1 Philipps-University Marburg, Department of Biology, Laboratory for Microbiology, D-35032 Marburg, Germany
2 Max-Planck-Institute for Terrestrial Microbiology, D-35043 Marburg, Germany
3 Ernst-Moritz-Arndt-University, Medical School, Laboratory for Functional Genomics, Walther-Rathenau-Str. 49A, D-17487 Greifswald, Germany
4 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Apartado 127, 2781-901 Oeiras Codex, Portugal
Correspondence
Uwe Völker
voelker{at}uni-greifswald.de
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ABSTRACT |
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The descriptions of all regulon members as well as complete sets of raw and normalized data are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.
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INTRODUCTION |
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Activation of F occurs in the prespore immediately after the polar division of the sporulating cell.
F controls gene expression during the early stages of prespore development, and directs transcription of the gene encoding
G, which replaces it during later post-engulfment stages of prespore development. Conversely,
E drives transcription of early mother-cell genes, including the structural gene for
K, which replaces
E following engulfment of the prespore by the mother cell. Activation of
F is coupled to the formation of the polar septum, and
F activity is required for the mother-cell-specific activation of
E (Errington, 2003
; Hilbert & Piggot, 2004
; Stragier & Losick, 1996
). The transcriptional activity of
E is then required for the activation of
G in the prespore, which is somehow coupled to the completion of the engulfment process (Partridge & Errington, 1993
; Sun et al., 2000
). Lastly,
G triggers a signalling pathway that activates
K in the mother cell (Losick & Pero, 1981
; Piggot & Losick, 2002
). These cellcell signalling mechanisms ensure that the forespore and mother-cell-specific programmes of gene expression are kept in pace and in register with the course of morphogenesis and are essential to ensure that the differentiation process takes place with high fidelity (Errington, 2003
; Hilbert & Piggot, 2004
; Stragier & Losick, 1996
).
Sporulation involves the expression of a large number of genes. Many loci have been identified following chemical mutagenesis of a sporulation-proficient strain, based on the property that on sporulation plates colonies of a wild-type (Spo+) but not those of many asporogenous (Spo) or oligosporogenous mutants produce a dark-brown pigment (Piggot & Coote, 1976). Sporulation loci have also been identified by transposon mutagenesis (Sandman et al., 1987
) or by the use of integrational plasmids for gene disruption, by reverse genetics, as encoding components of the spore or some of its structures (e.g. Donovan et al., 1987
; Kuwana et al., 2002
; Lai et al., 2003
), or by expression-based screens designed to find members of specific sporulation regulons (e.g. Beall et al., 1993
). Prior to sequencing of the B. subtilis genome, about 100 genes were listed as being involved in sporulation, and about 60 of those were known to be dependent on the activity of one of the four compartment-specific sigma factors (Stragier & Losick, 1996
). The availability of the B. subtilis genome sequence (Kunst et al., 1997
) has made possible the use of DNA arrays to study the profile of gene expression during sporulation at a genome-wide level. Three recent studies have employed DNA arrays and expression profiling to study sporulation. Fawcett et al. (2000)
have characterized the transcription profile of the early to middle stages of sporulation induced by nutrient exhaustion in Difco sporulation medium (DSM). These authors have compared transcripts present during growth, at the onset of sporulation, and 2 h after the initiation of sporulation of a wild-type strain, with transcripts present in mutants for spo0A and sigF. The transcription of 66 genes was found to be dependent on both Spo0A and
F, including several genes known to be under the control of
F or
E. The use of hidden Markov models trained to find known promoter elements allowed the assignment of 11 new genes to the
F regulon, and the assignment of 22 to control by
E (Fawcett et al., 2000
). Two studies have provided information on the composition of the
E regulon when sporulation is induced by growth and resuspension in a poorer synthetic medium. Eichenberger et al. (2003)
have reported transcriptional profiling and bioinformatics data to support their assignment of 253 genes to the
E regulon, including 181 new genes. Disruption of 12 of the newly identified genes produced a sporulation phenotype (Eichenberger et al., 2003
). Feucht et al. (2003)
have found a total of 171
E-dependent transcripts, 101 of which were previously unknown, and of these, mutations in about 10 diminished the efficiency of sporulation.
In the present study, we wanted to extend expression-profiling studies to the late-prespore and mother-cell-specific regulons (under G and
K control, respectively), and simultaneously provide an overall picture of the changes in the pattern of gene expression, over time, when sporulation is induced by growth followed by resuspension in a defined medium. We made use of DNA macroarrays to identify genes governed by
F,
E,
G and
K. Most of the previously characterized sporulation genes were found and assigned to the correct regulon. We report on the identification of a total of 439 sporulation genes, including 185 new genes. Our results strongly support the view that the different sporulation regulons are largely differentiated both temporally and spatially, with little overlap between consecutive prespore- or mother-cell-specific regulons (Li & Piggot, 2001
). The results also provide insight into the specific contributions of the prespore and mother-cell types to spore development and spore properties.
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METHODS |
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Cell lysis, RNA isolation and dot-blot/Northern analysis.
