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1 Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Saitama 338-8570, Saitama, Japan
2 Department of Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, 985 Sanzo, Higashimura-cho, Fukuyama, Hiroshima 729-0292, Japan
Correspondence
Yoshito Sadaie
ysadaie{at}molbiol.saitama-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase reporter assay. Heat-inducible, SigI-dependent transcription of the sigI-rsgI operon was stimulated greatly by disrupting rsgI. Yeast two-hybrid analysis showed direct interaction between the N-terminal portion of the presumed RsgI protein and SigI. Without RsgI function, induction of transcription of the sigI-rsgI operon upon transient heat stress depended on dnaK activity. However, transcription of the operon was induced during growth at prolonged higher temperature even without DnaK function. Without RsgI function, sigI-rsgI operon transcription was induced after the end of growth independent of any temperature shift in a sporulation medium and toward the end of growth in a rich complex medium. Furthermore, glucose addition resulted in a strong suppression of sigI-rsgI transcription. Therefore it is hypothesized that transcription of the sigI-rsgI operon of B. subtilis is negatively regulated by the putative transmembrane protein RsgI, which moderates SigI's sensitivity to heat shock or nutritional stress. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Wösten, 1998; Raivio & Silhavy, 2001; Helmann, 2002; Mittenhuber, 2002). These include the heat-shock factor RpoH and stationary-phase protein RpoS of Escherichia coli (Morita et al., 1999; Weber et al., 2005), the general stress-response protein SigB of Bacillus subtilis (Peterson et al., 2001; Price et al., 2001; Price, 2002) and ECF (extracytoplasmic function) sigmas such as SigE of Streptomyces coelicolor, RpoE of E. coli and SigW of B. subtilis (Lonetto et al., 1994; Raina et al., 1995; Rouviere et al., 1995; De La Penas et al., 1997; Wiegert et al., 2001).
These sigma factors usually exist at low concentrations or in an inactive form. Upon stress, in most cases, activation of these sigma factors and subsequent autoregulatory induction of transcription of their own genes leads to sufficient concentrations of sigma factor to transcribe their regulons. A translational activation process is involved in the case of RpoH of E. coli (Morita et al., 1999).
Some of the stress-responding sigma genes are associated with anti-sigma factor genes, whose products inhibit the activity of sigma factors through direct binding, which is disrupted upon stress through phosphorylation by anti-anti-sigma factors or digestion by endogenous proteases (Hughes & Mathee, 1998).
The sigI (ykoZ) operon of B. subtilis is thought to consist of sigI and rsgI (regulation of sigI, formerly ykrI) (Kunst et al., 1997), which encodes a 
70-type sigma factor, SigI, and a putative membrane protein, RsgI. Transcription of the sigI gene is induced by heat shock in a SigI-dependent manner, and the wild-type sigI gene is required for the growth at higher temperatures (Zuber et al., 2001). However, the role of rsgI is not known and little is known about the SigI regulons. As there is not a group I specific CIRCE sequence, a group II specific SigB-dependent promoter, or a group III specific CtsR-binding site within the upstream region of sigI, the sigI gene is proposed to be a member of group IV of the B. subtilis heat-shock genes (Zuber et al., 2001).
Orthologues (see http://bacillus.genome.jp/) of SigI and RsgI of B. subtilis are found in many species of the phylum Firmicutes. Their function in these organisms remains to be elucidated.
In this work, we studied the role of RsgI in the response to heat stress and in the expression of the sigI operon. To do this we introduced a heat-stable reporter gene into B. subtilis by constructing a strain carrying at the amyE locus the bgaB gene from Bacillus stearothermophilus encoding a heat-stable -galactosidase fused to the promoter of the sigI operon (Hirata et al., 1986; Yuan & Wong, 1995). Expression of bgaB and amounts of transcript of the sigI operon were examined in mutant strains with disrupted sigI or rsgI genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Cells of B. subtilis or E. coli were grown in Luria–Bertani broth or in a complex sporulation medium (Sambrook et al., 1989; Schaeffer et al., 1965). DNA manipulation, PCR, reagents and enzymes were as described elsewhere (Miwa et al., 2000; Ohshima et al., 2002).
