Promoter selectivity of the Bacillus subtilis response regulator DegU, a positive regulator of the fla/che operon and sacB
© Tsukahara and Ogura; licensee BioMed Central Ltd. 2008
Received: 21 August 2007
Accepted: 15 January 2008
Published: 15 January 2008
The response regulator DegU and its cognate histidine kinase DegS constitute a two-component system in the Gram-positive soil bacterium Bacillus subtilis. Unphosphorylated and phosphorylated forms of DegU are known to activate target gene transcription in B. subtilis. Although phosphorylated DegU (DegU-P) regulates more than one hundred and twenty genes, the targets of unphosphorylated DegU are unknown, except for comK.
We found that the fla/che (flagella and chemotaxis) operon is positively regulated by unphosphorylated DegU. The effect was most prominent in a strain bearing the functional swrAA gene, a positive regulator of fla/che. Unphosphorylated DegU bound to two regions in the fla/che regulatory region containing an inverted repeat-like sequence that resembles the inverted repeat (IR) in the comK promoter. Mutational analysis revealed that positive regulation of fla/che by SwrAA requires DegU-binding. An analysis of the DegU-P-regulated gene sacB (levansucrase gene) by footprint and mutational analyses revealed that DegU-P bound to a direct repeat (DR) of the DegU-recognition motifs, which has been shown to be functional in vivo, while unphosphorylated DegU did not. These results strongly suggest that the arrangement of the DegU-binding motifs determines whether unphosphorylated DegU or DegU-P binds to the sacB promoter. The hypothesis was confirmed by observing degS-independent expression when the DR in the sacB-lacZ fusion was changed to an IR, suggesting that unphosphorylated DegU regulates the sacB promoter through the newly created IR. This was confirmed by binding of unphosphorylated DegU to the IR in the sacB promoter.
This study demonstrated that DegU positively regulates flgB and sacB through its binding to the promoter regions. We demonstrated that DegU-P prefers binding to DR but not to IR in the sacB promoter.
To respond to environmental fluctuations, bacteria employ a large and elaborate family of two-component signaling systems. The classical two-component system consists of a sensor kinase and its cognate response regulator . In response to the signal input, the kinase phosphorylates its own histidine residue. The phosphoryl group is then transferred to a conserved aspartate residue on the cognate response regulator, which then acts as a transcription factor in most cases. Given the many studies on how response regulator regulates output response by phosphorylation, it is not surprising that variable strategies were found . Upon phosphorylation, some regulators dimerize to be activated or interact with other proteins or DNA , while other regulators are relieved from inhibition by their N-terminal domain . One such two-component system in the Gram-positive soil bacterium Bacillus subtilis consists of the response regulator DegU and its cognate histidine kinase DegS. DegU belongs to the NarL family, whose members have a helix-turn-helix structure at their C-terminus . The DegS-DegU system regulates many cellular processes, including exoprotease production and competence development [4–9]. It has also been reported to sense salt stress and to mediate appropriate responses [7, 10, 11]. In addition, it was found recently that the protein machinery for chromosome separation (SMC-ScpA-ScpB) forms a complex with DegS and inhibits its kinase activity . The activity of DegU itself has been shown to be finely tuned by several factors. The Rap-Phr systems are regulatory machinery to receive extracellular signals . DegU is negatively regulated by RapG since the protein inhibits its DNA-binding activity . RapG activity is in turn inhibited by its cognate extracellular pentapeptide PhrG after the peptide is taken up by the cell. Thus, the RapG-PhrG system functions as a positive regulatory mechanism for DegU. Moreover, the transcription of rapG is repressed by RghR .
Unphosphorylated DegU is required for competence development and binds to the promoter region of comK, which encodes a master regulator of competence development [16, 17]. Unphosphorylated DegU has also been reported to facilitate the binding of ComK to the comK promoter . Previously we identified a DegU-recognized incomplete inverted repeat (IR) on the comK promoter (GTCATTTA-N7-TAAATATC) by using various mutated comK-lacZ fusions . Additional targets of unphosphorylated DegU have not been identified.