RNA was isolated according to the acid phenol method described by Völker et al. (1994). Aliquots of the total RNA prepared for the DNA macroarray experiments were used for the dot-blot and Northern analysis of the expression profiles of the spoIIR, spoIID, spoIIID, sspE and gerE genes. Digoxigenin- (DIG) labelled anti-sense RNA probes were generated by in vitro transcription using a StripEZ-kit (Ambion) and gene-specific PCR products as templates. The following PCR products were generated for the production of antisense RNA probes: a 544 bp spoIIR fragment using primers spoIIR-for (5'-CTGGCAAACAGCGATAGTG-3') and spoIIR-rev (5'-TAATACGACTCACTATAGGGAGGTCGGAAATCCATTCG-3'), an 891 bp spoIID fragment using primers spoIID-for (5'-CACTATCCGTACTATGTGC-3') and spoIID-rev (5'-TAATACGACTCACTATAGGGAGGCCAAATCCTCTCGTC-3'), a 307 bp spoIIID fragment using primers spoIIID-for (5'-GTGGTGTGCACGATTACATC-3') and spoIIID-rev (5'-TAATACGACTCACTATAGGGAGGCGATTGCTGAACAGGCTC-3'), a 335 bp sspE fragment using primers sspE-for (5'-GAGAAAGCTTTACGATCACCTGCACATTC-3') and sspE-rev (5'-TAATACGACTCACTATAGGGAGGAGTGATTAGCTGTTTTGTTG-3') and a 186 bp gerE fragment using primers gerE-for (5'-TCGAAGCCGTCGCTAACG-3') and gerE-rev (5'-TAATACGACTCACTATAGGGAGGCTCTAGCTCACCCATTC-3'). Because, in each of the PCR reactions with chromosomal DNA from strain JH642, the reverse (rev) primers carried the sequence of the T7 promoter, the PCR fragments could be used for in vitro RNA synthesis with T7 RNA polymerase (Ambion). This yielded hybridization probes internal to the genes. Denaturing RNA electrophoresis on agarose gels, RNA transfer by diffusion onto a nylon membrane (NY13N; Schleicher & Schuell), hybridization to gene-specific probes and signal detection were performed as described by Scharf et al. (1998)
.
Preparation of labelled cDNA, array hybridization and DNA macroarray regeneration.
Prior to the cDNA labelling, the overall integrity of the total RNA preparation was verified by Northern-blot analysis with digoxigenin-labelled probes directed against known members of the four compartment-specific sporulation regulons. The DNA macroarray analyses employed commercially available Panorama B. subtilis DNA macroarrays from Sigma Genosys which carry duplicate spots of PCR products representing 4107 B. subtilis genes, as well as the corresponding commercial primer mix (Sigma Genosys), which consists of 4107 specific oligonucleotide primers complementary to the 3' ends of all mRNA-encoding B. subtilis genes. cDNA synthesis, probe hybridization and washing of the filters were performed as described by Steil et al. (2003). Arrays were exposed to storage phosphor screens (Molecular Dynamics) for two to four days, and subsequently scanned with a Storm 840/860 phosphorimager (Molecular Dynamics) at a resolution of 50 µm and a colour depth of 16 bit. Bound cDNA was stripped off the DNA-macroarray membranes by three washing cycles involving a short (1 min) washing step with 250 ml boiling buffer (5 mM sodium phosphate, pH 7·5, 0·1 % SDS) and an incubation in 250 ml fresh buffer at 95 °C for 20 min.
Data analysis.
Data analysis followed a three-step procedure. First, the ArrayVision software Version 6.1 (Imaging Research) was used for the quantification of the hybridization signals after direct import of the phosphorimager files. The analysis yielded the artifact-removed volumes (ARVol) and background values, calculated from the median of a line surrounding each group of eight spots on the array. These data were then used in a second step in Microsoft Excel to calculate, for every spot on the array, a quality score that reflected the ratio between the signal intensity and the background intensity (further details available at http://www.medizin.uni-greifswald.de/funkgenom/supplemental_material). This quality score was utilized to identify hybridization signals close to the detection limit, thereby avoiding artificially high induction ratios for those genes. Data normalization and data analysis were done in a third step with GeneSpring (Version 5.02) (Silicon Genetics). Gene expression for a particular comparison of conditions was considered to be changed when three criteria were fulfilled: i) expression of the gene had to exceed the background signal level by a threshold determined as described (further details available at http://www.medizin.uni-greifswald.de/funkgenom/supplemental_material); ii) changes in expression of the gene had to be statistically significant, as defined in a statistical group comparison of the values of the selected conditions with a parametric test (ANOVA) and a Benjamini and Hochberg False Discovery Rate correction with a P value cut-off of 0·05, as defined in the GeneSpring software package; iii) the change in expression had to exceed a factor of three. Calculations of ratios were done with means of the parallel spots on the filters.
Web access.
The complete dataset for all growth conditions investigated is available online (http://www.medizin.uni-greifswald.de/funkgenom/supplemental_material).
Construction of gfp transcriptional fusions inserted into the amyE locus.