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; Yuan & Wong, 1995). The sample was used to transform wild-type strain 168 to chloramphenicol resistance, to obtain a strain carrying at the amyE locus the bgaB gene with the promoter of the sigI operon in the plasmid pDLd backbone. The resulting gene fusion was designated amyE : : PsigI-bgaB.
BgaB activity.
Heat stable -galactosidase activity of the bgaB gene from B. stearothermophilus was measured at 62 °C as described elsewhere (Yuan & Wong, 1995; Platt et al., 1972; Sadaie, 1998). Samples were heated at 68 °C for 5 min before measuring BgaB activity, to inactivate the endogenous -galactosidase of the lacZ gene of the E. coli in the integrated pMutin plasmid.
Gene disruption.
Disruption of sigI and rsgI was performed as described elsewhere, with pMutin3 carrying DNA corresponding to a portion of each gene (Vagner et al., 1998). The DNA fragment cloned was obtained by PCR synthesis with a template DNA from a wild-type strain and primer pairs 5'-AAGAAGCTTCCAGTGCTTAGCCTTTTG-3'/5'-GGAGGATCCATTTTGTGCGCTTCTGGC-3' for sigI, and 5'-AAGAAGCTTCGTCACGTTGCTGACCC-3'/5'-GGAGGATCCATTTCGACGCTTGGATTG-3' for rsgI. Gene disruption with the Em cassette was performed as follows. Both side regions of the target gene were PCR amplified with a template DNA from a wild-type strain and primer pairs described below. The resulting DNA fragments were subjected to PCR amplification with a DNA carrying the em gene of pMutin to obtain a DNA fragment that contained em gene instead of the target gene. The resulting DNA was used to transform wild-type B. subtilis to erythromycin resistance to obtain an em-substituted strain. For sigI disruption, we used two primer pairs, 5'-TTGCGGCTTCCATGTTTGT-3'/5'-GCACTATCAACACACTCTTAAGTTATGCAGATCTTTATTGCCTTTTG-3' and 5'-GGAGCTAAAGAGGTCCCTAGGTGTATTATCATTACGGGTG-3'/5'-CTCGAGAGATTCCATGCGG-3'. For rsgI disruption, the primer pairs used were 5'-GACGGTTTCATCCGTTTG-3'/5'-GCACTATCAACACACTCTTAAGTTATCTTCTCATGAGTGCAGC-3' and 5'-GGAGCTAAAGAGGTCCCTAGCCGGCGAATAAAGACCTG-3'/5'-CTCAGACAGATCTGATGA-3'. For sigI-rsgI disruption, the primer pairs 5'-TTGCGGCTTCCATGTTTGT-3'/5'-GCACTATCAACACACTCTTAAGTTATGCAGATCTTTATTGCCTTTTG-3' and 5'-GGAGCTAAAGAGGTCCCTAGCCGGCGAATAAAGACCTG-3'/5'-CTCAGACAGATCTGATGA-3' were used. Underlined portions of the primers possess the same sequences as the 5' or 3' portion of the em gene of pMutin, which was amplified using pMutin DNA and the primer pair 5'-CTTAAGAGTGTGTTGATAGTGC-3'/5'-CTAGGGACCTCTTTAGCTCC-3'. The amplified 948 bp em gene contained the promoter sequence (–35, –10), SD sequence, and the em structural gene (encoding a 245 aa protein).
Construction of pMutinT3 : : sp insertional disruptant.
The em gene of pMutin was substituted with the sp gene of plasmid pEr : : sp (Lindow et al., 2002) by transformation of pMutin insertional disruptant BSU19 to Spr with linearized pEr : : sp DNA.
Construction of BSU28 (rsgI : : em spo0A
HB).
BSU17 was transformed to Trp+ Spo0A– with DNA from a strain that is a His+ Spo0A– transformant of NIG1121 (met his) (Sadaie, 1998) with DNA from UOT1611 (trpC2 lys1 aprE3 nprE18 nprR2 spo0AHB) (Asai et al., 1995).
Deletion of the promoter region of the sigI-rsgI operon.