Phosphorylated DegU (DegU-P) activates the expression of more than one hundred twenty genes, including aprE (which encodes alkaline protease) and sacB (which encodes levansucrase); it also represses wapA, which encodes a cell-wall associated protein [6–9, 20]. In addition, the expression of bpr, which encodes bacillopeptidase F, has been reported to be probably dependent on DegU-P [8, 21]. To date, the DNA recognition sequence of DegU-P has not been identified with the exception of aprE and bpr. Our analysis revealed that an important cis-factor for DegU-dependent aprE expression is a direct repeat (DR) of the downstream half of the DegU-recognized IR in the comK promoter with two-nucleotide spacing (-70 to -52 relative to the transcription start site), . In addition, we identified three DRs with zero or two-nucleotide spacing, which are important for DegU-binding to the bpr promoter region and DegU-P-dependent expression of bpr . Furthermore, overproduction of DegU or the degU32 mutation, which renders DegU-P resistant to dephoshorylation, resulted in a decrease in the expression of the fla/che operon encoding chemotaxis-related proteins and components of the flagella apparatus [5, 8, 9, 23, 24].
Since DegU-P stimulates the transcription of many genes and unphosphorylated DegU is required for comK transcription, DegU is regarded as a molecular switch that controls cell fate . However, what factor determines promoter selectivity of DegU-P and unphosphorylated DegU remains unclear.
In this paper, we found that flgB, which is the first gene of the 26-kb-long fla/che operon, is subject to direct positive regulation by unphosphorylated DegU through two DegU-binding sequences containing an IR-like sequence. In contrast, footprint analyses of the sacB promoter revealed that DegU-P bound to a DR within the DegU-binding sequence. Since unphosphorylated DegU bound to flgB, the arrangement of the DegU-binding sequence within promoter regions (DR or IR-like sequence) must dictate which form of DegU (phosphorylated or unphosphorylated) binds to them. This hypothesis was confirmed by expression analysis of sacB-lacZ fusions carrying an artificial IR within their DegU-binding sequence.
Unphosphorylated DegU binds to two IR-like sequences in flgB
DegU and SwrAA regulation of the expression of a F1D fusion carrying two BRs
DegU and SwrAA act on flgB expression through BR1
To determine the in vivo role of BR1 in the positive regulation of flgB by DegU, we constructed a fusion carrying BR1 alone (F1) and tested its expression in various genetic backgrounds (Figure 3B). As well as F1D, the expression of F1 was decreased in the degU but not degS background, suggesting positive regulation of F1 by unphosphorylated DegU. The reduced expression in the presence of the degU mutation and the increased expression in the presence of functional swrAA were abolished by introducing point mutations into the upstream and downstream half sites of the IR-like sequences in BR1 and the long spacing region (F1M1, F1M3 and F1M2, respectively) or by deleting BR1 (F2 and F3). These results demonstrated that unphosphorylated DegU binding to BR1 is required for the positive regulation of flgB by DegU, and for the enhancing effect of SwrAA. This result strongly suggested that at least part of the positive function of SwrAA is exercised through DegU. In fact, in the degU strain carrying F1D, F1DM or F1, SwrAA had little effect on the expression of flgB. Surprisingly, the introduction of the degU32 mutation into a strain carrying F1 and swrAA resulted in about a three-fold increase in the expression of F1, which is not consistent with the results of the expression analysis of F1D. This positive effect of degU32 in the swrAA strain was abolished by disrupting or deleting BR1, suggesting that higher cellular concentrations of DegU-P may positively regulate flgB expression through increased binding of DegU-P to BR1. However, this hypothesis is not in agreement with the result shown in Figure 1. We will discuss this discrepancy with respect to the binding of DegU to BR1 (Discussion).
