Transcriptional responses during outgrowth of Bacillus subtilis endospores

Malcolm J. Horsburgh1, Penny D. Thackray1 and Anne Moir1

Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1

Author for correspondence: Anne Moir. Tel: +44 114 222 2826. Fax: +44 114 272 8697. e-mail: A.Moir{at}sheffield.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The Bacillus subtilis 168 genome contains an array of alternative {sigma} factors, many of which play important roles in reprogramming expression during stress and sporulation. The role of the different {sigma} factors during outgrowth, when the germinated endospore is converted back to a vegetative cell, is less well characterized. The activity of the alternative {sigma} factors {sigma}B, {sigma}D and {sigma}H during endospore outgrowth was analysed by Northern blotting and lacZ reporter assays. While {sigma}D and {sigma}H were transcriptionally active during outgrowth, {sigma}B-dependent transcription was not observed until after the first cell division, when growth slowed. Using an IPTG-controllable copy of sigA, an optimal level of expression was required to maintain growth rate at the end of outgrowth. The genes encoding the putative extracytoplasmic function (ECF) {sigma} factors {sigma}I, {sigma}V, {sigma}W, {sigma}Z and YlaC were insertionally inactivated using pMUTIN4. These strains, together with sigM and sigX mutants, were tested to determine their role and measure their expression during endospore outgrowth. Transcripts or ß-galactosidase activity were observed for each of the ECF {sigma} factors early after germination. With the exception of MJH003 (sigM), which showed an exacerbated salt stress defect, inactivation of the ECF {sigma} factor genes did not affect outgrowth in the conditions tested.

Keywords: outgrowth, transcription, sigma factor, Bacillus subtilis

Abbreviations: ECF, extracytoplasmic function; NB, nutrient broth; SMM, Spizizen minimal medium


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Gram-positive soil bacterium Bacillus subtilis responds to nutrient starvation by initiating a cascade of gene expression that produces a dormant endospore at the expense of the mother cell. This process is regulated through the temporal and compartmentalized expression of alternative {sigma} factors ({sigma}H, {sigma}E, {sigma}F, {sigma}G and {sigma}K) that direct gene expression leading to the formation of a dormant endospore (Errington, 1993 ). Although still present during sporulation, the activity of the vegetative {sigma} factor {sigma}A is limited by an unknown mechanism, possibly by an anti-{sigma}A factor (Lord et al., 1999 ). Alternative {sigma} factors control motility, chemotaxis and autolysin functions ({sigma}D), and the expression of the general stress proteins ({sigma}B) in vegetative cells (Haldenwang, 1995 ; Hecker & Volker, 1998 ). In addition, the B. subtilis genome contains seven genes encoding extracytoplasmic function (ECF) {sigma} factors. Phenotypes for mutations in four, {sigma}I (Zuber et al., 2001 ), {sigma}M (Horsburgh & Moir, 1999 ), {sigma}W (Huang et al., 1998 , 1999 ; Turner & Helmann, 2000 ) and {sigma}X (Huang et al., 1997 , 1998 ; Huang & Helmann, 1998 ; Turner & Helmann, 2000 ) have been described in detail; sigI is sensitive to elevated temperature and induced by heat shock, a sigM mutant is unable to grow in high concentrations of salt, a sigW mutant is altered in resistance to cell wall biosynthesis inhibitors and a sigX mutant has reduced resistance to heat and oxidative insult.

Germination of an endospore in favourable, nutrient-rich conditions enables outgrowth to commence and leads to the restoration of a vegetative cell that is capable of normal cell division (Setlow, 1983 ). Since the dormant spore is essentially devoid of mRNA, protein synthesis during outgrowth is dependent on new transcription that begins in the first minutes of germination and precedes the synthesis of protein by several minutes. {sigma}A has been proposed to be involved in transcription immediately after germination of spores and is probably present in the mature spore (Sloma & Smith, 1979 ). While RNA and protein synthesis occur early after germination, the rapid synthesis of DNA does not start until 30 min later when chromosome replication begins (Garrick-Silversmith & Torriani, 1973 ). Protein synthesis during outgrowth has been studied by two-dimensional gel electrophoresis, revealing that many proteins are synthesized temporally during outgrowth (Mullin & Hansen, 1981 ; Wachlin & Hecker, 1982 ; Hecker, 1983 ; Hecker et al., 1984 ). The identity of these proteins is not yet known, but their temporal regulation was proposed to be transcriptional. To date no regulators have been found that exclusively regulate gene expression during outgrowth.

