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School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
Correspondence
Prashant Kodgire
pkodgire{at}bsd.uchicago.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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D form of RNA polymerase and is regulated by a group of proteins called transition state regulators (TSRs). Our studies show that hag transcription is negatively regulated by the transition state regulator ScoC, by binding to its promoter. Furthermore, ScoC, indirectly, also positively regulates hag by increasing the availability of D by downregulating the levels of the anti-D-factor FlgM. We further show that the positive regulation by ScoC predominates over the negative regulation.
Present address: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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D-dependent RNA polymerase (Mirel & Chamberlin, 1989
; Ordal et al., 1993). This gene is transcribed as a monocistronic mRNA and shows a temporal pattern of expression that peaks at the end of the exponential phase of growth followed by a decrease in transcript levels as sporulation proceeds (Mirel et al., 2000). In general, temporal regulation of many genes involved in post-exponential-phase processes such as motility, chemotaxis, extracellular enzyme synthesis and sporulation is coordinated by regulatory proteins called transition state regulators (TSRs) such as AbrB, ScoC and SinR (Smith, 1993; Strauch et al., 1989). Regulation of genes is also brought about by the interplay of sigma factors with their cognate anti-sigma factors (Hecker & Volker, 1998; Helmann, 1999; Hughes & Mathee, 1998). For example, in B. subtilis, the motility genes are transcribed at the post-exponential phase of growth by the D form of RNA polymerase. Until then, D is kept sequestered by the anti-D factor, FlgM; it is subsequently released to transcribe the motility genes (Helmann, 1999; Hughes & Mathee, 1998; Mirel et al., 1994). FlgM inhibits the activity of D RNA polymerase by the sequestration of D and also by destabilizing the existing D
holoenzyme (Bertero et al., 1999; Helmann, 1999; Hughes & Mathee, 1998). The transition state regulator ScoC is a negative regulator of protease production and sporulation that binds to a consensus DNA sequence, 5'-RATANTATY-3' (Henner et al., 1988; Kallio et al., 1991; Perego & Hoch, 1988). ScoC has also been reported to be a positive regulator of motility genes (Caldwell et al., 2001), which are mainly transcribed by the D-dependent RNA polymerase (Ordal et al., 1993). This conclusion has been derived from the transcription profiling of scoC mutant cells (scoC4), which showed that most of the motility genes, including hag, are transcribed at lower levels in the scoC mutant as compared to that in the wild-type (Caldwell et al., 2001). We were thus interested in understanding the regulation of hag by ScoC. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli DH5
was used as an intermediate host for plasmid constructions using standard cloning techniques (Sambrook et al., 1989). Both E. coli DH5 and B. subtilis cells were grown at 37 °C in LB medium unless specified otherwise. Transformations in B. subtilis were performed using the protoplast transformation protocol (Bron, 1990). Where necessary, antibiotics were added to the following final concentrations: for B. subtilis, chloramphenicol, kanamycin and spectinomycin were at 5, 10 and 100 µg ml–1, respectively; for E. coli, ampicillin was at 100 µg ml–1.
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) were synthesized at Microsynth AG, Balgach, Switzerland.
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) was PCR amplified from genomic DNA of B. subtilis 168 with primers KKR211 and KKR212 (Table 2
) (95 °C/1 min, 40 °C/1 min, 72 °C/40 s) and fused in translational frame to the E. coli lacZ gene, at the HindIII and BamHI sites, in the integrative plasmid pMUTIN4 (Table 1) (Vagner et al., 1998) to give pMhag'-lacZ (Table 1).
Construction of HA in pBluescript SK+.
To create the mutated H1 site, HA (5'-CCGCTGCAG-3'), two PCR products were obtained from pMhag'-lacZ with primers KKR211/KKR293 (95 °C/1 min, 42 °C/1 min, 72 °C/30 s) and KKR292/KKR212 (Table 2) (95 °C/1 min, 40 °C/1 min, 72 °C/30 s). The first product was digested with HindIII and PstI and cloned into pBluescript SK+. The second PCR product was digested with PstI and BamHI and cloned downstream of the first PCR product to give pBluescript SK+ containing HA. The mutation in HA was confirmed by PstI digestion as this site was introduced in the primers to create the mutation. This recombinant plasmid was used as template to amplify HA with primers KKR113/KKR114 (Table 2) (95 °C/1 min, 40 °C/1 min, 72 °C/20 s) for electrophoretic mobility shift (EMSA) studies.