The sigI promoters with deletions were made by fusing to the bgaB gene, in the amyE locus on the pDLd plasmid, DNA fragments PCR-synthesized using template DNAs from a wild-type strain and the primer pairs 5'-GAAGAATTCAAAAACACGCATAAAACCCCC-3'/5'-GGAGGATCCACTGGTTTCACCTCAGTTC-3' for the –35 deletion, 5'-GAAGAATTCTAGAAAGGCACGAAATCATGT-3'/5'-GGAGGATCCACTGGTTTCACCTCAGTTC-3' for the –10 deletion, and 5'-GAAGAATTCAGAACGTCAGAATGGTTTGTC-3'/5'-GGAGGATCCCTGGTTTCACCTCAGTTC-3' for the +1 deletion.
Conditions for primer extension and sequencing reactions.
RNA was prepared from cells of strains BSU15 (PsigI-bgaB), BSU14 ( PsigI-bgaB rsgI), BSU17 (PsigI-bgaB sigI) or BSU19 (PsigI-bgaB sigI rsgI) grown at 37 °C or 50 °C. The primer extension reaction was carried out as described elsewhere with digoxigenin-labelled primer DNA (10 pmol, 5'-CAAATTGAGGATAACACATTC-3', a sequence complementary to the region underlined with an arrow in Fig. 4) and reverse transcriptase (Invitrogen SuperScript II RNase H– Reverse Transcriptase) (Takamatsu et al., 2000). Sequencing of the sigI promoter region was performed with the DIG-labelled primer DNA described above and PCR-synthesized template DNA containing the promoter region of the sigI-bgaB operon. The latter DNA was PCR-synthesized with a template DNA from BSU15 (PsigI-bgaB) and the primer pair 5'-GAAGAATTCTGGGGTGTCTTAGCAG-3'/5'-CCTTGTCTAGCCATTCAAAG-3'(shown as dotted arrows in Fig. 4). The PCR product was purified by electrophoresis.
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Northern analysis.
This was performed as described elsewhere (Ohshima et al., 2002).
Cloning of sigI, rsgI and sigA into pGADT7 and pGBTK for yeast two-hybrid analysis.
Yeast two-hybrid analysis was performed as described elsewhere (Yoshimura et al., 2004). Plasmid vectors pGBTK and pGADT7 carrying cloned B. subtilis genes were used to transform mating-type yeast strains PJ69-4A and PJ69-4
, respectively. The sigI, sigA and rsgI genes, the 5'-terminal portion of rsgI and the 3'-terminal portion of rsgI were cloned into pGADT7 or pGBTK by inserting PCR-synthesized DNA fragments. PCR was performed with template DNA from a wild-type strain and the following primer pairs: 5'-GAAGAATTCATGAAACCAGTGCTTAGCC-3'/5'-GGAGGATCCTCATGAGTGCAGCACCCC-3' for sigI, 5'-GGAGGATTCATGAGAAGAGGGATTATAG-3'/5'-GGAGGATCCTTATTCGCCGGGGGCACTC-3' for rsgI, 5'-GGAGGATTCATGAGAAGAGGGATTATAG-3'/5'-GGAGGATCCTTAGCGCAGTTTAAAAAAATCAAAAAAG-3' for the rsgI N-terminal portion, 5'-GGAGGATTCTATGCGTATATGACAATCG-3'/5'-GGAGGATCCTTATTCGCCGGGGGCACTC-3' for the rsgI C-terminal portion, and 5'-GAAGAATTCATGGCTGATAAACAAACCC-3'/5'-GGAGGATCCTTATTCAAGGAAATCTTTCAAACG-3' for sigA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We confirmed the heat-inducibility of transcription of sigI by observing expression of the heat-stable BgaB activity of the bgaB gene of B. stearothermophilus fused to the sigI promoter (PsigI-bgaB) at the amyE locus (see Methods). Cells of B. subtilis strains carrying the PsigI-bgaB fusion were grown in LB medium at 37 °C and transferred to 50 °C. Induction of BgaB activity in the wild-type strain BSU15 was not observed following transient heat shock for 15 min at 50 °C (Fig. 1a), but was observed during sustained growth at 50 °C (Fig. 1b). Disruption of the rsgI gene by em displacement (rsgI : : em) in strain BSU17, on the other hand, stimulated greatly the expression of BgaB activity by transient heat shock (Fig. 1a) as well as in sustained growth at the higher temperature (Fig. 1b).