Attempts have been made to isolate mutants of B. subtilis that are temperature-sensitive during spore outgrowth as a means of identifying outgrowth-specific genes (Galizzi et al., 1973 ). Despite isolating temperature-sensitive outgrowth mutants, none of the loci identified in detail were outgrowth-specific; instead they were preferentially expressed during this phase. The outB mutation, originally characterized as producing a temperature-sensitive outgrowth phenotype at 46 °C in rich medium (Albertini & Galizzi, 1975 ) was located in a gene encoding an NH3-dependent NAD synthetase (NadE) (Nessi et al., 1995 ). However, each of the nadE (outB) mutants with reduced NAD synthetase activities was also impaired in growth at the permissive temperature of 35 °C, indicating the essential nature of this enzyme (Albertini et al., 1987 ). NadE was subsequently shown to belong to the family of {sigma}B-dependent general stress proteins (Antelmann et al., 1997 ).

The regulation of gene expression during outgrowth is still poorly characterized. We therefore surveyed the expression and activity of the alternative {sigma} factors {sigma}B, {sigma}D and {sigma}H and screened the seven potential ECF {sigma} factors in mutation and expression studies.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains, plasmids and growth conditions.
B. subtilis and Escherichia coli strains are listed in Table 1. E. coli was grown in Luria–Bertani (LB) medium. B. subtilis was grown at 37 °C with vigorous shaking in nutrient broth (NB) medium (Oxoid) or Spizizen minimal medium (SMM) (Anagnostopoulos & Spizizen, 1961 ) with 27·7 mM glucose, 15 mM L-glutamine and 0·25 mM L-tryptophan. When included antibiotics were added at the following concentrations: ampicillin, 100 mg l-1; neomycin, 10 mg l-1; spectinomycin, 100 mg l-1; erythromycin, 1 mg l-1; and lincomycin, 25 mg l-1. Sporulation was performed in CCY medium (Stewart et al., 1981 ) with incubation at 37 °C for 36 h and purification of spores by repeated washing with H2O and storage at -20 °C. For experiments with KH453 ({Delta}rpoD Pspac-rpoD), cells were grown overnight in NB or allowed to sporulate in CCY containing 30 µM IPTG; this concentration of IPTG provides approximately 100% of wild-type SigA protein expression intracellularly (Hicks & Grossman, 1996 ). For outgrowth, spores were heat-activated in H2O at 70 °C for 60 min then added to pre-warmed nutrient broth containing 0·5% (w/v) glucose, 0·5% (w/v) KCl and 10 mM L-alanine. Transformation of B. subtilis was performed as described by Kunst & Rapoport (1995) .


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Table 1. Bacterial strains, plasmids and primers used in this study

 
ß-Galactosidase assays.
Levels of ß-galactosidase activity were measured as described previously (Horsburgh & Moir, 1999 ). Briefly, 1 ml samples were harvested and cell pellets were resuspended in 0·5 ml AB buffer (60 mM K2HPO4, 40 mM KH2PO4, 100 mM NaCl) containing lysozyme (100 µg ml-1) and DNaseI (10 µg ml-1), and incubated at 37 °C for 10 min. Then 25 µl MUG (4-methylumbelliferyl ß-D-galactoside; 4 mg ml-1) was added. After incubation for 40 min at 30 °C the assay was stopped by addition of 0·5 ml 0·4 M Na2CO3. Fluorescence was measured using a DyNaQuant fluorometer (Hoefer) or a Victor plate reader (Wallac) with a 0·1 s count time; both instruments were calibrated with standard concentrations of MU (4-methylumbelliferone). One unit of ß-galactosidase activity was defined as the amount of enzyme that catalysed the production of 1 pmol MU min-1 (OD600 unit)-1. Assays were performed on duplicate samples and the values averaged. The results presented here were representative of three independent experiments that showed less than 20% variability.