Construction of HQ in pBluescript SK+.
To create the mutated H2 site, HQ (5'-CGGCCCGGG-3'), two PCR products were obtained from pMhag'-lacZ with primers KKR211/KKR322 (95 °C/1 min, 42 °C/1 min, 72 °C/30 s) and KKR321/KKR212 (Table 2) (95 °C/1 min, 40 °C/1 min, 72 °C/30 s). The first product was digested with HindIII and SmaI and cloned into pBluescript SK+. The second PCR product was digested with SmaI and BamHI and cloned downstream of the first PCR product to give pBluescript SK+ containing HQ. The mutation in HQ was confirmed by digestion with SmaI as this site was introduced in the primers to create the mutation. This recombinant plasmid was used as template to amplify HQ with primers KKR292/KKR320 (Table 2) (95 °C/1 min, 46 °C/1 min, 72 °C/20 s) for EMSA studies.
Construction of phag-lacZ.
To construct phag-lacZ, a DNA fragment of 361 bp (–189 to +172) containing the hag promoter and the first 27 codons of the hag ORF (Mirel & Chamberlin, 1989) was PCR amplified from pMhag'-lacZ (Table 1) with primers KKR211/KKR337 (Table 2) (95 °C/1 min, 42 °C/1 min, 72 °C/30 s). The amplified product was digested with HindIII/BamHI and fused in translational frame to the E. coli lacZ gene in the replicative multi-copy plasmid pRB381 (Bruckner, 1992) to give phag-lacZ (Table 1). Plasmid pRB381 is derived from plasmid pUB110 (Bruckner, 1992) and the copy number of pUB110 in B. subtilis is about 50 per chromosome (Bron, 1990).
Construction of phag-lacZ-HP.
To create HP (5'-GATATAGGG-3'), in which the 3'-end of H1 was mutated, two PCR products were obtained from pMhag'-lacZ with primers KKR327/KKR212 (95 °C/1 min, 40 °C/1 min, 72 °C/30 s) and KKR211/KKR328 (Table 2) (95 °C/1 min, 42 °C/1 min, 72 °C/30 s). The first product was digested with SmaI and BamHI and cloned into pBluescript SK+. The second PCR product was digested with HindIII and cloned upstream of the first PCR product to give HP in pBluescript SK+. The latter was used as the template to reamplify the hag promoter containing the HP site with primers KKR211/KKR337 (Table 2) and cloned in translational fusion with lacZ in pRB381 to give phag-lacZ-HP (Table 1). pBluescript SK+ carrying HP was also used to amplify HP with primers KKR113/KKR114 (Table 2) for EMSA with ScoC.
Construction of phag-lacZ-HQ.
The hag promoter containing the HQ site was amplified from the recombinant pBluescript SK+ plasmid carrying HQ (described above) with primers KKR211/KKR337 and cloned in translational fusion with lacZ in pRB381 to give phag-lacZ-HQ (Table 1).
Construction of pflgM-lacZ.
A 151 bp DNA fragment (–106 to +45 from the transcription start of yvyF) containing the PD-1 promoter and the ATG of yvyF (Mirel et al., 1994) was PCR amplified from genomic DNA of B. subtilis 168 with primers KKR296/KKR306 (Table 2) (95 °C/1 min, 48 °C/1 min, 72 °C/20 s), digested with HindIII and BamHI and fused in translational frame with the E. coli lacZ gene in pRB381 to give plasmid pflgM-lacZ (Table 1).
Construction of pIC-scoC.
A 1.65 kb DNA fragment containing the promoter, ORF and transcription terminator of scoC (Perego & Hoch, 1988) was amplified from genomic DNA of B. subtilis 168 with primers KKR262/KKR307 (Table 2) (95 °C/1 min, 48 °C/1 min, 72 °C/1 min 40 s) and cloned at the SalI site in plasmid pIC56 (copy no. 10) (Steinmetz & Richter, 1994) to give pIC-scoC (Table 1).
Assay for β-galactosidase in B. subtilis.