BR2 appears to prevent the positive regulation of flgB by DegU-P through BR1
To investigate the in vivo role of BR2, we constructed a F2D fusion carrying an incomplete BR1, BR2, and the core promoter, and tested its β-galactosidase activities in various genetic backgrounds. The levels of expression of F2D in all of the genetic backgrounds tested were similar to those of the F3 fusion, which should reflect strength of the core promoter of flgB. Moreover, the absence of effects of degU and functional swrAA in F1MD was reasonable, because F1MD is equivalent to F2D with respect to distribution of BRs. These results suggested that BR2 is not involved in positive regulation by SwrAA and unphosphorylated DegU. A comparison of the expression patterns of the F1D and F1DM fusions showed that there was essentially no difference except for the epistatic effect of the degU32 mutation. This suggested that BR2 could have some role to play in the negative effect of the degU32 mutation on F1D expression. In the presence of BR2, the degU32 mutation did not exhibit positive regulation of flgB through BR1, while, in the absence of BR2, the mutation resulted in increased fusion expression. Thus, BR2 appears to prevent the positive regulation of flgB by DegU-P through BR1, leading to downregulation of F1D in degU32 cells (Figure 3C). In addition, DegU-P bound to BR2 could not serve as a repressor, because there was no significant effect of the degU32 mutation on F2D expression. It should be noted that the slight reducing effect of the degU32 mutation on F1MD was observed in the swrAA background. This is due to an unknown reason.
Transcription start site of sacB
To characterize the interaction of DegU-P with its targets, we selected sacB, which is a well-characterized gene belonging to the DegU-P regulon. The DegU-P dependency of sacB expression has been reported previously . We determined the transcription start site of sacB by primer extension analysis (data not shown). A previous analysis found that the transcription start site is seven-base downstream of the newly determined site . This discrepancy is due to an unknown reason. The numbering of nucleotide of sacB was done based on our result hereafter.
Footprint analysis of the sacB promoter region
Confirmation of the in vivo function of the detected DR by lacZ fusion analysis
Changing the DR to an IR in the sacB promoter results in degS-independent but degU-dependent regulation
Another difference between the promoters of DegU-regulated and DegU-P-regulated genes is that the IR of comK and IR-like sequences of flgB (BR1 and BR2) have long spacing regions, whereas the DRs of aprE, bpr and sacB have short spacing regions. Thus, we examined whether a long spacer between the two half sites of sacB DR might alter its regulation. The introduction of a long spacer corresponding to that of the comK IR into the DR of sacB (SC-DR) did not affect degS-dependent positive regulation of sacB (Figure 7C). This suggests that spacing between two half-sites would not be a critical factor for the promoter selectivity of DegU.
This study demonstrated that while phosphorylation of DegU stimulates its binding to the DR in the sacB promoter, it inhibits DegU-binding to the artificial IR of the same promoter. These results strongly suggested that DegU-P prefers binding to DR but not to IR (Figure 9A). This hypothesis is consistent with the observation that phosphorylation of DegU abolished its binding to BR1 containing the IR-like sequence in the flgB promoter under the condition used. DegU-P, however, could bind to the BR2 in flgB and the degU32 mutation resulted in positive regulation of the F1 fusion through BR1, suggesting that DegU-P could bind to this region when cellular concentrations of DegU-P were high enough. We note that this positive regulation of the degU32 mutation was observed only in the swrAA background. The possible binding of DegU-P to the flgB promoter may be an exception to the hypothesis and remains to be elucidated.
Notably, DegU belongs to the NarL response regulator family, which is characterized by a classical helix-turn-helix domain that recognizes IRs of the same motifs . However, NarL has also been reported to bind to differently arranged motifs, namely, DRs . Indeed, another report has shown that the NarL-binding sites in various promoters are differently arranged as direct repeats, monomers, or inverted or divergent repeat . It is quite possible that the binding to differently arranged motifs requires different protein-protein interactions within the putative dimers of DegU, which might be regulated by phosphorylation. Global analysis of the DegU regulon has identified many genes other than the genes analyzed in this study [8, 9]. It would be of interest to determine how the DegU motif is arranged in the promoters of these genes as this may further enhance our understanding of DegU regulation.