Construction of strains.
The recombinant strains used in this study were constructed using PCR and standard cloning techniques (Sambrook et al., 1989 ) to generate derivatives of plasmid pMUTIN4 (an integrating plasmid conferring resistance to erythromycin and containing a promoterless lacZ; Vagner et al., 1998 ). Plasmids were constructed for disrupting sigV, sigW, sigZ, ylaC and sigI by PCR amplification of sigV (nt +25 to +334 relative to the translational start point), sigW (+150 to +359), sigZ (+19 to +294), ylaC (+9 to +225) and sigI (+2 to +270), respectively, using primers (Table 1) with a BamHI site at the 5' end and a HindIII site at the 3' end of the fragment, and inserted into the HindIII and BamHI sites of pMUTIN4 (Vagner et al., 1998 ; Table 1). All of these plasmids were integrated into the chromosome of B. subtilis 168 through homology with the sigV, sigW, sigZ, ylaC or sigI fragments by a Campbell-type event generating lacZ fusions and gene inactivation. The location and structural integrity of the DNA at the integration site was verified by Southern blotting in each case.

RNA isolation and analysis.
Total RNA was isolated from dormant and outgrowing spores of B. subtilis sampled at time points after the addition of spores to prewarmed NB medium containing germinants. The rapid liquid nitrogen chill method of Arnau et al. (1996) was used to ensure effective sampling at each time point. Frozen cell pellets, briefly stored at -70 °C, were thawed on ice and RNA rapidly extracted by cell disruption using the Fast-prep blue kit (Bio-101) and a Fast-prep system reciprocal shaker (Savant). Northern hybridization was performed using standard conditions (Sambrook et al., 1989 ) with DIG-labelled PCR fragments of sigH (-8 to +630 relative to the translational start point of sigH), sigM (+8 to +442), sigV (+23 to +495), sigW (+48 to +359), sigX (+30 to +510), sigY (-12 to +522), ylaC (+6 to +518), sigH (-8 to +630), ctc (+90 to +608), gspA (+92 to +839), hag (+91 to +865), lytD (+55 to +806) or outB (+26 to +798) synthesized using the oligonucleotide primers listed in Table 1. Sequence information was taken from the completed B. subtilis genome (Kunst et al., 1997 ). These labelled fragments were used to probe total RNA that had been separated on a 1% formaldehyde agarose gel and blotted onto Nylon membrane (Roche).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The level of SigA affects the rate of outgrowth
To investigate the effects of varying the level of {sigma}A on the rate of outgrowth we used strain KH453 ({Delta}rpoD Pspac-rpoD), in which the amount of {sigma}A can be changed over a relatively wide range in an IPTG-dependent manner (Hicks & Grossman, 1996 ). Spores were germinated and outgrown in rich (NB) medium containing germinants with varying levels of IPTG and their progress was monitored by OD560 (Fig. 1). Significant levels of {sigma}A protein were present in spores produced with IPTG in the sporulation medium, as the rate of outgrowth was not slowed until the end of outgrowth in medium to which no IPTG was added compared to outgrowth in the presence of 30 µM IPTG. This latter concentration of IPTG was previously determined to induce near wild-type levels of the protein as measured by immunoblotting (Hicks & Grossman, 1996 ). Continued expression of {sigma}A from the end of outgrowth onwards is required to maintain growth rates (Fig. 1). The addition of progressive levels of IPTG improved the growth rate and yield of the cells towards wild-type levels at around 100 µM. Above this concentration the rate of outgrowth was slowed, suggesting that the optimal level of {sigma}A protein within the cell had been exceeded.



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Fig. 1. The effect of varying the level of SigA during outgrowth of dormant endospores. After addition of prewarmed spores of JH642 (wild-type; filled symbols) or KH453 ({Delta}rpoD Pspac-rpoD; open symbols) to pre-warmed NB medium the rate of outgrowth was monitored by measuring changes in OD560 at the time points indicated. Spores were outgrown with IPTG present at the following concentrations: 0 ({circ}, {bullet}), 3 ({triangleup}), 30 ({lozenge}), 100 (+) and 300 µM ({square}, {blacksquare}). The closed circles and squares are superimposed. The presence of IPTG at 3, 30 and 100 µM did not affect the rate of outgrowth of JH642 (wild-type) (data not shown for clarity).