The strains were grown at 37 °C in Penassay broth to stationary phase (OD600 2.0) and the β-galactosidase activities were determined (Nicholson & Setlow, 1990). All the β-galactosidase assays presented in this work were performed in triplicate. The activities presented are the mean of triplicate values. At least three independent experiments were performed and a representative result is shown. We ensured that the lacZ results were not due to differences in copy numbers between the various strains. We compared the copy number of pRB381-derived plasmids by dot-blots, which were very similar in all the strains (data not shown).
Electrophoretic mobility shift assay.
EMSA was performed as described in the Roche gel shift manual (http://www.roche-applied-science.com/pack-insert/3353591a.pdf). End-labelling of the probe was carried out with digoxigenin-11-ddUTP (DIG-ddUTP) using terminal transferase in 20 µl reaction buffer containing 0.2 M potassium cacodylate, 0.25 M Tris/HCl, pH 6.6, 0.25 mg BSA ml–1 and 5 mM CoCl2 at 37 °C for 15 min, and then 2 µl 0.2 M EDTA (pH 8.0) was added to terminate the reaction. Binding reactions were carried out with 10 nM DIG-labelled probe and ScoC (1 µM) in 20 µl reaction buffer containing 20 mM HEPES, pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, Tween 20 (0.2 %, w/v), 30 mM KCl, 1 µg poly(dI-dC), and 0.1 µg poly-L-lysine at 37 °C for 15 min. For the competition reaction, protein was first incubated in presence of 100-fold molar excess of competitor DNA (unlabelled specific DNA) followed by incubation with the labelled probe. The bound product was electrophoresed on a 5 % polyacrylamide gel in 0.25x Tris/borate/EDTA buffer at 4 °C. The gel was electroblotted onto a nylon membrane and treated with anti-DIG–alkaline phosphatase conjugate. DNA was detected with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in AP buffer (100 mM Tris/HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) until visible bands were observed.
Cloning, expression and purification of FlgM, and raising of anti-FlgM antibodies.
The gene coding for FlgM (Mirel et al., 1994) was PCR amplified from genomic DNA of B. subtilis 168 with primers KKR311/KKR312 (Table 2) (95 °C/1 min, 45 °C/1 min, 72 °C/30 s) and cloned into plasmid pET28a (Novagen) at the NdeI and XhoI sites to give pFlgM. The cloning strategy was designed to provide a His-tag at the C terminus of the protein. pFlgM was transformed into E. coli BL21(DE3) and FlgM was induced with 0.1 mM IPTG. E. coli BL21(DE3)/pFlgM was inoculated in 5 ml LB broth and grown overnight at 37 °C in an orbital shaker at 200 r.p.m. Then 50 µl of the overnight grown culture was used to inoculate 5 ml fresh LB broth. The culture was grown at 37 °C in an orbital shaker at 200 r.p.m. for 2.5 h, until the OD600 reached 0.5–0.6. Freshly prepared IPTG was added to a final concentration of 0.1 mM and incubation continued for 40 min. Rifampicin was added to a final concentration of 150 µg ml–1 and incubation continued for 2.5 h. Cells were harvested by centrifugation and the pellet was washed with 1 ml buffer containing 25 mM Tris/HCl, pH 7.4. The pellet was resuspended in 500 µl buffer containing 25 mM Tris/HCl, pH 7.4, and cells were lysed by lysozyme treatment at 30 °C for 15 min, followed by sonication at 4 °C. The cell lysate was centrifuged to separate supernatant and pellet and samples were analysed by SDS-PAGE (data not shown).
FlgM was affinity purified from the supernatant on a Ni-NTA resin as described in the Qiagen Ni-NTA spin kit manual. Input sample was prepared in 50 mM phosphate buffer, pH 8.0, 0.3 M NaCl and 10 mM imidazole and loaded on a spin column. The column was washed with the above buffer containing 100 mM imidazole (six column volumes). The protein sample was eluted with 150 µl buffer containing 1 M imidazole. Samples were analysed by SDS-PAGE to determine purity and recovery of the protein. The purified protein, which showed a single band with purity greater than 95 % (data not shown), was used for raising polyclonal anti-FlgM antibodies in rabbits (by Bangalore Genei, Bangalore, India).
