Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Correspondence
Jeff Errington
jeff.errington{at}path.ox.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The late prespore-specific -factor,
G, is regulated at at least three levels. Firstly its gene sigG (spoIIIG) is transcribed by E
F (and later by E
G itself), thus restricting its localization to the prespore compartment (Fig. 1
; Sun et al., 1991
). The sigG gene is the distal element of the three-gene spoIIG operon, which comprises spoIIGA (encoding the pro-
E processing enzyme), sigE (spoIIGB) and sigG (Karmazyn-Campelli et al., 1989
). Transcription of the whole operon is under the control of the housekeeping
-factor,
A, and the sporulation-specific transcription factor Spo0A (Masuda et al., 1988
), and begins before asymmetric septation. However,
G is not translated from this polycistronic transcript due to the presence of a stemloop structure that blocks its ribosome-binding site (Masuda et al., 1988
). A promoter that is recognized by
F and
G itself is located immediately upstream of the sigG coding region, and it is from transcripts originating at this promoter that the
G protein is translated (Sun et al., 1991
). Secondly, once translated, the protein apparently does not become active until after the completion of engulfment of the prespore. In the presence of mutations in several different genes, including spoIIB, spoIID, spoIIM, spoIIP, spoIIIA and spoIIIJ,
G is synthesized but it does not become active (Errington et al., 1992
; Frandsen & Stragier, 1995
; Kellner et al., 1996
; Partridge & Errington, 1993
; Smith et al., 1993
). Four of the proteins, SpoIIB, SpoIID, SpoIIM and SpoIIP, are required for prespore engulfment (Frandsen & Stragier, 1995
; Smith et al., 1993
), suggesting that
G activity is coupled to this morphological event. Recently it has been shown that SpoIIIJ and the spoIIIA-encoded products are part of the signalling pathway that results in the activation of
G after completion of engulfment (Kellner et al., 1996
; Serrano et al., 2003
).
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Here we have investigated the E-dependence of sigG expression; we have identified a regulatory site within the sigG promoter by analysing mutant sigG promoters generated by site-directed mutagenesis and sigG promoters from other species. Replacement of the wild-type promoter with
E-independent promoters resulted in impairment of sporulation. Our data support the idea that
E activity is required for the transcription of sigG, probably by relieving the action of a repressor.
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METHODS |
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B. subtilis strains were transformed as described previously (Anagnostopoulos & Spizizen, 1961; Jenkinson, 1983
). Transformants were selected on Oxoid nutrient agar plates containing chloramphenicol (5 µg ml1) or kanamycin (5 µg ml1) as appropriate.
Resuspension and -galactosidase assay.
B. subtilis cells were induced to sporulate by the resuspension method (Sterlini & Mandelstam, 1969; Partridge & Errington, 1993
). Time zero (t0) was defined as the point at which the cells were resuspended in a starvation medium (SM). Samples (0·5 ml) were removed at intervals, pelleted and frozen in liquid nitrogen to be assayed for
-galactosidase activity.
-Galactosidase activity was assayed using a method described by Errington & Mandelstam (1986)
. One unit of
-galactosidase catalyses the production of 1 nmol 4-methylumbelliferone min1.
Construction of lacZ fusions to different lengths of the sigG promoter.
Plasmids pSG4742, pSG4731 and pSG4743 were constructed by amplifying sigG promoter fragments of, respectively, 338 bp, 143 bp and 82 bp by PCR from chromosomal DNA of wild-type strain SG38. The following primers were used for PCR: 4742 (5'-CCGGAATTCAAAAGCGCTTGA-3'), 4731 (5'-CCGGAATTCATGGTTAGAACC-3') or 4743 (5'-CCGGAATTCGCAGTGCATATT-3'), each of which introduced an EcoRI site, and H1 (5'-CGCCAAGCTTATTTCTCGACAC-3'), which introduced a HindIII site. The EcoRIHindIII-digested PCR products were subcloned into EcoRIHindIII-digested and gel-purified ptrpBGI (Shimotsu & Henner, 1986), thereby replacing the trp promoter with the sigG promoter and generating translational sigGlacZ fusions.
Site-directed mutagenesis.
Mutations were introduced into the sigG promoter by PCR amplification of plasmid pSG4732. Forward and reverse primers were designed to overlap completely and had the mutated base(s) in the middle. Pfu DNA polymerase was used to amplify the whole plasmid. Template DNA was degraded by DpnI digestion; the nicked circular products were then transformed into E. coli and the promoter then replaced into pSG4731. Each mutant promoter was sequenced before introduction into the B. subtilis chromosome at the amyE locus.