The gfp gene was amplified using primers gfpD (5'-CCCAAGCTTGGGGGATCCGGGAAAAGGTGGTGA-3') and gfpR (5'-GGCGAATTCTTATTTGTATAGTTCATCCATGC-3'), and plasmid pEA18 (a gift from Alan Grossman) as the template. The 744 bp PCR fragment was digested with HindIII and EcoRI and ligated to pMLK83 (Karow & Piggot, 1995) which had been digested with the same enzymes, yielding pMS157. To create transcriptional fusions of the yuiC, yhaX, yhcV and yxeE promoter regions to gfp, the following PCR products were first generated: a 468 bp fragment encompassing the yuiC promoter using primers yuiC-53D (5'-CATGCTGCTCGAGAATGTCTTGGATTATGGC-3') and yuiC-521R (5'-GTTCCTGGATCCCATTTTGACAAGTCCTTCGC-3'); a 516 bp fragment containing the yhaX promoter using primers yhaX-67D (5'-GGAAAACTCGAGATAATAACATTGAAAGCGCC-3') and yhaX-583R (5'-GGCATCAAGCTTTAGCGATTTCGC-3'); a 485 bp yhcV fragment with primers yhcV-30D (5'-AAATAACTCGAGTTATTACCAAGGAAC-3') and yhcV-515R (5'-CAACGGGGATCCGCCCCGACGTTATGC-3'); and a 409 bp fragment carrying the yxeE promoter using primers yxeE-42D (5'-GACCCTCGAGTGCTTTGGGAAATCACC-3') and yxeE-451R (5'-GTAAGGATCCTGCTGAGGCAGCTGAGGGC-3'). The PCR fragments carrying the yuiC, yhcV and yxeE promoter regions were digested with XhoI and BamHI and ligated to SalI- and BamHI-digested pMS157, to produce pMS174, pMS173 and pMS172, respectively (Table 1
). The fragment encompassing the yhaX promoter region was digested with XhoI and HindIII and ligated to pMS157 which had been digested with SalI and HindIII, yielding pMS175 (Table 1
). Samples of ScaI-digested pM172, pM173, pM174 and pM175 were used to transform the parental strain MB24, as well as a panel of congenic strains mutant for
F,
E,
G and
K, selecting for kanamycin resistance. AmyE transformants, the result of a double cross-over at the amyE locus, were kept for further analysis (see Table 1
).
Light microscopy and image processing.
Samples (0·5 ml) of cells in resuspension medium were collected throughout sporulation, and resuspended in the same volume of PBS supplemented with 10 µg ml1 4',6'-diamidino-2-phenylindole (DAPI). Microscope slides were prepared as described previously (Serrano et al., 2004). Images were acquired using a cooled charge couple device (Cooke) on a multi-wavelength wide-field three-dimensional microscopy system (63x/1·4 OIL Plan Apochromat objective, Zeiss 100M, Intelligent Imaging Innovation). Standard filters for fluorescein isothiocyanate (for green fluorescence protein, GFP) and DAPI were used.
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RESULTS AND DISCUSSION |
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Prior to the transcriptional profiling experiments, bona fide members of each regulon were selected, and their temporal expression was analysed by RNA dot-blot experiments in the B. subtilis wild-type strain JH642, in mutants lacking one of the four sporulation-specific sigma factors or the regulatory protein BofA, and in strains allowing artificial expression of active forms of the sigma factors in vegetative cells. This allowed us to determine the time points for the best discrimination among the four regulons (Fig. 1).
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Differentiation of the compartment-specific sporulation regulons
For their inclusion in the early prespore- or mother-cell-specific F or
E regulons, genes had to fulfil the threefold induction criterion at both 90 and 150 min, compared to 30 min, after initiation of sporulation in the wild-type (Figs 1a and 2a
), as well as for the comparison of the expression of the wild-type and the sigF mutant 90 min after initiation of sporulation (Figs 1a and 2b
). We were able to discriminate between the
F and
E regulons on the basis of their expression pattern in the sigE mutant. While genes assigned to the
E regulon displayed at least threefold higher expression at 90 min after initiation of sporulation in the wild-type compared to the sigE mutant, members of the
F regulon did not show this induction (Figs 1a and 2b
) but were instead required to display at least threefold higher expression at 90 min in the sigE mutant compared to the sigF mutant (Fig. 1a
).
