Interaction of Bacillus subtilis extracytoplasmic function (ECF) sigma factors with the N-terminal regions of their potential anti-sigma factors

Mika Yoshimura1, Kei Asai2, Yoshito Sadaie2 and Hirofumi Yoshikawa1

1 Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
2 Department of Biochemistry and Molecular Biology, Faculty of Sciences, Saitama University, Saitama 338-7507, Japan

Correspondence
Hirofumi Yoshikawa
hiyoshik{at}nodai.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Extracytoplasmic function (ECF) sigma factors constitute a diverse family of proteins, within the class of the sigma 70 subunit of RNA polymerase. Most members of the family studied to date are known to regulate gene expression in response to stress conditions. The Bacillus subtilis genome encodes at least 17 distinct sigma factors, seven of which are members of the ECF subfamily. Among these, five sigma factors, namely SigV, SigW, SigX, SigY and SigM, are encoded by the first genes of the cognate sigma operons. Disruption or repressed expression of the downstream gene(s) resulted in transcriptional activation of the cognate sigma operon. Moreover, in vivo protein–protein interaction analyses by yeast two-hybrid experiments indicated that these immediate downstream gene products bind the cognate ECF sigma factor, suggesting that they function as anti-sigma factors by capturing sigma factor on the inner surface of the cytoplasmic membrane. Interaction with other sigma factors was not observed. The results presented here also show that these anti-sigma factors interact with ECF sigma factors through their N-terminal region, implying that the N-terminal domain resides inside the cytoplasmic membrane.


Abbreviations: ECF, extracytoplasmic function


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The extracytoplasmic function (ECF) sigma factors are found in a diverse range of bacteria and many are activated to transcribe their regulons in response to a change in environmental conditions (Helmann, 2002; Lonetto et al., 1994; Raivio & Silhavy, 2001). For example, ECF sigma factors regulate iron uptake and heat-shock responses in Escherichia coli (Braun, 1997; De Las Penas et al., 1997), alginate biosynthesis and exotoxin secretion in Pseudomonas aeruginosa (Hershberger et al., 1995; Ochsner et al., 1996), carotenoid biosynthesis in Myxococcus xanthus (Gorham et al., 1996) and expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor (Kang et al., 1999; Paget et al., 1998).

In many cases, the ECF sigma factor is co-transcribed with one or more negative regulators. Often, these include a trans-membrane protein with an extracytoplasmic sensory domain and an intracellular inhibitory domain functioning as an anti-sigma factor that binds and inhibits the cognate sigma factor. Although only a limited number of examples have been shown, direct interaction with sigma factor and anti-sigma factor has been reported for E. coli SigE and RseA (Campbell et al., 2003; Raivio & Silhavy, 2001), FecI and FecR (Enz et al., 2000), P. aeruginosa AlgU and MucA (Rowen & Deretic, 2000), Rhodobacter sphaeroides SigE and ChrR (Newman et al., 2001), S. coelicolor SigR and RsrA (Li et al., 2002) and Myxococcus xanthus CarQ and CarR (Browning et al., 2003).

Bacterial genome sequencing has revealed numerous new members of this class of sigma factor including seven in Corynebacterium diphtheriae (http://www.sanger.ac.uk/Projects/C_diphtheriae/), 10 in Mycobacterium tuberculosis (Cole et al., 1998), 16 in Mesorhizobium loti (Kaneko et al., 2000) and 41 in Streptomyces coelicolor A3(2) (Bentley et al., 2002). The ECF sigma factors retain many of the conserved domains of principal sigma factors, but show significant divergence from other members of the family. The functions and mechanisms of regulation for most of these newly described potential ECF sigma factors remain unknown.