 
SigD and SigH, but not SigB, are active during outgrowth of endospores
To determine whether the alternative {sigma} factors, {sigma}B, {sigma}D and {sigma}H are active during outgrowth, RNA was extracted from spores throughout outgrowth (10–90 min) and after the first cell division (100–110 min) using a rapid chill method that stabilized RNA and ensured representative sampling for each time point. Using Northern blots the isolated RNA samples were probed for {sigma}B-(ctc, gspA) and {sigma}D-dependent (hag) transcripts (Fig. 2b, c). ctc is transcribed from two promoters, PA and PB, during the exponential and stationary phases of B. subtilis growth, respectively (Hilden et al., 1995 ). Two transcripts were observed (Fig. 2b); a 3 kb gcaD-prs-ctc transcript from PA and a 0·6 kb ctc transcript from PB. Expression of the latter transcript was not observed until 110 min, when outgrowth was complete and the growth rate slowed. Similarly gspA (Fig. 2c), which is a member of the {sigma}B regulon, was only transcribed at the end of outgrowth. This demonstrates a lack of {sigma}B-dependent transcriptional activity during outgrowth. In contrast, {sigma}D activity was observed during outgrowth. A transcript from the {sigma}D-dependent gene hag, which encodes flagellin, was observed throughout outgrowth. To determine whether {sigma}H was also active, strain KH590 (ftsZP2-lacZ), which contains a lacZ fusion to a {sigma}H-dependent promoter of ftsZ, was assayed for ß-galactosidase activity during outgrowth (Fig. 3). Levels of ß-galactosidase expressed from this {sigma}H-dependent reporter increased initially before becoming relatively constant, indicating that {sigma}H was active during endospore outgrowth. Transcription of the sigH gene was confirmed by Northern blotting (Fig. 4).



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Fig. 2. (a) Isolation of intact RNA from dormant and outgrowing endospores of B. subtilis. The arrow labelled with an asterisk indicates an abundant species of RNA which migrates slightly faster than the 23S rRNA species in dormant and early outgrowth time point samples. Transcripts of the {sigma}B-dependent genes ctc (b) and gspA (c), and the {sigma}D-dependent gene hag (d) during outgrowth in NB medium were detected by Northern blotting. RNA extracted from dormant spores was probed with DIG-labelled PCR fragments amplified with the primers detailed in Table 1. ctc was transcribed (b) as a {sigma}A-dependent 3 kb gcaD-prs-ctc mRNA during outgrowth and a 0·6 kb {sigma}B-dependent ctc mRNA when exponential growth ended.

 


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Fig. 3. Activity of {sigma}H, measured in the reporter strain KH590 (ftsZP2-lacZ), during outgrowth of B. subtilis spores in NB medium. Outgrowth was monitored by changes in OD560 ({square}) and samples were removed at the times indicated and assayed for ß-galactosidase activity ({bullet}).

 


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Fig. 4. Northern blots of RNA isolated during outgrowth, probing for transcription of the sigH gene, and sigM, sigV, sigW, sigX, sigY and ylaC, the genes encoding six of the eight B. subtilis ECF {sigma} factors. Five micrograms RNA was loaded for each time point.

 
Transcription of ECF {sigma} factors during outgrowth
We sought to determine which of the seven ECF {sigma} factor genes were transcribed during outgrowth. Northern blots were probed for transcripts corresponding to sigI, sigM, sigV, sigW, sigX, sigY, sigZ and ylaC genes. Of these, transcripts were only observed for the sigM (1·9 kb), sigV (1·5 kb), sigW (1·3 kb), sigX (1·8 kb), sigY (1·4 kb) and ylaC (2 kb) genes (Fig. 4). Despite repeated attempts, transcripts of the remaining ECF {sigma} factor genes, sigI and sigZ, were not observed in blots of outgrowth RNA; the RNA preparations used were the same for each of the sig gene blots.