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In addition to recording the temporal expression in the wild-type strain and the effect of individual sigma-factor knock-outs on the expression profile, we also tested induction of genes following expression of the four sporulation-specific sigma factors during exponential growth. This series of experiments employed strains in which expression of the sigma-factor-encoding genes was governed by the IPTG-inducible promoter PSPAC. Activation of the mother-cell-specific sigma factors E and
K during sporulation requires their proteolytic processing from inactive pre-proteins, and thus this study made use of specific sigE and sigK alleles that are active without processing (Oke & Losick, 1993
; Stragier et al., 1988
). Before our stringent selection criteria were applied to the data derived from the artificial induction of the sigma factors, we utilized the group of sporulation genes previously characterized by biochemical and genetic approaches as test set. This analysis yielded quite different reassignment rates for the four different sporulation regulons. Whereas 76 % and 75 % of the 21
F- and 57
K-dependent genes discovered by non-chip approaches displayed at least threefold higher expression upon induction of the respective sigma factor allele during growth, only 20 % and 43 % of the 59
E-dependent and 46
G-dependent genes were reassigned using this procedure. Furthermore, the induction ratios observed after artificial induction during growth were in general much lower than those observed in sporulating cells. These lower induction ratios might be a reflection either of the leakiness of the PSPAC promoter or of the only partial activity of the sigma factor alleles used. In the case of sigE, the limited activity of a sigE copy from which pro-amino acid sequences have been removed has been observed before (Eichenberger et al., 2003
). Failure to induce the whole set of sporulation genes during growth is not unexpected because many sporulation genes might require other regulatory inputs that are not provided during growth. As a consequence of this analysis of a well-defined screening set of known sporulation genes, we decided to utilize the data of the artificial induction experiments merely as supportive information (column PSPACIPTG/co in Supplementary Tables S1S4, available online as supplementary data with the online version of this paper at http://mic.sgmjournals.org), but not as discriminative information, in order to avoid large numbers of false-negative candidates.
The fact that we were able to find conditions that permitted the separation of most of the genes in consecutive regulons expressed in different compartments of the sporulating cell, as for F and
E (Fig. 2b
),
E and
G (Figs 1a, b and 2
) or
G and
K (Fig. 2d
), or in the same compartment, as for
F and
G in the prespore (Fig. 2a
), or
E and
K in the mother cell (Fig. 2c
), supports the conclusion of an earlier study which indicated that sporulation-specific gene expression is largely compartmentalized, both temporally and spatially (Li & Piggot, 2001
). Nevertheless, as illustrated by the group of genes lying almost at the diagonals of the comparisons depicted in Fig. 2a, c
, some genes displayed an ambiguous behaviour. This group of genes could result from limitations of our experimental analysis, or could reflect a biological property of the system, for example, that the expression of some genes is governed by more than one
factor (see also below).
Temporal differentiation within the F and
G regulons
In addition to the key role of the four compartment-specific sigma factors in establishing the overall pattern of temporal and compartment-specific gene expression during sporulation, gene expression within each regulon can also be classified in several epistatic classes, in part because of the influence of ancillary transcription factors that may function as repressors or activators (Errington, 2003; Hilbert & Piggot, 2004
). For example, some
F-dependent genes are expressed soon after asymmetric division of the sporangial cell and the concomitant prespore-specific activation of
F (Karow et al., 1995
; Londono-Vallejo & Stragier, 1995
; Londono-Vallejo et al., 1997
; Wu & Errington, 2003
), while expression of the dacF or spoIIIG genes, for example, appears delayed relative to the first wave of
F-directed genes (Karow et al., 1995
; Partridge & Errington, 1993
; Schuch & Piggot, 1994
; see below). Therefore, we wanted to test whether or not, based on our data, we could discriminate between groups of genes with a common expression profile within each of the main regulons. Based upon their temporal pattern of expression, we were able to differentiate the 55 genes of the
F regulon into two classes (Fig. 3a
and Supplementary Table S1). Class 1 comprises 36 genes whose expression peaked at around 90 or 150 min and decreased thereafter, suggesting that their transcription is switched off. Class 2 included 19 genes whose main period of expression was centred 270 min after the onset of sporulation. The spoIIR, spoIIQ, lonB and rsfA genes were assigned to class 1, in agreement with experimental data (Karow et al., 1995
; Londono-Vallejo & Stragier, 1995
; Londono-Vallejo et al., 1997
; Serrano et al., 2001
; Wu & Errington, 2000
), whereas dacF, gpr, spIVB, sspN and tlp (Cabrera-Hernandez et al., 1999
; Schuch & Piggot, 1994
; Sussman & Setlow, 1991
) were assigned to class 2 (Supplementary Table S1).
F and
G have overlapping promoter specificities, and therefore some
F-dependent genes are only recognized by
F, whereas other genes are recognized by both
F and
G (Amaya et al., 2001
; Haldenwang, 1995
; Helmann & Moran, 2002
). The class of late
F-dependent genes, which presumably coincides with class 2 as defined here (Fig. 3
and Supplementary Table S1), appears to include genes under the dual control of
F and
G, such as gpr and dacF (Schuch & Piggot, 1994
; Sussman & Setlow, 1991
). Most of the
F-dependent genes that deviate significantly from the y axis towards the diagonal in Fig. 2a
, which depicts the separation of the
F and
G regulons, also cluster in class 2, which has a temporal definition, and in that sense defines a partial overlap between the
F and
G regulons.
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It should be noted that the mechanism by which expression of certain F genes is delayed relative to the first class of
F-dependent genes is not clear (Errington, 2003
; Hilbert & Piggot, 2004
). Expression of the late
F-governed gene spoIIIG appears to require the activity of
E in the mother cell (Partridge & Errington, 1993
). Presumably,
E activity could lead to the activation of a prespore-specific factor required for spoIIIG transcription, or otherwise cause the inactivation or removal of a repressor. However, this putative signalling pathway has not been examined in detail, and it is not known whether other class 2 genes are also
E-dependent. Recently, the
F-dependent gene rsfA has been shown to be involved in the control of prespore-specific
F-dependent gene expression (Wu & Errington, 2000
). Disruption of rsfA had different effects on class 1 genes: it caused increased expression of spoIIR, but had no effect on the expression of spoIIQ or of rsfA itself (Wu & Errington, 2000
). Evidently, rsfA does not appear to be the main regulatory factor in the temporal differentiation of the
F regulon, or in specifying
F only or dual
F/
G control.