Construction of strains with sigG under the control of E-independent promoters.
The PBt and PsigG-14 mutant promoters were cloned in place of the wild-type promoter at the sigG locus. The resulting arrangement of genes is such that sigE and sigG are still in tandem with the aphA-3 gene inserted between them, in the opposite orientation to prevent read-through.
pSG4739.
pSG4738 carries the 3' coding region of sigE up to 10 bp downstream of the stop codon. A 500 bp segment of the sigG gene from 11 bp downstream of the sigE stop codon was amplified by PCR [using primers Eco(IIIG) (5'-CGGAATTCTGGTTAGAACCCCTTGATTTTAC-3') and SigGSphIrev (5'-AAACATGCATGCGTAAGCGATGTCCCGG-3'] from SG38 chromosomal DNA and inserted into pSG4738 along with the aphA-3 cassette generated by BamHI/EcoRI digestion of vector pAM1.
pSG4740 and pSG4741.
The Bacillus thuringiensis promoter was amplified from B. thuringiensis chromosomal DNA by PCR using primers 5'-CGAATTCGTAGGCTGGTCTTATTC-3' and 5'-GGACTAGTTTCCCTCCTATCGGGAGTTGC-3' and digested with EcoRI and SpeI. The coding region of sigG was amplified by PCR of SG38 chromosomal DNA using primers 5'-GACTAGTGTCGAGAAATAAAGTCGAAATC-3' and 5'-AAACATGCATGCGTAAGCGATGTCCCGG-3' and digested with SpeI and SphI. The two PCR products and the aphA-3 fragment from EcoRI and BamHI digestion of pAM1 were ligated all at once into pSG4738 cut with SphI and BamHI, resulting in plasmid pSG4740. The PsigG-14 mutation was introduced into pSG4739 using the same method for mutagenesis of pSG4731 described above.
Each plasmid was sequenced, then B. subtilis SG38 was transformed with the resulting plasmids, giving strains 2806 (wild-type promoter), 2807 (PBt promoter) and 2808 (PsigG-14 promoter).
Expression of F in vegetative growth.
An IPTG-inducible copy of spoIIAC, encoding F, carried on plasmid pSG635 was integrated by single crossover into the spoIIA operon of strains 2803-PsigG, 2803-PsigG-14, 2803-PBco-sigG and 2803-PBt-sigG. The resulting strains (2821, 2829, 2822 and 2823 respectively) express spoIIAA and spoIIAB from their wild-type promoter whereas spoIIAC is controlled by the IPTG-inducible Pspac promoter. Cells were grown at 37 °C to OD600 0·25 in CH medium (Sterlini & Mandelstam, 1969
); at this point the culture was split into two and
F expression induced in one half by the addition of 1 mM IPTG. Samples (0·5 ml) were taken over 3 h, pelleted, frozen in liquid nitrogen and assayed for
-galactosidase activity as described above.
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RESULTS |
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It appeared that the mutant promoters were of similar overall strength to the wild-type. Nevertheless, to exclude the possibility that the mutant promoters were simply stronger than the wild-type an IPTG-inducible copy of F was introduced into strains 2803-PsigG and 2803-PsigG-14. Expression of
F was induced during vegetative growth and
-galactosidase activity from the sigG promoters was measured in the absence of any sporulation-specific regulation. As shown in Fig. 4(d)
, both promoters were expressed similarly, indicating that the increased promoter activity seen in the sigE background is not simply due to increased promoter strength.
sigG promoters from most other spore-forming species are not dependent on E in B. subtilis
As an alternative means of further analysing the sigG promoter and to examine how conserved this level of regulation is, we isolated and examined sigG promoters from other Bacillus species and from Clostridium acetobutylicum. By using forward and reverse primers specific to the 3' of sigE and the 5' of the sigG coding regions from B. subtilis respectively, the intergenic region was amplified by PCR from B. thuringiensis, B. licheniformis, B. coagulans and B. polymixa chromosomal DNA. The PCR products were sequenced, and new primers were designed to clone the promoters into pSG4731 in-frame with lacZ. The C. acetobutylicum sigEsigG intergenic region was amplified by PCR from plasmid pSE1 (Sauer et al., 1994; kindly provided by P. Dürre, University of Ulm) and inserted in place of the B. subtilis promoter between the EcoRI and HindIII sites of pSG4731. Each plasmid was then integrated into the amyE locus of strains 2803 and 2804.
