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
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ABSTRACT |
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INTRODUCTION |
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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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METHODS |
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Yeast two-hybrid analysis.
Yeast strains used in this analysis were PJ69-4A (MATa trp1-901 leu2-3, 112 ura3-52 his-200 gal4 gal80
LYS2 : : GAL1HIS3 GAL2ADE2 met2 : : GAL7lacZ) and PJ69-4
(MAT
trp1-901 leu2-3, 112 ura3-52 his-200 gal4
gal80
LYS2 : : GAL1HIS3 GAL2ADE2 met2 : : GAL7lacZ), 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 YhdKN, was PCR-amplified from B. subtilis genomic DNA using Proof Start DNA polymerase (Qiagen) and cloned into pGBTK or pGADT7. Since the fragment of YhdKN 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 (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.
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RESULTS |
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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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DISCUSSION |
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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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ACKNOWLEDGEMENTS |
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Received 15 August 2003;
revised 11 November 2003;
accepted 4 December 2003.