In Bacillus subtilis, seven putative ECF sigma factors (SigV, SigW, SigX, SigY, SigZ, SigM and YlaC) have been identified (Kunst et al., 1997). Among them, SigX has been reported to contribute in survival at high temperature (Huang et al., 1997). Brutsche & Braun (1997) demonstrated and confirmed the anti-sigma activity and the predicted membrane localization of RsiX (YpuN), the product of the downstream gene. The sigW gene is co-transcribed with its downstream gene ybbM (Huang et al., 1998) which is postulated to be anti-sigma factor RsiW (Helmann, 2002), although no sufficient experimental evidence has been shown. Expression of sigM is up-regulated in cells growing in medium of high-salt concentration, and a sigM mutant failed to grow in such medium (Horsburgh & Moir, 1999). The two downstream genes yhdL and yhdK are known to negatively regulate the activity of SigM and, interestingly, yhdL is essential for normal growth (Horsburgh & Moir, 1999).

We employed a yeast two-hybrid analysis to examine specific interactions between putative anti-sigma proteins and their cognate sigma factors. Of the seven ECF sigma candidates, six genes are encoded within operons; sigZ is mono-cistronic (Sorokin et al., 1997), as illustrated in Fig. 1. The sigV, sigW and sigX operons appear to be bi-cistronic (Liu et al., 1997; Sorokin et al., 1993, 1997), and the products of the downstream genes each contain one trans-membrane domain (see information described in the Bacillus subtilis Genome Database web site at http://bacillus.genome.ad.jp). The putative sigM operon includes three genes, of which two encode negative regulators (Horsburgh & Moir, 1999). The sigY gene is the first gene of a putative hexa-cistronic operon (Yoshida et al., 1996). The ylaC gene is the third gene in a putative tetra-cistronic operon (Kunst et al., 1997). We analysed the genes located in the putative ECF sigma operons, as well as yrpG, yrpE and yxlH, as primary candidates for screening of potential anti-sigma factors since these genes reside near the ECF sigma genes (Fig. 1). Here, we report at least five ECF sigma factors (SigV, SigW, SigX, SigY and SigM) that interact with the products of respective downstream genes as judged by a yeast two-hybrid analysis. The interactions appear to be highly specific as no cross-talk was observed.



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Fig. 1. Gene organization of the ECF sigma operons in B. subtilis. ECF sigma genes are shown as solid arrows. Stem and loop structures represent putative transcription terminators. Vertical grey lines in the genes indicate probable membrane-spanning domains, predicted by the program SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html).

 

   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of strains to assay for sigma-directed expression of lacZ.
B. subtilis derivative strains of 168 (trpC2) harbouring the ECF sigma promoter–lacZ fusion in the amyE locus were constructed previously to monitor the expression of each sigma gene (Asai et al., 2003). The cloned promoter region for each operon was -270 to +49 for sigV, -216 to +116 for sigY and -318 to +34 for ylaA, where +1 represents the first nucleotide of the initiation codon of each gene. The promoters were fused transcriptionally to lacZ through cloning into the EcoRI and BamHI sites of pDL2, as described previously (Fukuchi et al., 2000). The resulting strains, BSU32, BSU35 and BSU37, are summarized in Table 1.


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Table 1. Strains used in this study

 
Disruption or overexpression of anti-sigma factor candidate genes using pMUTIN derivatives.
pMUTIN derivative plasmids pMUTINT3{Delta}Z and pMUTIN2{Delta}Z, which were devoid of the E. coli lacZ gene, were constructed from plasmids pMUTINT3 (Moriya et al., 1998) and pMUTIN2 (Vagner et al., 1998), through excision of ClaI–SacI fragments. Putative anti-sigma factor genes yrhM and ylaD were disrupted with pMUTINT3{Delta}Z harbouring DNA fragments internal to the coding region, which were PCR-amplified using the primer sets listed in Table 2. Resulting strains were ASK441 and ASK446, respectively (Table 1). The cognate promoter activities for the sigV and ylaA operons were assayed with these disruptants by constructing strains ASK4001 and ASK4002 by using the chromosomal DNA of strains BSU32 and BSU37 (Table 1). Strain ASK431 was constructed for the overexpression experiment in which the yxlC gene was placed under the control of the spac promoter (Pspac) using pMUTIN2{Delta}Z through cloning of the 5'-upstream region of yxlC. PCR primers used to amplify the region are also listed in Table 2.