Expression from the five foreign sigG promoters was measured during sporulation (Fig. 5). The timings of
-galactosidase production showed that all five promoters are indeed recognized by B. subtilis E
F, but only the B. licheniformis promoter (Fig. 5b
) was dependent on
E activity; for the other species, expression appeared to be independent of
E.
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The reduction in sporulation frequency in strain 2808 compared to strain 2806 could not be due to different expression levels from the two promoters, because the level of transcription from the PsigG-14 promoter was almost identical to that of the wild-type (Fig. 4d). Moreover, replacement of the wild-type promoter with either weak (PsigG-14) or strong (PBt)
E-independent promoters decreased the sporulation frequency similarly.
Surprisingly, the reduction in sporulation frequency was not as severe as that observed when F was transcribed prematurely (Arigoni et al., 1996
; Feucht et al., 1999
), suggesting multiple levels of control to ensure the timely synthesis and activation of
G. Therefore, we examined how the uncoupling of sigG transcription from
E activity affected the timing of
G activation.
G activity was measured in these strains by the introduction of the
G-dependent spoVAlacZ fusion. As shown in Fig. 8
, strain 2806, where insertion of KanR separated spoIIG from spoIIIG, showed similar
-galactosidase activity to SG38. However, with the PBt (strain 2807) and PsigG-14 promoter (strain 2808), expression of
-galactosidase was not earlier, as might be expected, but slightly delayed. A similar pattern was observed using an sspAlacZ fusion as an alternative
G-dependent reporter (data not shown). This suggests that when transcription from the sigG promoter is uncoupled from the activity of
E, the
G protein is held inactive until a later stage than normal.
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DISCUSSION |
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Preliminary data suggest that SpoVT (Bagyan et al., 1996), a transcriptional regulator of prespore-specific genes, is not required for
E-dependence of sigG transcription. In addition, attempts to identify the putative regulator by random mutagenesis of the B. subtilis chromosome have so far proved unsuccessful (L. Evans, unpublished).
Expressing G early (i.e. from a
E-independent promoter) led to a slight but reproducible decrease in sporulation efficiency, emphasizing the importance of co-ordinating the developmental programmes of the two cells. The reduction in sporulation frequency was not as drastic as that observed when
F was activated prematurely (Arigoni et al., 1996
; Feucht et al., 1999
), suggesting the existence of other levels of regulation for
G. Indeed, when the expression of two
G-dependent lacZ fusions was measured we found that when sigG was expressed from a
E-independent promoter, activation of
G was only slightly delayed (Fig. 8
). Stragier & Losick (1996)
reported a similar finding: premature expression of sigG from a strong
F-dependent promoter did not affect the timing of
G activity. Taken together, the results are in agreement with the notion that multiple levels of control act upon the synthesis and activation of
G (see Introduction).
Endospore formation by some Gram-positive bacteria belonging to the genera Bacillus and Clostridium seems to be a highly conserved process despite the triggers for the induction of sporulation being different. Comparison of the genomes of B. subtilis, B. anthracis, B. stearothermophilus, C. acetobutylicum and Clostridium difficile showed that not only the sporulation-specific -factors but also the so far known regulatory pathways leading to activation and coordination of their activity in the two compartments have been conserved (Stragier, 2002
). This is supported by the finding that sigE, sigG and sigK of C. acetobutylicum have been found to be expressed in the same order as in B. subtilis (Santangelo et al., 1998
). Also, the comparison of the promoters from several Bacillus species and C. acetobutylicum (Fig. 6
) shows that there is a high degree of conservation in the 10 and 35 regions; thus they are all recognized and transcribed by B. subtilis E
F (Fig. 5
). The finding of such a high degree of similarity raises the interesting question of whether the genes are subject to the same
E-dependent regulation. However, only the B. licheniformis promoter was expressed in a
E-dependent manner (Fig. 5b
). B. licheniformis is one of the species more closely related to B. subtilis, so it is possible that in more distant species promoter sequences may have diverged sufficiently for the B. subtilis regulatory protein no longer to recognize them. It is also possible that in these other species sigG expression is not dependent on
E, although this would seem less likely given the degree of conservation of the sporulation process across these species (Stragier, 2002
).
It is unclear why the E-independent promoters are expressed to such high levels compared with the B. subtilis promoter. The experiment where
F was induced in vegetative growth (Fig. 7
) suggests that the heterologous promoters are intrinsically stronger. However, they do not appear to be any closer to the
F consensus sequence than the
E-dependent ones are.
A major challenge now is to identify the proteins that consitute the signal transduction pathway that couples the activation of E in the mother cell with the transcription of sigG in the prespore.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 November 2003;
revised 16 February 2004;
accepted 5 April 2004.
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