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), but was not observed when both sigI and rsgI were disrupted in strain BSU19 (rsgI : : pMutinT3 : : sp sigI : : pMutinT3) (Fig. 1c). Furthermore, an increase in BgaB activity during sustained growth at higher temperature (50 °C) was not observed in the sigI-disrupted strain (data not shown in Fig. 1b). An independent experiment showed -galactosidase activity of 
4.6 Miller units in the sigI-disrupted strain. This was also confirmed in the primer extension experiment described in a later section. Therefore transcription of the sigI gene is SigI-dependent, i.e. auto-regulated.
Detection of the mRNA of the complete sigI-rsgI operon
Heat-shock induction of sigI was confirmed by Northern blot analysis of RNA extracted from cells treated with heat. The expected transcript of more than 1896 nt from the predicted sig-rsgI operon was not detected upon transient heat shock (50 °C for 15 min) in the wild-type strain 168 (Fig. 2a). However, heat shock induced greatly a transcript hybridizing to the sigI probe in the rsgI-disrupted (rsgI : : em) strain BSU13 (Fig. 2b). The sizes of the wild-type sigI and rsgI genes are 752 nt and 1142 nt respectively, and the em-substituted disrupted rsgI gene is 967 nt long. The expected size of the transcript from the sigI-rsgI : : em gene is slightly longer than 1719 nt. The heat-induced signal detected showed a transcript of 1500 nt, which is longer than the 752 nt transcript expected from sigI or the 967 nt transcript expected from rsgI : : em. Thus, the signal detected must be the transcript from the entire sigI-rsgI operon, possibly quickly processed to give a shorter mRNA.
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, lanes 1–4, and Fig. 4). Experiments using cells of both strains grown at the higher temperature gave stronger signals (Fig. 3, lanes 2 and 4) than those from cells grown at the lower temperature (Fig. 3, lanes 1 and 3). The rsgI-disrupted strain showed a slightly stronger signal (Fig. 3, lanes 1 and 2) than the wild-type strain (Fig. 3, lanes 3 and 4). This transcription was SigI-dependent, as the sigI disruptant BSU18 (sigI : : pMutinT3) and the sigI rsgI double disruptant strain BSU19 (sigI : : pMutinT3 rsgI : : pMutinT3 : : sp) gave no signals at either temperature (Fig. 3, lanes 5, 6, 9 and 10).
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Deletion analysis of the promoter region was performed to confirm identification of the above transcription initiation site, because a paper reported that transcription of the sigI operon starts from two different sites, 18 and 23 bases downstream of the initiation site described above (Zuber et al., 2001). Deletion of the upstream region of the promoter (BSU23) did not strongly reduce sigI operon transcription (Figs 4 and 5), while deletion of the –35 region (BSU24) resulted in a background level of transcription, i.e. similar to transcription in the sigI-disrupted strain. Deletion of the –35 region should not result in reduced transcription if transcription starts 18 or 23 bases downstream of the predicted start site. Therefore our deletion analysis seemed to support the transcription start site determined in this study. Deletion of the –35 and –10 sequences completely eliminated transcription from the sigI promoter.
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, the full-length SigI and RsgI proteins interacted when SigI protein was fused to the DNA-binding domain and RsgI protein was fused to the activation domain. Furthermore, the full-length SigI protein and N-terminal portion (1st to 58th aa) of the RsgI protein interacted directly, as fusions either to the binding domain or to the activation domain. On the other hand, SigI did not show interaction with the C-terminal domain (90th to the final 381st aa) of RsgI. As RsgI is assumed to possess one transmembrane domain (67th to 89th aa predicted by SOSUI: http://bp.nuap.nagoya-u.ac.jp/sosui/), our results suggest that the cytoplasmic N-terminal portion (1st to 58th aa) of the RsgI protein must interact directly with the SigI protein. We also found that SigA did not interact directly with RsgI.
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), and rsgI in strain BSU22 (dnaK : : sp rsgI : : em) resulted in no stimulation (for 30 min) of the transient heat response of the sigI-rsgI operon which was observed in the rsgI-disrupted strain BSU17 (rsgI : : em) (Fig. 7a). However, prolonged heat induced sigI-rsgI operon transcription even in the dnaK rsgI double disruptant BSU22 (Fig. 7b). Disruption of rsgI by the insertional plasmid pMutinT3 gave the same result (data not shown). These results suggest that transient heat activation of SigI depends upon DnaK.