We observed that His-tagged unphosphorylated DegU bound to the promoter regions of aprE and sacB (Figure 5) [14, 19]. This might mean that His-tagged unphosphorylated DegU can bind to DR. This characteristic is different from that observed for intact unphosphorylated DegU. It was reported that His-tagged Spo0A was phosphorylated in E. coli, resulting in an isolation of Spo0A-P without any phosphorylation reaction . Thus, it is possible that His-tagged DegU purified from E. coli might undergo phosphorylation and serve as DegU-P. The possibility is unlikely, however, because a half-life of DegU-P has been reported to be less than 90 min due to an intrinsic phosphatase activity of DegU . It has been shown that the addition of the His-tag to the C-terminus of Salmonella PhoP response regulator affects its biochemical properties and conformation with respect to its dimer formation and DNA-binding ability . Addition of His-tag at the N-terminus of DegU might alter its conformation as in the case of Salmonella PhoP.
In our analysis of the mechanisms regulating flgB expression, we observed that SwrAA requires DegU-binding to BR1 to enhance flgB expression. While the exact function of SwrAA is not yet understood, it is known that swrAA is required for swarming motility [26, 35], γ-poly-glutamic acid synthesis  and enhancing of the transcription of the large fla/che operon in a non-laboratory strain . The result that SwrAA stimulates the fla/ che transcription in a DegU-dependent manner led us to a speculation that SwrAA might facilitate DegU-binding to its target promoter or modulate the DegU function. We noted that if SwrAA functions to enhance the expression of some DegU-dependent genes, it remains to be determined how SwrAA focuses specifically on its targets among the many genes that are regulated by DegU.
Amati et al. showed that the downstream region from the flgB transcription start site containing BR2 did not interact with DegU . This result is inconsistent with our footprint data (Figure 1 and Figure 2). We do not know the reason for this discrepancy. In addition, they claimed that DegU-binding to the upstream region resulted in repression of ylxF, which is the ninth gene of the fla/che operon. Our result clearly showed that the expression of the F1 fusion containing only BR1 was increased in degU32 cells (Figure 3B). The different interpretation between their results and ours might be due to the use of ylxF-lacZ in their analysis. DegU-P bound to BR1 could be somehow dysfunctional in the presence of BR2. The regulatory mechanism involving the two BRs remains to be solved, although we speculate that DegU-P bound at each region might interact together through DNA-looping, leading to the abolishment of the positive effect of DegU-P through BR1. As a result, in the regulatory region carrying both regions, the degU32 mutation results in a decrease in flgB expression. This could be caused by a decrease in the cellular concentrations of unphosphorylated DegU, but not in the repressor function of DegU-P at BR2, as BR2 did not serve as a functional cis-acting site in the absence of BR1. DegU-P at BR2 might rather work as an anti-activator.
We found that unphosphorylated DegU is a positive regulatory factor of the flgB promoter, which is the first gene in the fla/che operon (including the sigD gene that encodes sigma D factor). It was reported that disruption of degU decreases the expression of sigD , which is governed by the upstream flgB promoter [23, 38, 39]. Our findings are consistent with this observation.
Recently it has been reported that swarming motility and the flgB expression in the non-laboratory strain requires DegS-independent low-level of DegU-P [40, 41]. These observations may be consistent with our findings, since unphosphorylated DegU (or DegU phosphorylated at a low level) may positively regulate the expression of the fla/che operon. To further characterize the DegS-independent phosphorylation of DegU, a study using a DegU mutant lacking the phosphorylation site should be needed.
This study demonstrated that DegU positively regulates flgB and sacB via the BRs containing the IR-like sequences and the DR present in their promoter regions, respectively (Figure 9). In, addition, we showed that DegU-P prefers binding to DR but not to IR in the regulatory region of the sacB promoter.
