diluted with LB medium to OD600 
0.1 and cultivated with shaking to OD600 0.4. The three-times concentrated cell suspensions were mixed in equal proportions, plated onto LB plates and incubated overnight at room temperature. The mixed bacterial growth was scraped from the plates, resuspended in 2 ml sterile physiological salt solution and spread on LB agar plates supplemented with Nal, Cm and Tc in the case of the Ye10 recipient strain and Nal, Cm, Km and Tc for AR5.
SDS-PAGE analysis of outer-membrane proteins.
Outer-membrane proteins were isolated from Y. enterocolitica strains grown to late exponential phase at 25 °C. The cultures were centrifuged at 8000 g for 10 min and cell pellets were resuspended in PBS (pH 7.2) containing 1 mM PMSF (Sigma). The cell suspensions were sonicated five times for 30 s with a 30 s incubation on ice between sonication steps, broken debris was removed by low-speed centrifugation (10 min at 6000 g) and membrane fractions were pelleted by centrifugation at 40 000 g for 1 h. Inner membrane proteins were solubilized by resuspension in 10 mM Tris buffer (pH 7·4) containing 0.1 % Triton X-100 for 30 min, then discarded in the supernatant following further centrifugation. The remaining outer-membrane pellets were resuspended in electrophoresis sample buffer, dissolved by boiling for 10 min and proteins were separated by electrophoresis on 10 % SDS–polyacrylamide gels. Proteins were visualized by Coomassie blue staining and destaining.
Overproduction and purification of the OmpR protein.
A 717 bp fragment containing the entire coding sequence of ompR without the ATG start codon was amplified by PCR with primers RR1 (5'-CAAGAGAATCACAAGATTTTGG-3') and RF2 (5'-TCATGCTTTACTGCCGTCCG-3'). This was cloned into the pDrive cloning vector, then subcloned into expression vector pQE30, digested with SalI and PstI, to create plasmid pQR1 encoding a 6His-tagged OmpR protein. The final construct was verified by DNA sequencing. The pQR1 plasmid was introduced into E. coli M15 [pREP4] (Qiagen) to express 6His-tagged OmpR under the control of an IPTG-inducible promoter. The M15 [pREP4] strain harbouring pQR1 was grown to mid-exponential phase (OD600 0.5–0.7) in 500 ml LB medium containing 100 µg ampicillin ml–1. IPTG was then added to a final concentration of 0.5 mM, and incubation continued for 4 h at 37 °C. After induction, the cells were harvested and resuspended in 5 ml buffer A [50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 55 µM PMSF, 5 mM imidazole, 10 mM 2-mercaptoethanol, 0.1 % Tween 20]. A crude protein extract was obtained by sonication and insoluble cell material was pelleted by centrifugation at 25 000 g. His-tagged OmpR protein was purified from the supernatant fraction by immobilized-metal affinity chromatography using Ni2+-NTA agarose (Qiagen). The protein extract was mixed with 3 ml Ni2+-NTA slurry and incubated with gentle agitation for 1 h at 4 °C. After loading the mixture onto a column, the matrix was washed four times with 3 ml washing buffer I [50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 20 mM imidazole, 55 µM PMSF] and five times with 3 ml washing buffer II [50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 5 mM imidazole, 55 µM PMSF, 10 % glycerol]. The 6His-OmpR protein was eluted from the column in 2 ml portions of elution buffer [50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 10 % glycerol] containing increasing concentrations of imidazole (50–300 mM). Purification was monitored by 10 % SDS-PAGE. The selected eluate fractions were dialysed against elution buffer without imidazole.
Electrophoretic mobility shift assays.
OmpR-binding studies were performed using a 114 bp fragment of the inv promoter region, encompassing the putative OmpR binding site, generated by PCR using Y. enterocolitica Ye9 chromosomal DNA as the template with primers 5'-GCAAGCTAATATTACCATGATG-3' and 5'-CACTAACAATACAATATAATAGC-3' with a Taq/Pfu polymerase mixture (EURx). The unrelated competitor DNAs were a 324 bp fragment of Paracoccus aminophilus DNA amplified with primers 5'-CGAATTCTTCAGCCTCACCGTCAGACA-3' and 5'-TGGATCCTATTCGTACCAGTCGGCGCT-3', and sonicated salmon sperm DNA. The PCR fragments were 3' end-labelled with 11-DIG-dUTP as recommended by the supplier (DIG oligonucleotide 3' end labelling kit, 2nd generation, Roche). The labelled inv DNA fragments (100 fmol) and varying amounts of OmpR (with or without competitors) were mixed in a binding buffer containing 10 mM Tris/HCl (pH 7.4), 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 5 mM DTT, 5 % glycerol and 50 µg BSA ml–1 in a final volume of 20 µl. For phosphorylation of OmpR, 10 mM acetyl phosphate (Sigma) was added to the reaction mixture. OmpR phosphorylation and DNA binding were performed at 23 °C for 35 min. After incubation, the OmpR/DNA mixtures were subjected to electrophoresis on a 0.5xTBE, 5 % non-denaturing polyacrylamide gel for 2 h at 120–200 V at 4 °C. After electrophoresis, the DNA was blotted onto a positively charged nylon membrane (Sigma), fixed under UV light and developed using a chemiluminescence kit according to the manufacturer's protocol (Roche). The signal was recorded by placing the membrane against X-ray film (Kodak).