As for F, the 113 genes assigned to the
G regulon could be divided into two classes. Class 1 groups 73 genes whose expression is increased at 150 min and tends to decrease at later time points, whereas class 2 includes 40 genes whose expression is induced by 210 min (Fig. 3c
). Class 1 includes genes such as gerAA, gerAB and spoVT, previously characterized as
G dependent (Bagyan et al., 1996
; Feavers et al., 1990
), as well as the spoIIIG gene (Sun et al., 1991
). That spoIIIG was found in the
G and not in the
F regulon (Supplementary Tables S1 and S3) is consistent with the report that most spoIIIG transcription stems from an auto-catalytic loop in which, following its activation,
G recognizes the promoter for its own gene (Sun et al., 1991
). This positive feedback regulatory scheme is thought to result in a rapid increase in the cellular level of
G triggered by its activation following completion of engulfment. Note however that some
F-dependent transcription of spoIIIG does occur (Sun et al., 1991
), albeit not to sufficient levels to pass our stringent criteria for its inclusion in the
F regulon (see above). Class 2 includes, for example, sspA (Mason et al., 1988
), most of the genes of the spoVA operon (Mouldover et al., 1994
), and sspF, previously shown to be transcribed about one hour later than other genes in the
G regulon (Panzer et al., 1989
). There are two known transcriptional regulators within the
G regulon, SpoVT and SplA (Bagyan et al., 1996
; Fajardo-Cavazos & Nicholson, 2000
). The role of the TRAP-like SplA protein may be limited to the modulation of the level of expression of the gene encoding the SplB spore photoproduct lyase (Fajardo-Cavazos & Nicholson, 2000
), whereas the spoVT gene has been shown to encode an AbrB-like transcription factor with a more global role in the control of
G-controlled gene expression (Bagyan et al., 1996
). Expression of the spoVT gene itself, as well as that of spoIIIG and the gerA operon, is normally repressed in a spoVT-dependent manner, whereas expression of the spoVA operon and of sspA requires spoVT (Bagyan et al., 1996
). Prolonged expression of several genes, including spoIIIG and gerA, was observed in cells of a spoVT mutant, suggesting that SpoVT may normally shut off transcription of these genes. These observations are in agreement with the overall expression profile of the genes grouped in class 1. Presumably, class 1 mostly includes genes whose expression is not critically dependent on SpoVT or that are repressed by SpoVT, whereas class 2 includes genes whose expression depends to various extents on SpoVT.
Temporal differentiation within the E and
K regulons
A similar analysis of the 154 E-dependent genes allowed the differentiation of three distinct temporal classes (Fig. 3b
and Supplementary Table S2). Class 1 includes 41 genes whose expression is rapidly induced and peaks at 90 min, to decrease thereafter. As for the first class of
F-dependent genes, this suggests that transcription of this group of genes is switched off (see above). In contrast, the 93 genes in class 2 show a slower rate of induction, and prolonged expression, which peaks between 150 and 210 min. A third class includes 20 genes which are maximally induced around 210 min, and whose expression persists at later times in development. spoIID (Rong et al., 1986
) and the SpoIIID-repressed spoIIIA operon (Illing & Errington, 1991
) are both found in class 1. Expression of the spoIIIA operon occurs transiently, prior to the accumulation of SpoIIID to high cellular levels (Illing & Errington, 1991
), which fits well with the overall pattern of class 1 genes, and suggests that other genes in this class may be subjected to repression. In contrast, the spoIIID gene itself and spoIVCB (encoding the N-terminal half of
K), both of which are known to be SpoIIID dependent (Kunkel et al., 1989
; Sato et al., 1994
; Stevens & Errington, 1990
), are found in class 2. Presumably, efficient expression of the class 2 genes requires the accumulation of SpoIIID above a certain threshold level, as was suggested for spoIIID and spoIVCB (Kunkel et al., 1989
; Sato et al., 1994
; Stevens & Errington, 1990
). Class 3 includes the coat morphogenetic gene cotE, which is expressed from two tandem
E-dependent promoters, one of which (P2) is additionally dependent on SpoIIID (Zheng & Losick, 1990
), spoVJ, known to be expressed from tandem
E- and
K-dependent promoters (Foulger & Errington, 1991
), and at least one gene, csk22, previously reported to be under
K control (Henriques et al., 1997
). The distinction between classes 1 and 2 may be attributable mostly to the effects of the regulatory protein SpoIIID upon
E-dependent gene expression (Zheng & Losick, 1990
). SpoIIID-independent genes are expressed early, and SpoIIID-dependent genes are expressed later, while the expression of some of the early genes is repressed or switched off (Halberg & Kroos, 1994
; Illing & Errington, 1991
; Kroos et al., 1989
; Kunkel et al., 1989
). However, class 3 may represent an overlap between the
E and
K regulons, either because genes in this class have multiple promoters utilized by
E or
K, or because atypical
E-type promoters can also be recognized by
K (Helmann & Moran, 2002
). In any case, class 3 represents a partial overlap between the mother-cell-specific
E and
K regulons.