Bacterial strains and culture media
Strains and plasmids used for this study.
Reference or source
trpC2 thrC ::swrAA N (Spr)
trpC2 leuC7 degU (Kmr)
trpC2 leuC7 ΔdegS (in frame deletion)
trpC2 ΔdegS (in frame deletion, yvyE ::Cmr::Emr)
trpC2 amyE ::flgB-lacZ 1D (Cmr)
trpC2 amyE ::flgB-lacZ 1D (Cmr) thrC ::swrAA N (Spr)
trpC2 amyE ::flgB-lacZ 1D (Cmr) degU (Kmr)
trpC2 amyE ::flgB-lacZ 1D (Cmr) ΔdegS (Emr)
trpC2 amyE ::flgB-lacZ 1D (Cmr) thrC ::swrAA N (Spr) degU (Kmr)
trpC2 amyE ::flgB-lacZ 1D (Cmr) thrC ::swrAA N (Spr) ΔdegS (Emr)
trpC2 amyE ::flgB-lacZ 1D (Cmr) degU32
trpC2 amyE ::sacB-lacZ S-WT (Cmr)
trpC2 amyE ::sacB-lacZ S-WT (Cmr) degU (Kmr)
trpC2 amyE ::sacB-lacZ S-WT (Cmr) ΔdegS (Emr)
trpC2 amyE ::sacB-lacZ SC-IR2 (Cmr)
trpC2 amyE ::sacB-lacZ SC-IR2 (Cmr) degU (Kmr)
trpC2 amyE ::sacB-lacZ SC-IR2 (Cmr) ΔdegS (Emr)
Reference or source
pDG1731 carrying swrAA N
pCA191 carrying a part of yvyE and a promoter of degSU
Ampicillin and erythromycin resistance
pTYB2 carrying degU, ampicillin resistance
pET28b carrying degS, kanamycin resistance
Plasmid carrying T5 promoter-His6-degU, ampicillin resistance
Insertion vector to amyE, chloramphenicol resistance, lacZ
pIS284 carrying a promoter region of flgB
pIS284 carrying a promoter region of sacB
Synthetic oligonucleotides were commercially prepared by the Tsukuba Oligo Service (Ibaraki, Japan). The plasmids and oligonucleotides used in this study are listed in Table 1 and Additional file 1, respectively. We used total DNA from B. subtilis 168 as the PCR template unless otherwise indicated. To construct pDG-swrAN, a PCR product produced by using swrA-FA and swrA-FB and total DNA from B. subtilis IFO3335 was digested by Bam HI and Eco RI and cloned into PDG1731 treated with the same restriction enzymes . To construct pTYB-degU, a PCR product produced by degU-Chi-N and degU-Chi-S was digested by Nde I and Sma I and cloned into pTYB2 treated with the same restriction enzymes. To construct pCa-yvyE, a PCR product produced by using yvyE-F1 and yvyE-R1 was digested by Hin dIII and Bam HI and cloned into pCA191 treated with the same restriction enzymes . To construct pIS284-flgB and pIS284-sacB, PCR products were prepared by using the primer pairs, flgB-F2 and flgB-DD, and sacB-U and sacB-D, respectively. The DNA fragments were digested with Bam HI and Hin dIII and ligated into the similarly-digested pIS284 plasmid. The resultant plasmids were transformed into 168 after linearization by Pst I digestion, thereby generating the strain carrying F1D and S-WT, respectively. The del series of sacB-lacZ fusions, the F1 to F3 derivatives, and F2D were constructed by generating PCR products using oligonucleotides corresponding to different 5'-termini or 3' termini of the fusions and cloning these fragments into pIS284. The M series of flgB-lacZ and sacB-lacZ fusions and the SC series of sacB-lacZ fusions as well as S-IR were constructed by cloning mutagenized PCR products into pIS284. Site-directed mutagenesis was performed by an oligonucleotide-based PCR method as described previously . S-del4 was constructed by site-directed mutagenesis using oligonucleotides sacB-del-1, sacB-del-8, sacB-U and sacB-D. S-M3 was obtained by PCR error during the construction of S-WT. The resulting fusion-bearing plasmids were used to transform 168. The sequences cloned into all plasmids were confirmed.