In silico analysis of the inv promoter sequence.
The nucleotide sequence of the region upstream of the inv ORF was scanned for potential OmpR binding sites. The analysis was based on the sequence of inv from Y. enterocolitica W1024 serotype O : 9 (GenBank accession no. Z48169) and on the OmpR binding site consensus sequence (Egger et al., 1997). Alignments were carried out using BLAST software (NCBI).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), was used as an external control. The results shown in Fig. 1(a) demonstrate that the levels of mRNA transcripts for inv were very different in the tested strains. The inv mRNA level was slightly higher in the wild-type Y. enterocolitica strain Ye9 at 25 °C compared with 37 °C. In the ompR null mutant AR4 the amount of inv transcript was markedly increased compared with the Ye9 wild-type strain when grown at 25 °C, but significantly decreased at 37 °C. This result at the higher temperature was quite surprising and suggests high complexity in the regulation of inv expression, particularly at the level of transcription. Densitometry of the ethidium bromide-stained PCR product bands was used to quantitatively evaluate the expression level of the inv and gapA genes. The density of inv bands was calculated relative to those of gapA and these ratios are presented in Fig. 1(b). These data suggest that the OmpR protein may inhibit invasin synthesis at 25 °C by lowering the inv mRNA level in the wild-type strain.
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). At 25 °C a higher level of activity was obtained in the ompR mutant background compared with the wild-type strain grown in LB medium. The lack of the OmpR protein resulted in a sixfold increase in inv expression. In both tested strains there was less inv expression at 37 °C compared with the level at 25 °C. This reduction at the higher temperature was twofold in Ye10 cells and ninefold in AR5, but still the level of inv expression in the ompR mutant was slightly higher than in the wild-type. However, the higher level of inv' : : lacZYA expression in the ompR mutant (AR5) at 37 °C compared with the wild-type strain (Ye10, active OmpR) seems to contradict the RT-PCR analysis findings, where the level of inv transcript at 37 °C was lower in the ompR mutant (AR4) than in the wild-type strain (Ye9). This discrepancy might be due to the different stabilities of the inv and inv' : : lacZYA transcripts at 37 °C. These results imply a negative role for the OmpR protein in the regulation of inv expression. However, the strong derepression of inv at 25 °C in the absence of OmpR was not observed at 37 °C, indicating that temperature-mediated repression of inv is OmpR-independent.
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, in cultures grown at 25 °C, high osmolarity decreased, albeit slightly, expression from the inv promoter in the strain with active OmpR (Ye10) but had no effect on inv expression in the ompR mutant (AR5). It appears that inv expression in AR5 was no longer responsive to high osmolarity.
Complementation tests
Complementation analyses were carried out using the plasmid pBR3 harbouring the gene encoding His-tagged OmpR, introduced into Y. enterocolitica strains Ye10 and AR5 carrying the chromosomal inv' : : lacZYA fusion. Initially, complementation was verified by analysis of the OmpR-dependent porin profile in Y. enterocolitica strains. Resolution of Y. enterocolitica Ye10 (wild-type) outer-membrane proteins by SDS-PAGE revealed the presence of a single band at the position corresponding to the porin proteins with a molecular mass of 38–39 kDa. Our previous detailed studies on porin expression in Y. enterocolitica O : 9 suggest that this band corresponds to two porins: YompC and YompF (Brzostek & Raczkowska, 2007). In contrast, the protein profile of the outer-membrane fraction prepared from the mutated AR5 strain, devoid of the osmotic regulator OmpR, lacked this band (Fig. 3). When the ompR gene (pBR3) was introduced into strain AR5, a thick band, most probably composed of YompF/YompC, appeared on the gel (Fig. 3, lane 3). This result indicates that the His-tagged OmpR protein expressed from the gene supplied in trans is active and restores the wild-type porin profile in the ompR null mutant strain. Introduction of the pBR3 plasmid carrying the ompR allele into the OmpR+ strain Ye10 also produced an effect on the outer-membrane protein profile. The porin protein band was noticeably stronger than in the outer-membrane preparation from wild-type cells without the added ompR transgene (Fig. 3, lane 4). Finally, detailed analysis of the outer-membrane protein profile suggested that the protein migrating just below the porins might also be affected by the increased level of the OmpR regulator (Fig. 3, lanes 3 and 4). When an additional copy of ompR was introduced into both the mutant and the wild-type strain an increase in the amount of this unidentified protein was observed.