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Table 2. Primer pairs and oligonucleotides used for cloning in this study

Restriction sites that were added artificially are underlined.

 
Xylose-inducible-promoter insertion.
Using the primer set for sigY listed in Table 2, a PCR-amplified fragment containing the ribosome-binding site and the 5' end of the sigY ORF was cloned into plasmid pX2 (Mogk et al., 1997), an integrational vector harbouring a xylose-inducible promoter. The amplified fragment was treated with BamHI prior to cloning in vector pX2. The chloramphenicol-resistance gene of strain BSU35, carrying the PsigY–lacZ construct into the amyE locus, was substituted with a spectinomycin-resistance gene, using the integrational plasmid pCm : : Sp (Bacillus Genetic Stock Center, OH, USA). Finally, this strain was transformed with the pX2-derived plasmid to construct a strain, ASK4016, which was chloramphenicol-resistant and expressed sigY under the control of a xylose-inducible promoter (Table 1 and Fig. 2a). To determine the effect of overexpression of putative anti-sigma factor YxlC and downstream genes on the transcription of the sigY operon, strain ASK4016 was transformed with chromosomal DNA of strain ASK431 to obtain strain ASK4017 (Table 1 and Fig. 2b). The genetic structures of these constructs were verified through PCR.



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Fig. 2. Effect of overexpression of putative anti-sigma factor on transcription of its cognate sigma factor gene. SigY activity was monitored as {beta}-galactosidase activity (Miller units) using PsigY–lacZ inserted in the amyE locus, with the original sigY operon fused to the xylose-inducible promoter. Samples were withdrawn from the culture at two points during exponential growth (OD600 0·5 and 0·8) and analysed (a). The downstream genes of sigY were directed by an IPTG-inducible promoter and the SigY activity was measured as above. Note that the expression of downstream genes was repressed without IPTG due to the terminator located in front of Pspac (b).

 
Cell culture conditions and {beta}-galactosidase assay.
An aliquot (1 ml) of cells growing in LB medium with or without IPTG or xylose was harvested by centrifugation, and the {beta}-galactosidase activity was assayed according to the methods described by Youngman et al. (1985). Unit of enzyme activity is expressed as nmol ONPG hydrolysed min-1 (mg protein)-1. Protein concentration was determined using a Bio-Rad Protein Assay Kit.

Yeast two-hybrid analysis.
Yeast strains used in this analysis were PJ69-4A (MATa trp1-901 leu2-3, 112 ura3-52 his-200 gal4{Delta} gal80{Delta} LYS2 : : GAL1–HIS3 GAL2–ADE2 met2 : : GAL7–lacZ) and PJ69-4{alpha} (MAT{alpha} trp1-901 leu2-3, 112 ura3-52 his-200 gal4{Delta} gal80{Delta} LYS2 : : GAL1–HIS3 GAL2–ADE2 met2 : : GAL7–lacZ), obtained from Philip James (James et al., 1996). Plasmid pGBTK, a GAL4 DNA-binding domain fusion vector, is a derivative of pGBT9 (Clontech) in which the ampicillin-resistance gene is replaced with the kanamycin-resistance gene of pGBKT7 (Clontech). Plasmid pGADT7, a GAL4 activation domain fusion vector, was purchased from Clontech. Oligonucleotides used for yeast two-hybrid analysis are listed in Table 2. Each fragment, except for YhdK–N, was PCR-amplified from B. subtilis genomic DNA using Proof Start DNA polymerase (Qiagen) and cloned into pGBTK or pGADT7. Since the fragment of YhdK–N is very small, the whole region was synthesized with restriction-site linkers (Table 2). Inserts of the resultant plasmids were verified by sequence determination.