We tested the ability of the anti-FlgM antibodies to detect FlgM in extracts of B. subtilis 168. The cells were grown at 37 °C for 6 h and the presence of FlgM was tested by Western blot analysis (Sambrook et al., 1989) with the anti-FlgM antibodies. A single band of 10 kDa was clearly detected in wild-type cells that was missing in the flgM mutant 1A764 (Mirel et al., 1994), in which 80 of the 88 amino acids have been deleted (see Fig. 4A).
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3.0, of B. subtilis 168 and 168scoC strains from an overnight culture were seeded into 25 ml Penassay broth and grown with shaking at 37 °C. The growth rates of both strains were identical. Samples (5 ml) corresponding to equal numbers of cells were harvested at 5 h (t1) and 6 h (t2) relative to t0 (4 h of growth; time of entry into stationary phase) and centrifuged. The pellet was heated at 95 °C for 15 min in 30 µl SDS-loading buffer, loaded onto a 15 % SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The membrane was probed with rabbit anti-FlgM antibodies (raised against purified FlgM; see above); bands were detected using an alkaline phosphatase-linked secondary antibody and NBT/BCIP (Sambrook et al., 1989). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was fused to the E. coli lacZ gene in an integrative plasmid, pMUTIN4 (Vagner et al., 1998), to give pMhag'-lacZ (Table 1) and transformed into B. subtilis 168 and 168scoC, a scoC disruptant (Kodgire et al., 2006), to yield 168 : hag'-lacZ and 168scoC : hag'-lacZ (Table 1). The strains were grown with shaking at 37 °C in Penassay broth to stationary phase OD600 2.0) and the β-galactosidase activities were determined (Nicholson & Setlow, 1990). We observed an almost twofold increase in the hag promoter activity in 168scoC : hag'-lacZ (435±15 Miller units) as compared to that in wild-type (225±10 Miller units) (Table 3A), indicating that ScoC negatively regulates hag transcription. This result was in contrast to that observed from the transcription profiles of B. subtilis 168 and scoC4, which demonstrated decreased hag transcription in scoC4 (Caldwell et al., 2001).
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), we searched for putative ScoC binding sites using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). This search revealed the presence of two sites located between –11 and –2 (H1, 5'-GATATTAAT-3') and between +103 and +112 (H2, 5'-CACAATATT-3') (Fig. 1) with an 8/9 and 7/9 match with the consensus ScoC binding site (5'-RATANTATY-3'), respectively (Caldwell et al., 2001). The H1 site overlaps with the –10 region of the bipartite hag promoter and the H2 site is located in the hag ORF (Fig. 1). We therefore tested the ability of ScoC to bind to these sites by EMSA.
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). Fig. 2(A, B) shows that both the DNA probes, containing the H1 and H2 sites respectively, were retarded in the presence of ScoC (lane 2) as compared to in its absence (lane 1). The retardation was significantly reduced with a 100-fold molar excess of unlabelled probe (lane 3), showing the specificity of binding. Thus, we conclude that ScoC is capable of binding to both the sites in the hag promoter region.
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, lane 4 shows that mutation of either of the ScoC binding sites significantly reduced the binding of ScoC to the hag promoter region. Furthermore, the mutated probes could not compete for binding of ScoC to the respective wild-type probes (Fig. 2A, B, lane 5), indicating that H1 and H2 are bona fide binding sites for ScoC. Thus, our results are strongly in support of a direct transcriptional regulation of hag by ScoC.
Mutation of the H1 or H2 site in the hag promoter region relieves repression
To investigate whether mutation of either the H1 or H2 site led to relief from repression, we made three constructs, phag-lacZ, phag-lacZ-HP and phag-lacZ-HQ (Table 1), carrying the wild-type hag promoter and mutated H1 and H2 sites respectively. To construct phag-lacZ, a DNA fragment of 361 bp (–189 to +172) containing the hag promoter and the first 27 codons of the hag ORF (Mirel & Chamberlin, 1989) was cloned in translational frame to the E. coli lacZ gene in the replicative multi-copy plasmid pRB381 (Table 1) (Bruckner, 1992). The constructs phag-lacZ-HP and phag-lacZ-HQ were identical to phag-lacZ in all respects except for the mutation in the H1 (HP) or H2 (HQ) site, respectively. Unlike HA, in which all the bases of H1 were mutated and which was used for the binding experiments with ScoC, the HP site in phag-lacZ-HP contained mutations in only four bases from the 3'-end of H1 (5'-GATATAGGG-3'). This was done so as to keep the –10 sequence of the promoter intact (Fig. 1). Binding studies confirmed that, similar to HA, ScoC is unable to bind HP (Fig. 2A, lane 6) and the partially mutated probe could not compete for binding of ScoC to the wild-type probe (Fig. 2A, lane 7).
