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
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ABSTRACT |
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Keywords: outgrowth, transcription, sigma factor, Bacillus subtilis
Abbreviations: ECF, extracytoplasmic function; NB, nutrient broth; SMM, Spizizen minimal medium
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INTRODUCTION |
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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.
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
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 factors
B,
D and
H and screened the seven potential ECF
factors in mutation and expression studies.
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METHODS |
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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).
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RESULTS |
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Mutation analysis of ECF factors
To determine whether any of the ECF 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
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
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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DISCUSSION |
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To understand the programmed gene expression of B. subtilis during outgrowth we examined the role and activity of the alternative factors.
B-dependent transcription of gspA and ctc was notably absent during outgrowth. In contrast,
D- (lytD and hag) and
H-dependent (ftsZP2) promoters were transcribed, demonstrating the activity of these alternative
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 factor,
A, is important for the timing of sporulation, due to competition with the sporulation
factor,
H. When the level of
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
D and
H were shown to be transcriptionally active during outgrowth, an increased level of
A may titrate these alternative
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
A, possibly an anti-
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 factors were identified as a subset of alternative
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
factor family in B. subtilis was important for successful outgrowth. Differing levels of expression of each of the ECF
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
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.
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ACKNOWLEDGEMENTS |
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Received 2 April 2001;
revised 19 June 2001;
accepted 9 July 2001.