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). The high level of β-galactosidase activity previously observed in the AR5 mutant strain was dramatically decreased at both 25 °C and 37 °C after complementation, reaching levels even lower than those of strain Ye10. OmpR-dependent repression of inv promoter activity had been restored in the ompR-mutated strain by complementation. Moreover, we observed a strong inhibitory effect produced by an additional copy of ompR introduced on the plasmid pBR3 into the Ye10 strain (OmpR+). The sixfold decrease in β-galactosidase activity at 25 °C indicated that overexpression of OmpR results in enhanced inhibition of inv transcription. Introduction of the pBBR1 MCS-3 vector alone into strains Ye10 and AR5 had no effect on the regulation of inv expression.
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Interaction of OmpR with the inv promoter region
In silico examination of the nucleotide sequence upstream of the inv ORF of Y. enterocolitica (Fig. 5a) led to the identification of a sequence with a high level of identity (78 %) to the consensus binding site for OmpR (Fig. 5b). To determine whether OmpR actually binds to this sequence, DNA mobility shift assays were performed. A 114 bp fragment of the inv promoter was DIG labelled and mixed with increasing amounts of purified phosphorylated His-tagged OmpR protein. The results presented in Fig. 6 demonstrate a single shifted OmpR–DNA complex when the 114 bp inv regulatory region fragment interacts with 1.5 and 2.0 µg of the protein. DNA binding was specific for the inv promoter fragment because the shifted band disappeared with the addition of an excess of unlabelled fragment (Fig. 6a, lane 9), but not in the presence of salmon sperm DNA (lane 8). The specificity of OmpR for the inv promoter fragment was confirmed in a competitive mobility shift assay with a non-related control DNA fragment from P. aminophilus (324 bp). The mobility of this heterologous fragment was unaltered in the presence of different amounts of purified OmpR-P, whereas the inv fragment was clearly shifted (Fig. 6b, lanes 2 and 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pepe et al., 1994).
The OmpR protein is part of the OmpR/EnvZ two-component regulatory system that senses osmolarity. Thus, we investigated the possibility that invasin synthesis might be regulated via this pathway. To analyse the molecular mechanism responsible for osmoregulation of inv we used strains with or without the active OmpR protein. An ompR deletion mutant of Y. enterocolitica O : 9 was previously constructed using inverse PCR (Brzostek & Raczkowska, 2003). The pattern of inv expression at different temperatures was initially investigated by RT-PCR, which demonstrated a significant increase in inv mRNA at 25 °C in Y. enterocolitica lacking the OmpR protein. These unexpected results were an incentive to study the properties of OmpR in detail. The activity of the inv promoter was examined in strains with or without the ability to express OmpR using a chromosomal transcriptional inv' : : lacZYA fusion. In LB medium at 25 °C, sixfold higher inv expression was detected in the strain carrying an ompR null mutation (AR5) than in the wild-type strain (Ye10). This effect, i.e. the strong derepression of inv at 25 °C, was not observed at 37 °C, indicating that temperature-mediated repression of inv is OmpR-independent.
Considering that OmpR participates in the osmoregulation of inv, we measured the effect of high osmolarity on inv transcription in the wild-type and ompR mutant strains. The level of inv expression remained unchanged in conditions of high osmolarity in the ompR mutant background, compared with the decreased expression of inv seen in the wild-type. In other words, inv expression at 25 °C was no longer responsive to high osmolarity. Therefore, we propose that OmpR in Y. enterocolitica negatively regulates inv expression at the transcriptional level at 25 °C and that high osmolarity enhances the inhibitory effect of this regulatory protein.
It has been shown previously that phosphorylation of E. coli OmpR activates this regulatory protein. Phosphorylation of OmpR by EnvZ in response to high osmolarity enhances the affinity of OmpR-P for its target DNA by approximately 10-fold without altering its specificity (Lan & Igo, 1998; Russo & Silhavy, 1991). Changes in invasin synthesis in Y. enterocolitica in response to osmolarity suggest that the effect of a high concentration of salts in the growth medium involves the phosphorylation of OmpR by EnvZ. On the other hand, it is likely that under normal osmolarity conditions (found during growth on LB medium), some fraction of OmpR would be available for phosphorylation by other phospho donors, which might result in OmpR activation. In E. coli, some portion of OmpR seems to be phosphorylated by acetyl phosphate (McCleary & Stock, 1994).