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(a), BgaB activity resulting from PsigI-bgaB at the amyE locus increased 1 h after the onset of sporulation in the wild-type strain BSU15 or the rsgI disruptant BSU17 (rsgI : : em) grown at 37 °C in a complex sporulation medium. This activity was dependent on SigI, as the sigI-rsgI double disruptant strain BSU19 (sigI : : pMutinT3 rsgI : : pMutinT3 : : sp) did not show such an increase (data not shown). Disruption of the dnaK gene in addition to rsgI disruption in BSU21 (rsgI : : pMutin dnaK : : sp) delayed the start of induction of BgaB activity by about 60 min (data not shown). DnaK must be required toward the end of growth as well as for the response to heat stress, to activate free SigI protein in the rsgI disruptant strain.
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). SigI expression may be induced by direct activation of free SigI protein induced by phosphorylation of Spo0A protein, or by derepression or activation of the sigI promoter by spo0A-P, AbrB or SigH. However, in the spo0A mutant strain BSU28 (rsgI : : em spo0A), we did not observe any reduction of the above increase of BgaB expression from PsigI-bgaB upon sporulation (Fig. 8a). Therefore it was considered that sigI induction is not related to sporulation.
Activation of sigI-rsgI operon transcription in strain BSU15 or BSU17 (rsgI : : em) was also induced toward the end of growth even at 37 °C in LB broth (Fig. 8b), which does not support efficient sporulation. Induction was transient and showed a maximal value near the end of growth. This induction depended on SigI, because the rsgI disruptant (BSU17) showed strong induction but the rsgI sigI double disruptant BSU19 (sigI : : pMutinT3 rsgI : : pMutinT3 : : sp) showed reduced induction (data not shown). Furthermore, this induction was not dependent on DnaK, as the rsgI and dnaK double disruptant BSU21 (rsgI : : em dnaK : : sp) showed the same induction as BSU17.
Glucose repression of sigI-rsgI operon expression
Addition of glucose strongly suppressed activation of sigI-rsgI operon transcription in strain BSU17 (rsgI : : em) grown at 37 °C in a complex sporulation medium (Fig. 8a) or in strain BSU15 grown at 50 °C in LB broth as described below. Glucose causes repression of gene expression by CcpA through the catabolite fructose biphosphate (Deutscher et al., 2002). Although the CcpA target-like sequence (complementary cre-like sequence of ATGAAAACGCTTCAA) was found upstream of the –35 region (Fig. 4) (Miwa et al., 2000), deletion of this target sequence resulted in glucose-sensitive sigI-rsgI expression as shown by strain BSU23. Rapidly growing cells of BSU15(PsigI-bgaB) or BSU23(P(–35)sigI-bgaB) in LB broth at 37 °C were transferred to 50 °C for 60 min with or without glucose (0.5 %) addition. BSU15 showed BgaB activity of 16.5 Miller units (MU) without glucose and 3.5 MU with glucose, while BSU23 showed BgaB activity of 6.2 MU without glucose and 1.7 MU with glucose.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase gene (bgaB) fused to the sigI promoter enabled measurement of transcription of the sigI-rsgI operon. Furthermore, reverse transcription of transcripts derived from PsigI-bgaB, using a primer complementary to bgaB, allowed determination of a unique, heat-inducible and SigI-dependent transcription initiation site. The predicted RsgI protein has a transmembrane sequence, and a long C-terminal sequence containing an aspartic-acid-rich region (BSORF: http://bacillus.genome.jp/). The N-terminal portion (first 58 aa excluding transmembrane domain) of RsgI interacted with SigI. Deletion or pMutin-insertional inactivation of the rsgI gene resulted in effects on heat-inducible SigI activation. The latter insertion left the N-terminal sequence including the transmembrane sequence intact. Therefore, even if SigI-interacting sequences and the transmembrane sequence of RsgI were retained in vivo, the SigI-interacting sequence did not function. This suggests that RsgI requires the long C-terminal domain to negatively regulate SigI factor, or to be properly localized into the membrane.
Although we did not examine interaction between DnaK and SigI or RsgI, free SigI protein in the rsgI : : em dnaK : : sp double disruptant strain BSU22 requires heat to become active. DnaK facilitates activation of heat-shocked SigI protein. Prolonged heat activates SigI in the absence of DnaK. During sporulation, free SigI protein in the rsgI : : em dnaK : : sp double disruptant strain became active without heat.