The two plasmids, phag-lacZ-HP and phag-lacZ-HQ, were introduced into B. subtilis 168 to give 168/phag-lacZ-HP and 168/phag-lacZ-HQ (Table 1) and the promoter activities were compared with that in 168/phag-lacZ (phag-lacZ in B. subtilis 168, Table 1). The promoter activities in 168/phag-lacZ-HP and 168/phag-lacZ-HQ were 16 500±150 and 17 000±150 Miller units, respectively, as compared to 2000±50 Miller units in 168/phag-lacZ (Table 3B), clearly indicating that the mutation of either the H1 or the H2 site led to loss of repression.
The level of relief from repression upon mutation of either the H1 or the H2 site was almost eightfold higher as compared to a twofold relief in 168scoC : hag'-lacZ. Similar twofold relief was observed when the multicopy plasmid phag-lacZ was introduced into 168scoC and hag reporter expression was compared with that in 168/phag-lacZ (data not shown). This suggested to us that hag is subject to both negative and positive regulation and that in 168scoC : hag'-lacZ the extent of relief in repression is blunted by the loss of positive regulation.
Based on the transcription profiling of scoC4, in which motility genes were shown to be downregulated, it was proposed that ScoC positively regulates the availability of D by downregulating FlgM activity (Caldwell et al., 2001). FlgM has previously been shown to be a negative regulator of hag expression (Fredrick & Helmann, 1996). We confirmed that FlgM indeed negatively regulates hag transcription by measuring hag expression in an flgM mutant background (1A764, Table 1) and compared its activity with that in the isogenic wild-type strain, JH642 (Table 1). We measured the effect of FlgM on hag expression in the JH642 background instead of in the 168 background, avoiding intricacies of moving the in-frame flgM deletion into 168 without any selectable marker, since strain 1A764, an flgM mutant, was created by deleting 80 of the 88 amino acids in the FlgM ORF (Mirel et al., 1994). The plasmid phag-lacZ was introduced into B. subtilis JH642 and B. subtilis 1A764 to give JH642/phag-lacZ and 1A764/phag-lacZ (Table 1), respectively, and the promoter activities were determined. The β-galactosidase activity in 1A764/phag-lacZ was 3700±50 Miller units, more than twofold higher as compared to JH642/phag-lacZ (1700±25 Miller units; Table 3C). A similar increase in hag promoter activity in the flgM mutant background was previously reported by Fredrick & Helmann (1996).
flgM is transcriptionally regulated by ScoC
To show experimentally that ScoC downregulates FlgM, we assessed flgM transcription in wild-type B. subtilis 168 and 168scoC. flgM is the second gene of an operon that contains four ORFs, yvyF (orf 139), flgM, yvyG (orf 160) and flgK, and is transcribed from a single promoter, PD-1 (Mirel et al., 1994). The PD-1 promoter and the ATG of yvyF (hereafter referred to as the flgM promoter) was cloned in translational frame with the E. coli lacZ gene in pRB381 (Bruckner, 1992) to give plasmid pflgM-lacZ (Table 1). pflgM-lacZ was transformed into B. subtilis 168 and 168scoC to yield 168/pflgM-lacZ and 168scoC/pflgM-lacZ (Table 1) and the β-galactosidase activities were determined (Nicholson & Setlow, 1990) between 4 h (t0) and 6 h (t2) of growth (t0 corresponds to entry into stationary phase), as flgM expression has been observed to be maximal during this stage of growth (Liu & Zuber, 1998). The growth rates of both strains were identical. The promoter activity in 168scoC/pflgM-lacZ was at least twice that in 168/pflgM-lacZ (Fig. 3), indicating that ScoC negatively regulates flgM expression. The increase in expression of flgM in 168scoC is also reflected in the levels of FlgM protein as determined by Western blot analysis using polyclonal anti-FlgM antibodies raised against purified FlgM. Fig. 4(B) shows that the level of FlgM is at least two to three times higher in 168scoC as compared to that in wild-type cells when measured at 5 h (t1) and 6 h (t2). We conclude that the negative regulation of flgM is also reflected in the levels of the FlgM protein. These results thus provide experimental support that ScoC positively regulates hag expression by downregulating the levels of FlgM, leading to enhanced D activity. The fourfold difference observed between the relief from repression when either of the sites is mutated and the relief in repression in 168scoC : hag'-lacZ also suggests that positive regulation of hag by ScoC predominates.