Additional regulators may contribute to the fine-tuning of gene expression within the E regulon. For example, Wu & Errington (2000)
reported that the
E-dependent gene ylbO, which other studies also placed in the
E regulon (Eichenberger et al., 2003
; Feucht et al., 2003
; Supplementary Table S2), encodes a putative transcriptional regulator, highly similar to RsfA. YlbO may work together with
E in the mother cell, in much the same way that RsfA regulates
F-dependent gene expression in the prespore (Wu & Errington, 2000
). Unfortunately, the effects of a ylbO mutation on mother-cell-specific gene expression have not yet been examined. Moreover, in addition to SpoIIID and, hypothetically, to YlbO, the expression of some
E-dependent genes may be influenced by other factors. Several known or putative transcriptional regulators have been assigned to the
E regulon: purR, encoding the repressor of the purine operons; birA, a biotin acetyl-CoA-carboxylase synthetase and transcriptional regulator (Eichenberger et al., 2003
); yhgD (TetR/AcrR family) and ytzE (DeoR family) (Feucht et al., 2003
). Note, however, that none of these four genes could be assigned to the
E regulon under our experimental conditions (Fig. 4b
and Supplementary Table S2). In yet another example, the expression of the mmg operon, which encodes proteins with similarity to fatty-acid-metabolizing enzymes, is under
E control, but is subjected to catabolite repression in a ccpA-dependent manner (Bryan et al., 1996
). It is not known whether CcpA regulates other mother-cell-specific genes.
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As noted above for the E regulon, the expression of the
K regulon may be subject to additional levels of control. Recently, the yjcC gene (renamed spoVIF; Supplementary Table S4) was found to be required for the formation of heat- and lysozyme-resistant spores, and it has been suggested that it could play a role in modulating the expression of other
K-governed genes (Kuwana et al., 2003
). Moreover, our analysis identifies a transcriptional regulator of the MarR family (ysmB), upstream of the gene (racE) encoding a glutamate racemase, both in class 2 (Fig. 4d
and Supplementary Table S4). It is not known whether expression of the racE gene can be modulated in response to the availability of glutamate or some other factor via YsmB. However, these observations suggest that, even at a late stage in spore morphogenesis, the mother-cell-specific line of gene expression is responsive to environmental stimuli.
An overview of the four compartment-specific regulons
The F regulon.
Several regulatory functions can be attributed to F-directed gene expression. First, transcription of the spoIIR gene is required to signal
E activation in the mother cell (Karow et al., 1995
; Londono-Vallejo & Stragier, 1995
), and transcription of bofC and spoIVB is required for proper signalling of
K activation (Cutting et al., 1991b
; Gomez & Cutting, 1996
).
F also drives expression of at least two genes involved in the control of its own activity, rsfA and lonB (Serrano et al., 2001
; Wu & Errington, 2000
), of the spoIIIG gene (Sun et al., 1989
, 1991
) and of the spoIIQ gene, which is required for efficient expression of spoIIIG (Londono-Vallejo et al., 1997
; Sun et al., 2000
). In addition to its regulatory role, spoIIQ is also involved in the engulfment process, although only under certain nutritional conditions (Sun et al., 2000
). With the exception of the spoIIIG gene, which did not pass our selection criteria (see above), all these regulatory genes were found in the present study (Supplementary Table S1 and Fig. 4a
). Several genes in the
F regulon have functions in spore protection or spore germination: the katX-encoded catalase, for example, is implicated in spore protection against hydrogen peroxide (Bagyan et al., 1998
); the mutTA gene encodes an antimutator 8-oxo-dGTPase (Ramirez et al., 2004
); the sspN and tlp genes, which code for small acid-soluble spore proteins (SASP), act by shielding the prespore chromosome (Cabrera-Hernandez et al., 1999
); gerD is required for efficient germination in response to L-alanine and to a mixture of glucose, fructose, L-asparagine and KCl (Kemp et al., 1991
); and gpr encodes a protease involved in SASP protein degradation during spore germination (Sussman & Setlow, 1991
). Interestingly, our analysis identified the gene (yyaC) for a second possible GPR-like protease in the
F regulon (Fig. 4a
and Supplementary Table S1), suggesting a scenario of partial redundancy. Another function of the
F regulon may be to contribute to the morphogenesis of the spore protective layers. For example, the dacF gene encodes a penicillin-binding protein (PBP), with D-alanyl-D-alanine carboxypeptidase activity, which is involved in regulating the degree of cross-linking of the spore peptidoglycan (Popham et al., 1999
). No obvious alteration in spore peptidoglycan structure was found for a dacF single insertional mutant (Wu et al., 1992
), but our study identifies a second PBP-encoding gene (yrrR, in an operon with a gene of unknown function, yrrS) in the