Plasmid vectors pGBTK (TRP1) and pGADT7 (LEU2) were used to transform mating-type yeast strains PJ69-4A (for pGBTK) and PJ69-4{alpha} (for pGADT7) using the method essentially described by Gietz & Schiestl (1995) and as modified by Ito et al. (2000). Briefly, yeast strains were cultured in YPAD (1 % yeast extract, 2 % peptone, 0·00002 % adenine sulfate, 2 % glucose) medium, and competent cells were prepared without the addition of 10 % DMSO. Transformants were mated in appropriate liquid media in flat-bottomed 96-well plates. After mating, cultures were collected and washed with sterile water, then spotted onto a synthetic complete (SC) agar plate lacking Leu and Trp (SC-LW) for selection of LEU2 and TRP1 diploid cells. These cells were replica-plated onto selection media – SC lacking His (SC-LWH) and/or SC lacking adenine (SC-LWHA) – agar plates supplemented with 1 or 5 mM 3-aminotriazole, to inhibit auto-activation of the HIS3 reporter gene.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transcriptional regulation of ECF sigma operon by downstream gene(s)
As described above, some of the ECF sigma factors, SigW, SigX and SigM, have already been demonstrated to be regulated by their respective downstream gene products. Here, we analysed the remaining sigma factors, namely SigV, SigY and YlaC, but not SigZ, since it is mono-cistronic (Asai et al., 2003). If each anti-sigma is inactivated and the corresponding sigma is derepressed, then the transcription of the cognate operon will be activated because the SigV, SigY and YlaC operons appear to be positively autoregulated by RNA polymerases bearing the corresponding sigma factor (Asai et al., 2003). Thus, we tried to inactivate the downstream genes as primary candidates for anti-sigma factor genes through insertion of exogenous plasmid DNA, as described in Methods. Disruption mutant strains of yrhM and ylaD were constructed in which sigV–lacZ and ylaA–lacZ activity could be compared with that of the relevant wild-type strain (Table 3). As expected, inactivation of yrhM elevated the expression of a sigV–lacZ fusion (strain ASK4001). In contrast, disruption of ylaD had little effect on the expression of ylaAlacZ (ASK4002), suggesting that YlaD is not a negative regulator of expression of ylaC. We carried out similar experiments for the sigW, sigX and sigM operons and confirmed previously reported results (data not shown).


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Table 3. Effect of mutation in putative anti-sigma factor genes on sigma-directed expression of lacZ

 
In the case of the sigY operon, yxlC or yxlD gene disruptants showed abnormal colony morphology (data not shown). As an alternative approach, we inserted a xylose-inducible promoter upstream of the sigY gene to drive expression of the operon, which was not detectable under normal conditions, and we fused an IPTG-inducible promoter upstream of the yxlC gene to see the effect of expression of downstream genes on sigY–lacZ expression. Addition of xylose only slightly enhanced sigYlacZ expression (Fig. 2a), but high levels of expression were obtained upon xylose induction when downstream gene expression was repressed (Fig. 2b). Addition of IPTG induced the expression of yxlC and downstream genes and resulted in total impairment in sigY–lacZ expression (Fig. 2b).

Specific interactions of SigW, SigX and SigY with their possible anti-sigma factors
To examine direct interactions between ECF sigma factors and other gene products, we adopted a yeast two-hybrid analysis. We first cloned the seven sigma genes into pGBTK to obtain DNA-binding domain fusions and cloned all the other genes shown in Fig. 1 into pGADT7 for activation domain fusion proteins. After mating, interactions between fusion proteins in yeast cells were screened through the expression of the reporter genes HIS3 and/or ADE2. As shown in Fig. 3, positive interactions were clearly observed in pairs of SigW/YbbM, SigX/YpuN and SigY/YxlC. No other combinations yielded positive colonies.



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Fig. 3. Interaction of ECF sigma factors with possible anti-sigma factors. Diploid strains were constructed by mating PJ69-4A containing pGBTK or its derived plasmids harbouring ECF sigma factors with PJ69-4{alpha} containing pGADT7 or its derived plasmids harbouring anti-sigma factor candidates. Two-day-old cells that had been grown on a plate were suspended in culture medium, and 30 µl each of bait and prey plasmid-harbouring cells were mixed with 140 µl of media per well, allowing the cells to mate during overnight incubation at 30 °C. Cells were washed twice in distilled water and resuspended in 20 µl distilled water; a 5 µl aliquot was then spotted onto an SC-LW plate. Plates were incubated at 30 °C for 2–3 days, followed by replica plating of diploid cells onto selective SC-LWHA plates containing 5 mM 3-aminotriazole using a replicator and incubation at 30 °C for another 7 days. AD, Activation domain; BD, DNA-binding domain.