Mutation analysis of ECF {sigma} factors
To determine whether any of the ECF {sigma} factors were important for successful outgrowth, the sigV, sigW, sigZ, ylaC and sigI genes were inactivated using the suicide vector pMUTIN4 (Vagner et al., 1998 ). Correct insertion of the plasmid and inactivation of the corresponding gene were confirmed by Southern blotting. None of the strains had a vegetative growth defect in either rich or minimal media, as noted by Turner & Helmann (2000) . Spores of ECF {sigma} factor mutants MJH101 (sigX; Huang et al., 1997 ), MJH003 (sigM) and the reporter strain MJH004 (sigM-lacZ sigM+) (Horsburgh & Moir, 1999 ), MJH010 (sigV), MJH011 (sigW), MJH012 (sigZ), MJH013 (ylaC) and MJH014 (sigI) were examined by light microscopy and the optical density change measured during outgrowth in rich (NB) and minimal (SMM) media. In addition, samples were taken at each time point and assayed for ß-galactosidase activity. For each of the mutants, with the exception of MJH003 (sigM), outgrowth was not impaired and wild-type morphological changes were observed in each medium (data not shown). MJH003 (sigM), in contrast, had a pronounced outgrowth defect in NB medium containing germinants (Fig. 5a), and outgrowing spores of MJH003 (sigM) failed to elongate properly and swelled, forming large amorphous cells compared to the long rod-shaped cells of 1604 (wild-type) (Fig. 6). The salt sensitivity of a sigM mutant in NB medium would appear to be more pronounced in outgrowth than that reported during vegetative growth (Horsburgh & Moir, 1999 ). During outgrowth of MJH003 (sigM) in NB containing 700 mM NaCl, the spores formed large round cells that failed to elongate (data not shown). A similar morphological defect during outgrowth was observed for a pbpA mutant of B. subtilis (Murray et al., 1997 ). However, MJH002 (sigM) did not have altered expression of pbpA, compared to 1604 (wild-type), when measured using a pbpA-lacZ fusion (data not shown). During outgrowth, ß-galactosidase activity was similar for both MJH003 (sigM) and MJH004 (sigM-lacZ sigM+), indicating that {sigma}M does not contribute significantly to expression of its own gene under these conditions (Fig. 5b). This contrasts with vegetative growth, where expression from both promoters (PM and PA) contributes to overall sigM yhdL yhdK operon transcription (Horsburgh & Moir, 1999 ).



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Fig. 5. (a) Endospore outgrowth of 1604 (wild-type; {square}) and MJH003 (sigM; {bullet}) in NB medium. MJH003 (sigM) was retarded in outgrowth compared to 1604 (wild-type). (b) In vivo expression driven by the sigM promoters (PAPM) in MJH003 (sigM-lacZ; {bullet}) and MJH004 (sigM-lacZ sigM+; {square}) during outgrowth in NB medium.

 


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Fig. 6. (a) Endospore outgrowth of 1604 (wild-type) and (b, c) MJH003 (sigM) in NB medium. Cells were harvested at 90 min (a, b) or 165 min (c), concentrated by centrifugation and fixed for electron microscopy. The normal rod-shaped bacilli of 1604 (wild-type) contrast with the shortened, swollen and irregular cells of MJH003 (sigM) at 90 and 165 min. Cells of 1604 (wild-type) at 165 min were long rods (2–4 cells long) (data not shown). Bars, 1 µm.

 
Expression of the sigV, sigW, ylaC and sigI genes, assayed using the lacZ reporter fusions, was observed during outgrowth in both rich (NB) and minimal (MSSM) media (Fig. 7). ß-Galactosidase activity, which reflects nett accumulation of the reporter enzyme, increases in an approximately linear fashion during the early stages of outgrowth. Levels of sigZ expression were very low and only just detectable above background in rich medium; sigI expression was markedly higher in minimal medium. These observations are consistent with the failure to detect transcripts from these genes in RNA preparations from NB cultures. The relative levels of expression of the ECF {sigma} factors, compared in Fig. 7, may be an underestimate since inactivation of these {sigma} factors may have eliminated expression from upstream ECF {sigma}-dependent promoters. Such promoters have been observed for sigM (Horsburgh & Moir, 1999 ), {sigma}W (Huang et al., 1998 ) and sigX (Huang et al., 1997 ).