F regulon (Fig. 4a
and Supplementary Table S1). It will be interesting to analyse a yrrR mutant, as well as a strain doubly mutant for dacF and yrrR. The finding of ripX, which encodes a site-specific integrase/recombinase involved in proper chromosome partitioning (Sciochetti et al., 1999
), and yqhH, predicted to code for an SNF2-type helicase (Supplementary Table S1), suggests that their products may prepare the spore for the resumption of growth following germination and outgrowth. Several genes encode proteins that were found in the proteomics study of Kuwana et al. (2002)
to be under
F control and to encode spore components, including yfhE and yfhD, yhcM, ytfI and ytfJ (Supplementary Table S1). Detailed functional studies have not yet been reported for any of these genes. With 55 genes, the
F regulon is the smallest of the four compartment-specific sporulation regulons; 14 genes were found in the region of the chromosome that is trapped in the prespore compartment following asymmetric division (Fig. 4a
) (Wu & Errington, 1998
). Positional information has been shown to have an important regulatory function in the context of the
F regulon. First, spoIIR is required to signal the prompt activation of
E in the mother cell (Khvorova et al., 2000
; Zupancic et al., 2001
). Second, the absence of lonB from the prespore of a mutant deficient in the function of the SpoIIIE translocase which transfers the remaining 70 % of the chromosome into the prespore leads to increased activity of
F (Serrano et al., 2001
). A previous study has found 66 genes expressed during the initial stages of sporulation that required both Spo0A and
F, but the group could also include
E-dependent genes (Fawcett et al., 2000
). Therefore, this is the first report on the definition of the
F regulon. Our results strengthen the view that
F plays an important regulatory role in the synthesis and activation of the late prespore regulator
G and in signalling the activation of the mother-cell-specific factors
E and
K (Hilbert & Piggot, 2004
), and that most of the morphogenesis and spore protection is the function of these other regulators.
The E regulon.
E plays a decisive role in spore morphogenesis. The spoIID, spoIIM and spoIIP genes are essential for the engulfment process, and also act to prevent a second polar division of the sporulating cell (Abanes-De Mello et al., 2002
; Eichenberger et al., 2001
; Frandsen & Stragier, 1995
; Lopez-Diaz et al., 1986
; Pogliano et al., 1999
; Rong et al., 1986
; Smith et al., 1993
; Smith & Youngman, 1993
). The initial stages in assembly of the spore coat require expression of the spoIVA, cotE, spoVID and safA genes, whose products are morphogenetic proteins which guide the assembly of the several coat structural components (Beall et al., 1993
; Driks et al., 1994
; Ozin et al., 2000
; Takamatsu et al., 1999
; Zheng et al., 1988
), and several genes which encode spore coat components with no obvious morphogenetic functions are also under the control of
E (e.g. Henriques et al., 1995
); spoVE, spoVD, dacB, spoVR, spmAB and murF (Supplementary Table S2), among others, are required for synthesis or modification of the spore cortex peptidoglycan (Beall & Moran, 1994
; Daniel et al., 1994
; Henriques et al., 1992
; Popham et al., 1995
). A poorly defined function of the
E regulon may be to maintain appropriate metabolic conditions in the mother cell in order to sustain proper spore morphogenesis. This role has been inferred through the identification and analysis of
E-dependent metabolic operons, such as mmg and exu (see also above). Our results, as well as those of Eichenberger et al. (2003)
and Feucht et al. (2003)
, strengthen this view, through the finding of a large number of genes which could influence metabolism in the mother cell. These include, for example, the genes for acyl-CoA dehydrogenase (acdA), butyrate-acetoacetate CoA transferase (yodR) and 3-oxoadipate CoA transferase (yodS), long-chain acyl-CoA synthetase and AMP-binding enzymes (yng operon), malate dehydrogenase (yjmC), and a sugar dehydrogenase (ywqF), among others (Supplementary Table S2). Also noteworthy is the presence in the
E regulon of several genes for putative transporters, including components of ABC-type transporters (e.g. the gln operon for glutamine transport and the possible gene for a di-tripeptide ABC transporter, yclF) and ywcA (encoding a possible Na+-dependent symporter). This suggests that extensive biochemical transactions with the surrounding medium take place during the period in which
E is active. Also, the finding of opuD (glycine betaine transporter) suggests that the cell may require active protection against osmotic stress, and the inclusion of cypA (encoding a cytochrome p450-type enzyme), sodF (encoding a superoxide dismutase) and ydjP (encoding a putative chloroperoxidase), together with the previous identification of the CotJC Mn2+-dependent catalase (Henriques et al., 1995
), suggests that these genes may play a role in detoxification and protection against oxidative stress (Supplementary Table S2).