Confirmation of the effect of a lack of functional OmpR protein emerged from the complementation experiments. Genetic complementation of the ompR mutation in strain AR5 (
ompR : : Km, inv' : : lacZYA) with a plasmid expressing 6His-OmpR (pBR3) restored the physiological characteristics of the wild-type strain. The complementation affected both the outer-membrane porin profiles and inv expression. In terms of the porin profile, the results demonstrated that the His-tagged OmpR protein is active and that OmpR-dependent porin expression in Y. enterocolitica resembles that seen in E. coli. The AR5/pBR3 strain also showed a fully restored wild-type phenotype with respect to inv promoter activity. When the 6His-OmpR protein was expressed from pBR3 in the Ye10 strain (OmpR+), an enhanced inhibitory effect of this protein was observed.
The OmpR protein in E. coli has been characterized as a DNA-binding protein that is able to directly modify gene expression by binding within promoter regions (Egger et al., 1997; Jubelin et al., 2005; Yamamoto et al., 2000). Inspection of the DNA sequence in the region upstream of the transcription start site of inv revealed the presence of a putative OmpR specific binding site at –15 to –33 nucleotides, which exhibits 78 % homology to the OmpR consensus binding sequence. To characterize the OmpR binding properties of this sequence, we performed electrophoretic mobility shift assays with a DNA fragment from the inv promoter carrying the putative binding site. The mobility shift assays with purified phosphorylated 6His-OmpR protein revealed its affinity for the inv promoter fragment, which led to the formation of an OmpR–DNA complex. The assays were performed with OmpR phosphorylated by acetyl phosphate since it has been previously shown that the phosphorylation of E. coli OmpR enhances its affinity for its binding site (Shin & Park, 1995). In view of these findings, OmpR might directly interact with its specific binding site in the inv promoter to prevent RNA polymerase association and, consequently, to switch off inv expression. Thus, OmpR fits the criteria for a repressor of inv in Y. enterocolitica.
The Y. enterocolitica inv gene is subject to regulation by a number of factors including RovA, YmoA and H-NS. Although the roles of H-NS and YmoA in inv regulation have been difficult to study due to the inability to inactivate the hns and ymoA genes, it has recently been demonstrated that these factors probably form an inhibitory complex on the inv promoter (Ellison & Miller, 2006a, b; Ellison et al., 2003). RovA, described until recently as a transcriptional activator, seems to act as a derepressor of H-NS/YmoA inv inhibition in Y. enterocolitica (Ellison & Miller, 2006a, b) and H-NS-mediated inv repression in Y. pseudotuberculosis (Heroven et al., 2004; Nagel et al., 2001) by competing with the other factors for binding to inv. However, in vitro studies indicate that interaction of RovA with the Y. pseudotuberculosis inv promoter occurs even when H-NS is bound to this region. Furthermore, the analysis of RovA, H-NS and YmoA expression in bacterial cells grown at 26 and 37 °C demonstrated that the cellular level of RovA is a key determinant in the thermoregulation of inv in Y. enterocolitica (Ellison & Miller, 2006b).
Based on current knowledge we hypothesize that OmpR could act as an additional repressor of inv. It is possible that RovA, induced in response to environmental stimuli, binds to DNA upstream of the inv gene, disturbing OmpR-P binding to the inv promoter, which results in the activation of transcription. When bacteria encounter environmental conditions that fully activate OmpR, such as phosphorylation by EnvZ under high-osmolarity conditions, OmpR-P might bind efficiently and repress inv (observed under these conditions) even though RovA is still bound to the inv promoter. Whether OmpR competes with H-NS (YmoA) for binding to DNA is an open question. However, we cannot rule out the possibility that Y. enterocolitica OmpR also modulates expression of inv through interactions with other inv regulators.
To summarize, the OmpR protein, acting as a repressor of inv (encoding the virulence factor of Y. enterocolitica), might play a role in coordinating the expression of invasin during the process of Y. enterocolitica pathogenesis. This process may be fine tuned in response to changing environmental conditions through a complex network of interactions between the multiple regulatory factors involved.
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ACKNOWLEDGEMENTS |
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Edited by: P. van der Ley
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Received 6 October 2006;
revised 15 February 2007;
accepted 12 March 2007.
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