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Fig. 7. ß-Galactosidase expression in the pMUTIN4 ECF {sigma} factor mutant strains, sigV ({blacksquare}), sigW ({triangleup}), sigX ({bullet}), sigZ ({blacktriangleup}), ylaC ({circ}) and sigI ({lozenge}), and wild-type 1604 (+) assayed during outgrowth in (a) rich (NB) or (b) minimal (SMM) media. Each strain outgrew at a similar rate and a representative OD560 reading (x) is shown. The 1604 assay control data (+) are at the base of each graph and were lower than those of sigZ ({blacktriangleup}), in each case.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Outgrowth is an important developmental stage in the life cycle of B. subtilis that rapidly enables a dormant spore to make the transition back to vegetative growth. Since the dormant endospore contains no functional mRNA, gene expression must start afresh. During this time the cell must sense the environment into which it has entered and rapidly adapt should the conditions become unfavourable. The inability of the outgrowing spore to completely protect itself from environmental assault is demonstrated by its vulnerability to increased salt concentrations and to the cell-wall antibiotics nisin and tylosin (Gould, 1964 ; Vinter, 1970 ).

To understand the programmed gene expression of B. subtilis during outgrowth we examined the role and activity of the alternative {sigma} factors. {sigma}B-dependent transcription of gspA and ctc was notably absent during outgrowth. In contrast, {sigma}D- (lytD and hag) and {sigma}H-dependent (ftsZP2) promoters were transcribed, demonstrating the activity of these alternative {sigma} factors. From this it can be proposed that outgrowth is not inherently a stressed state. Melly & Setlow (2001) observed that expression of heat-shock genes, including ctc, were not induced by wet heat damage to dormant spores prior to germination.

The level of the housekeeping {sigma} factor, {sigma}A, is important for the timing of sporulation, due to competition with the sporulation {sigma} factor, {sigma}H. When the level of {sigma}A is increased, entry into sporulation is delayed and spore yield drops (Hicks & Grossman, 1996 ). In our experiments, a reduced growth rate at the end of outgrowth was observed when IPTG was either omitted or added at elevated concentrations, suggesting that the level of SigA was important for optimal outgrowth. Since both {sigma}D and {sigma}H were shown to be transcriptionally active during outgrowth, an increased level of {sigma}A may titrate these alternative {sigma} factors in an analogous manner to that described by Hicks & Grossman (1996) . Multiple promoters contribute to sigA expression during different stages of the B. subtilis life cycle (Wang & Doi, 1987 ; Carter et al., 1988 ; Qi & Doi, 1990 ; Qi et al., 1991 ; Hicks & Grossman, 1996 ) and an inhibitor of {sigma}A, possibly an anti-{sigma} factor, has been proposed as the mechanism for inhibiting vegetative gene expression during sporulation (Lord et al., 1999 ). The nature of this putative inhibitor remains to be determined. Activation of SigA is extremely rapid during germination and outgrowth, since many of the transcripts were readily identifiable just 5 min after the addition of spores to NB medium (data not shown).

The ECF family of {sigma} factors were identified as a subset of alternative {sigma} factors that are predominantly involved with the transcription of genes in response to one or more extracellular signals (Lonetto et al., 1994 ). We tested whether the expression of any of the members of the ECF {sigma} factor family in B. subtilis was important for successful outgrowth. Differing levels of expression of each of the ECF {sigma} factor genes (sigI, sigM, sigV, sigW, sigX, sigY, sigZ and ylaC) was shown by Northern blotting and/or lacZ expression. This suggests they are therefore available for activation in outgrowing cells. Inactivation of the ECF {sigma} factors did not affect outgrowth, except sigM, which was important for outgrowth in medium containing increased concentrations of salt. Outgrowing spores have been demonstrated to be more sensitive to salt than vegetative cells (Gould, 1964 ; Vinter, 1970 ) and the exacerbated defect of the sigM mutant during outgrowth may reflect this.


   ACKNOWLEDGEMENTS
 
We thank Alan Grossman, John Helmann and Peter Setlow for their kind gifts of strains. This research was funded by a BBSRC/DTI Link/Celsis Connect project grant.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 2 April 2001; revised 19 June 2001; accepted 9 July 2001.