The expression or activity of BgaB may be altered by heat stress through translational or post-translational processes. However, BgaB activity from the bgaB gene fused to three promoters (sigW, divIB or thiCK) that do not respond to heat shock was stimulated only by a factor of 1.6. Introduction of dnaK disruption altered this factor to 1.3. Therefore the activity of BgaB was not stimulated strongly by heat, and DnaK did not seem to be required in the activity of BgaB at 50 °C. The heat induction and DnaK-dependence of BgaB activity derived from the bgaB gene fused to the sigI promoter described in the text was considered to reflect sigI-rsgI expression.
When a primer extension experiment was performed with a primer (5'-GATAGGGATTCGCAAAGGAG-3') hybridizable to the original sigI promoter region, there were two weak but significant bands which were different from the unique band shown in Fig. 3. They seemed to indicate two transcription initiation sites at T or A residues located two and one bases upstream of the unique start site described in the text. They were heat-shock- and SigI-dependent. Even in this experiment, we did not obtain the results indicating transcription initiation sites described elsewhere (Zuber et al., 2001).
In the primer extension experiment with a primer hybridizable to bgaB, there was another transcript (not shown in Fig. 3) from the A residue 23 bases upstream of the sigI initiation codon GTG. It was SigI-independent and heat (50 °C) sensitive. On the other hand, as shown in Fig. 5, in the sigI-disrupted strain BSU16, wild-type promoter fusion showed residual activity at 50 °C. A truncated promoter fusion with only the –10 region showed similar residual activity, and a promoterless fusion showed complete loss of BgaB at 50 °C. We did not analyse further whether the above transcript might be related to the sigI promoter. Nor did we further study the possibility that some sigma factor other than SigI might be involved in the residual BgaB activity described above.
Expression of the sigI-rsgI operon appears to be regulated at least at the transcriptional level. Initiation of transcription depended on the presence of active SigI. Transcription was strongly suppressed by glucose addition, but the presumed CcpA target-like site in the promoter region was not involved. The glucose effect was observed even in the absence of RsgI. The glucose effect described above explains the previous observation that sigI transcription is not observed in a minimal-glucose medium (Zuber et al., 2001). Operon transcription was transiently induced at near the end of growth in a rich complex medium independent of DnaK function, and was gradually induced 1 h after the end of growth in a complex medium which supports efficient sporulation. As the spo0A gene was not involved, sigI induction was not related to sporulation, and nutrients such as glucose seemed to suppress sigI-rsgI transcription.
Summarizing the above results, a model of the activation process of the SigI protein is shown in Fig. 9. The N-teminal domain (58 aa) of the transmembrane anti-sigma protein RsgI interacts directly with the SigI protein and keeps it inactive. Upon prolonged heat exposure, or a strong transient heat shock, SigI protein is released from RsgI and activated through heat- and DnaK-mediated process before integrating into the SigI-RNA polymerase core complex. Inactivation of the rsgI gene releases free but inactive SigI protein which is easily activated by transient heat shock in a DnaK-dependent fashon. Heat of long duration, however, activates free but inactive SigI independently of DnaK. Transient or prolonged heat may release SigI protein from some interacting negative factor(s).
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ACKNOWLEDGEMENTS |
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Edited by: T. Msadek
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Received 22 June 2006;
revised 18 August 2006;
accepted 23 September 2006.
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, 
) and rsgI : : em-disrupted strain BSU17 (
, 
). Filled symbols, heat-shocked cells; open symbols, cells incubated at 37 °C without heat shock. (b) Cells were grown at 37 °C, then transferred to 50 °C at time 0. BgaB activity from PsigI-bgaB was measured in the wild-type strain BSU15 (
, 
). Filled symbols, heat-shocked cells; open symbols, cells incubated at 37 °C without heat shock.
, 
). Filled symbols, heat-shocked cells; open symbols, cells incubated at 37 °C without heat shock. (b) Rapidly growing cells were transferred from 37 °C to 50 °C at time 0. BgaB activity was measured for the bgaB gene fused to the sigI promoter in BSU16 (rsgI : : pMutinT3, 
HB, 