Purification of soluble His-tagged DegS, His-tagged DegU and intact DegU proteins
The recombinant His-tagged DegS, His-tagged DegU and chitin-binding domain (CBD)- and intein-fused DegU proteins were induced in E. coli BL21 carrying pET-degS, M15 carrying pRep4 and pDG-His-degU, and BL21 carrying pTYB-degU by previously described methods . His-tagged DegS was produced as insoluble protein and its pellets were resuspended in 2.5 ml of buffer A [6 M guanidine-HCl, 150 mM NaCl, and 50 mM Tris-HCl (pH 7.6)] and left for 1 hr at room temperature. Two ml of 50% Ni-NTA resin (Qiagen) equilibrated by buffer A were added to the sample, which was then shaken gently for 45 min at room temperature. The resultant suspension (about 5 ml) was packed in a mini-column, washed first with 10 μl of buffer A and then with 5 ml of buffer B [150 mM NaCl and 50 mM Tris-HCl (pH 7.6)] containing 8 M urea. Renaturation of His-tagged DegS was performed by removing urea, which was done in a step-wise fashion by passing 5 ml each of buffer B containing 6 M, 4 M and 2 M urea through the column. Finally, the column was washed with 5 ml of buffer B containing 10 mM imidazole. The protein was then eluted with buffer B containing 0.5 M imidazole. His-tagged DegU was produced as a soluble protein in E. coli and purification was performed by step-wise elution from a Ni-affinity column with imidazole as described previously . CBD- and intein-fused DegU was purified by using chitin-coupled resin and autoactivating intein by DTT according to the manufacturer's recommendations (New England Biolab inc). After SDS-PAGE analysis of the fractions, the purified proteins were dialyzed against TEDG buffer . Aliquots of the purified proteins were stored at -80°C.
Footprint assays using DegU-P
Phosphorylation of DegU and the binding of DegU-P to the probe DNA were carried out by incubating intact DegU and His-tagged DegS at 25°C for 30 min (molar ratio 3:1) with a biotinylated DNA probe, i.e., in a buffer containing protein solution (TEDG buffer). The final concentration of the reaction mixture (65 μl) for the footprint analysis was as follows; 10 mM (NH4)2SO4, 1 mM DTT, 0.2% Tween20, 5 mM MgCl2, 31 mM Tris-HCl (pH 7.5), 0.3 mM EDTA, 44 mM KCl, 1 μg poly dI-dC and 1 mM ATP. Immediately at the end of the reaction, 4 units of DNase I (Roche, Indianapolis, USA) in 7. 5 μl buffer containing 6% glycerol, 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM DTT and 1 mg bovine serum albumin was added. The reaction mixture was left at room temperature for 5 min and then subjected to phenol extraction and subsequent ethanol precipitation after the addition of stop solution (0.1% SDS, 20 mM EDTA, 200 mM NaCl, 40 μg/ml tRNA). After the addition of a loading dye, the samples were applied onto a 6% polyacrylamide gel. The probes were prepared by PCR using the oligonucleotide pairs indicated in Additional file 1. Detection of biotinylated DNA and preparation of sequencing ladder were done by using the methods described previously .
We thank K. Kobayashi for communicating unpublished results and the kind gift of plasmids. We also thank P. Stragier and I. Smith for the kind gift of plasmids. We are indebted to K. Shimane and T. Ohsawa for technical assistance. This work was supported by a Grant-in-aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and research grants from KANPOU Science Foundation and Takano Agricultural Chemistry Foundation.
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