 
ECF sigma factors interact with the N-terminal region of anti-sigma factors
In other organisms, most of the anti-sigma factors investigated so far are integral cytoplasmic membrane proteins (Helmann, 2002; Rowen & Deretic, 2000). If this is also the case for B. subtilis, then in at least either side of the membrane-spanning domain, anti-sigma proteins should possess a cytoplasmic domain where sigma factors interact. Therefore, we analysed separately the N-terminal and C-terminal domains of the possible anti-sigma factors without the membrane-spanning domain, to verify the interaction with ECF sigma factors, since a defined region often gives a clear result in yeast two-hybrid analysis. The N-terminal regions of YrhM, YbbM, YpuN, YxlC and YhdL each interacted with their cognate ECF sigma factor (Fig. 4). These results strongly suggest that the N-terminal region of these putative anti-sigma factors is localized in the cytoplasm to interact with the sigma factor.



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Fig. 4. Interaction of ECF sigma factors with fragments of possible anti-sigma factors devoid of the membrane-spanning domains. Bait constructs were the same as for Fig. 3. For the prey constructs, the N-terminal region or the C-terminal region of the anti-sigma candidates without putative membrane-spanning domains was cloned in pGADT7. The amino acid residues of the anti-sigma candidate protein regions are: YrhM–N, 1–37; YrhM–C, 61–285; YbbM–N, 1–84; YbbM–C, 108–208; YpuN–N, 1–45; YpuN–C, 69–368; YhdL–N, 1–71; YhdL–C, 95–358; YxlC–N, 1–49; YlaA–C, 278–648; YlaB–N, 1–64; and YlaD–M, 17–72. Diploid strains for testing were grown and spotted as described in Fig. 3 except that an SC-LWHA plate containing 1 mM 3-aminotriazole instead of 5 mM 3-aminotriazole was used. AD, Activation domain; BD, DNA-binding domain.

 
Interactions among sigM operon proteins
As shown in Fig. 4, we detected an interaction between SigM and the N-terminal region of YhdL. It has been postulated that the activity of SigM is negatively regulated by the downstream gene products YhdL and YhdK (Horsburgh & Moir, 1999). To determine the role of YhdK in the SigM–YhdL interaction, we analysed the possible interactions among SigM operon proteins and obtained evidence that YhdK interacts with YhdL (Fig. 5a). Since these proteins have integral membrane-spanning domains, in order to eliminate the possibility of the non-specific hydrophobic interaction frequently observed in the two-hybrid analysis, we tested YhdK and YhdL with other proteins containing one or two membrane-spanning regions. Results included in Fig. 5(a) demonstrate a highly specific interaction between YhdK and YhdL.



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Fig. 5. Specific interaction of YhdK with YhdL. Genes encoding proteins containing probable membrane-spanning domains were cloned in both bait and prey plasmids, and diploid strains for testing were analysed as described in Fig. 2 using an SC-LWH plate containing 5 mM 3-aminotriazole (a). To identify the interaction domain of YhdK and YhdL, DNAs for N-terminal or C-terminal fragments of these proteins with or without membrane-spanning domains (designated M) were cloned in bait and prey plasmids and assayed as described in Fig. 3 using an SC-LWHA plate containing 1 mM 3-aminotriazole and 14 days incubation (b). These constructs are represented schematically (c). Open rectangles indicate the putative membrane-spanning domains of YhdL and YhdK and the numbers represent the amino acid residues.