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), encoding ScoC, to give 168 : hag'-lacZ/pIC-scoC, and the promoter activity of hag was compared with that in 168 : hag'-lacZ. The hag promoter activity of 168 : hag'-lacZ/pIC-scoC was 410±20 Miller units and was twice that of 168 : hag'-lacZ (225±10 Miller units) (Table 3A). This clearly supports the contention that the positive effect of ScoC is predominant. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This is a testament to the crucial role played by ScoC in the regulation of motility genes in B. subtilis. To date, the exact mechanism for this regulation is not known, although it was thought that ScoC regulates these genes by indirectly regulating the availability of D via a pathway that involves SinR (Caldwell et al., 2001). SinR is expressed from a di-cistronic operon, sinIR. The product of the first gene, SinI, post-translationally antagonizes the activity of SinR by converting SinR from its active tetrameric state to an inactive SinI-SinR heterodimer. ScoC represses sinI transcription (Kallio et al., 1991; Shafikhani et al., 2002), resulting in elevated levels of active SinR and thus leading to increased levels of D (Caldwell et al., 2001), probably via decreasing FlgM activity. This model is supported by the observation that deletion of flgM could restore motility in a sinR null mutant (Fredrick & Helmann, 1996). However, we have demonstrated that ScoC downregulates FlgM and thereby positively controls the availability of D and therefore motility gene expression.
Our observation that ScoC is a repressor of hag expression is contrary to the report of Caldwell et al. (2001), wherein, from transcriptional profiling experiments, they reported decreased hag expression in a scoC mutant background. In general microarray results are quite reliable; however, accurate measurement of absolute expression levels is often not possible for various reasons such as suboptimal design or choice of probes, inconsistent sequence fidelity of the spotted microarrays, variability of differential expression and discrepancy in fold-change calculation (Kothapalli et al., 2002; Draghici et al., 2006). Our results clearly suggest that ScoC specifically binds to two sites in the hag promoter region and that repression of hag is relieved in the absence of ScoC. The presence of two ScoC binding sites in the hag promoter region suggests that, as in the case of the nprE, aprE and sinI promoters (Kallio et al., 1991), ScoC may manifest its function through the formation of a bent repression loop as proposed by several investigators (Smith, 1993; Strauch & Hoch, 1992, 1993), thereby affecting promoter clearance. In addition ScoC may regulate transcription initiation of hag by preventing open complex formation, as the H1 site overlaps with the –10 region of the promoter (Fig. 1). CodY, another regulator of hag, monitors the general nutritional state of the cell by sensing intracellular GTP levels (Fisher, 1999; Ratnayake-Lecamwasam et al., 2001). It is known to repress hag expression in rich media, and repression of hag is relieved approximately twofold in a codY mutant background. Interestingly, the H2 site of hag overlaps with the CodY binding site (Bergara et al., 2003). Therefore, it is possible that mutation of H2 might affect the binding of CodY to the hag promoter region and additionally contribute to the relief in repression of hag.
The difference in the extent of relief from repression in 168scoC as compared to that in 168/phag-lacZ-HP and 168/phag-lacZ-HQ suggests that hag transcription is both negatively and positively regulated by ScoC. The positive regulation is manifested by increased availability of D as a result of decreased levels of FlgM. A model for hag regulation is presented in Fig. 5. We envisage that such a dual control by ScoC is most likely aimed at ‘fine-tuning’ hag expression levels to suit the cell's needs in different environmental conditions, such as efficient nutrient acquisition, avoidance of toxic substances, translocation to optimal colonization sites and dispersal in the environment.
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ACKNOWLEDGEMENTS |
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Edited by: T. Msadek
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Received 3 July 2008;
revised 18 September 2008;
accepted 29 September 2008.
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) and 168scoC/pflgM-lacZ (
). t0 is the time of entry into stationary phase. Data shown (means±SE from triplicate values) are from a representative experiment of at least three independent experiments.