 
To investigate further the role of the membrane-spanning domain of these proteins, various fusion proteins with or without the membrane-spanning domain were constructed and analysed. As clearly summarized in Fig. 5(b, c), only proteins possessing a membrane-spanning domain exhibited positive interaction, implying that this domain might be an essential component for the interaction of these proteins to occur, probably in the cytoplasmic membrane.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
It is widely believed that ECF sigma factors regulate the expression of their own gene/operon in response to external signals, i.e. so-called autoregulation (Helmann, 2002). This is also the case for B. subtilis, as shown by DNA microarray analyses of transcription stimulated by B. subtilis ECF sigma factors (Asai et al., 2003). Taking these factors into consideration, our strategy was to monitor sigma factor activity through measuring the cognate promoter activity. Experimentally, we analysed the effects of anti-sigma factor gene disruption on the expression of the cognate promoter–lacZ fusion gene, measured as {beta}-galactosidase activity. In the case of the bi-cistronic operons sigV, sigW and sigX, our transcriptional activation experiment for sigV and sigW, as well as previous reports on sigX, indicate that these three sigma factors are negatively regulated by the second gene products of the cognate operon (Table 3, and Huang et al., 1997, 1998). On the other hand, we find the hexa-cistronic sigY operon to be rather complicated. Interaction with SigY was observed only against YxlC, but disruption of the yxlD gene produced a similar colony morphology to that of the yxlC disruptant (data not shown). This may suggest some co-operation between YxlC and YxlD. Cao et al. (2003) reported that yxlC could be disrupted and YxlD, not YxlC, might regulate SigY activity. Further investigation needs to be carried out.

Direct interaction between ECF sigma factor and anti-sigma factor has been demonstrated in several Gram-negative bacteria and Streptomyces (see Introduction). In this study, using a yeast two-hybrid system, we have shown that B. subtilis ECF sigma factors SigM, SigV, SigW, SigX and SigY interact with proteins encoded by the immediate downstream genes, an interaction highly specific only for those within the operon. SigM seems to be also bound to the N-terminal domain of YhdL, an observation that has been made for other bi-cistronic gene products. However, the third gene product, the small hydrophobic protein YhdK, appears to interact with the trans-membrane domain of YhdL, suggesting some specific role for YhdK in the anti-sigma function of YhdL. YxlC is a SigY-binding protein, but the function(s) of the other gene products in the cognate operon needs further investigation.

Interestingly, all interactions observed between sigma factors and anti-sigma factors occurred through the N-terminal regions of the latter, implying that the N-terminal domain is configured intracellularly (Fig. 6). The involvement of the N-terminal region has been reported in the above-mentioned Gram-negative examples, RseA, FecR and MucA (Campbell et al., 2003; Enz et al., 2000; Rowen & Deretic, 2000). The involvement of a number of membrane proteins in the ECF sigma operon, as seen in the sigY, sigM and ylaC operons, suggests participation of these proteins in the signal transduction (Figs 1 and 6). It is noteworthy that the rpoE operon of Thermoanaerobacter tengcongensis, and LMO2228 and the downstream genes of Listeria monocytogenes share homology, at least in part, with the B. subtilis sigY operon. YbbM and YlaD contain the conserved HxxxCxxC motif and have previously been classified as members of the ZAS family of anti-sigma factors by Paget et al. (2001). Moreover, some extended overall homologies are found among YbbM, Bacillus halodurans BH0264 and T. tengcongensis TTE0873, as well as between YhdL and T. tengcongensis TTE1558. More understanding of the functionalities of these anti-sigma factors may shed light on their evolutionary roles.



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Fig. 6. Model of the interactions between ECF sigma factors and putative anti-sigma factors. Hatched boxes indicate predicted trans-membrane domains. Other interactions suggested in this article are also included. CM, cytoplasmic membrane.

 


   ACKNOWLEDGEMENTS
 
We are grateful to W. Schumann (University of Bayreuth) for providing plasmid pX2. We also thank N. Ogasawara (Nara Institute of Science and Technology) for critical reading of the manuscript. This work was supported by a Grant-in-Aid for Science, Research on Priority Area (C) of the Ministry of Education, Science, Sports and Culture, Japan.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 15 August 2003; revised 11 November 2003; accepted 4 December 2003.