In addition to its function in driving morphogenesis and influencing metabolism, the E regulon has important regulatory functions. Mutations in several
E-governed genes that block the engulfment process at various stages also prevent the activation of
G in the prespore, emphasizing the link between morphogenesis and gene expression (Errington, 2003
; Hilbert & Piggot, 2004
; Stragier & Losick, 1996
). However, the completion of engulfment is not sufficient for the activation of
G, which also requires expression of the spoIIIA octacistronic operon in the mother cell (Illing & Errington, 1991
; Kellner et al., 1996
; Piggot & Coote, 1976
). Hence, signalling of
G activation following the completion of engulfment is an important function of
E. Moreover,
E also drives synthesis of the SpoIIID regulatory protein, which modulates gene expression within the
E regulon, and our analysis indicates that several other transcriptional regulators are predicted in this regulon (see above; see also Supplementary Table S2). In addition to conducting the sporulating cell to the completion of engulfment,
E also drives synthesis of pro-
K, the precursor for the late mother-cell-specific regulator
K, and also drives synthesis of the regulatory proteins that from the mother-cell side control
K activation following completion of the engulfment process (reviewed by Errington, 2003
; Hilbert & Piggot, 2004
).
Because of the stringent criteria imposed by the multiple approach, the E regulon, as defined here (154 genes), is smaller than that reported by Eichenberger et al. (2003)
(253 genes) or Feucht et al. (2003)
(171 genes), but is in any case larger than any of the other compartment-specific regulons (Figs 2 and 4b
and Supplementary Tables 25). The size and diversity of the
E regulon emphasizes its central role in spore morphogenesis. In any case, the three studies agree well, with a common core of 93
E-dependent genes. The differences found highlight the impact of precise experimental conditions, including the type of arrays used, in the definition of a specific regulon.
The G regulon.
Several functions can be assigned to the G regulon. Production of active
G is one. The evidence suggests that the main period of spoIIIG transcription follows the activation of
G after the completion of engulfment (Errington, 2003
; Hilbert & Piggot, 2004
; see above).
G then drives expression of the spoVA operon, required for the uptake of dipicolinic acid from the mother cell into the prespore (Tovar-Rojo et al., 2002
); spoVT, a modulator of
G-dependent gene expression (Bagyan et al., 1996
); bofC and spoIVB, required for signalling pro-
K activation in the mother cell (Cutting et al., 1991a
; Gomez & Cutting, 1996
, 1997
; Wakeley et al., 2000
); the gerA, gerB and gerK operons, required for efficient spore germination (Corfe et al., 1994
; Feavers et al., 1990
; this work, Supplementary Table S3); pdaA, involved in the formation of muramic
-lactam in the spore cortex peptidoglycan, and indirectly in efficient spore germination (Fukushima et al., 2002
); and sleB, encoding a cortex lytic enzyme activated during germination (Boland et al., 2000
). An important function of the
G regulon lies in spore protection. This is evidenced by the
G-dependent transcription of the splB gene, encoding a spore photoproduct lyase (Fajardo-Cavazos & Nicholson, 2000
; Pedraza-Reyes et al., 1997
), and yqfS, encoding a type IV apurinic/apyrimidinic endonuclease (Urtiz-Estrada et al., 2003
). The SASP proteins, some of which bind to the DNA and participate in resistance to heat, UV radiation and desiccation, are abundant spore proteins (Setlow, 1995
). The SASP proteins also serve as a source of amino acids during spore germination and resumption of growth (Setlow, 1995
). With the exception of sspG, which is under
K control (Bagyan et al., 1998
), all other ssp genes (A to P and Tlp, encoded by the second cistron of the sspN operon) are controlled by
G (Cabrera-Hernandez et al., 1999
; Cabrera-Hernandez & Setlow, 2000
; Mason & Setlow, 1987
). Our study reinforces the functions that have been assigned to the
G regulon. The ywjD, ykoU and dnaN genes, for example, code for a putative UV-nuclease, an ATP-dependent DNA ligase and the
subunit of DNA polymerase III, respectively, all of which could contribute to DNA protection and repair. Also, the yndDEF and yfkQR operons code for proteins with similarity to those encoded by the gerA, gerB and gerK operons and the spoVAF cistron (Tovar-Rojo et al., 2002
), suggesting roles in spore germination (Fig. 4c
and Supplementary Table S3). Interestingly, our results suggest that, apart from bofC and spoIVB, the
G regulon may contribute in yet another way to the signalling pathway which leads to
K activation in the mother cell. The ctpB gene was recently characterized as a member of the
E regulon, which encodes a protease with a role in the correct timing of
K processing (Pan et al., 2003
). Our analysis places the ctpB gene in the
E regulon, but also unequivocally in the
G regulon (Supplementary Tables 3 and 4), a result that we confirmed by primer extension analysis (to be published elsewhere). In fact, ctpB is the only single gene whose expression was found to occur in the two compartments of the sporulating cell at consecutive stages of spore development. It will be interesting to determine whether or not the involvement of ctpB in the
K checkpoint requires its expression in the mother cell, in the prespore, or in both cellular compartments. Our analysis also suggests a new function for the
G regulon. We found that the yraGDydhDEF cluster encodes proteins with similarity to the N- (yraG and yraD) and C-terminus (yraE and yraF) of spore coat protein CotF (Cutting et al., 1991c
) (A. J. Ozin and others, unpublished results). This suggests that
G may be involved in the synthesis of some spore coat proteins, which in that case would have to cross the two prespore membranes to be incorporated into the spore coat structure (Henriques & Moran, 2000
). The suggestion that
G is involved in spore coat assembly is supported by the finding that a spoVT mutant produces an abnormally thick coat, in particular near the spore poles, that protrudes into the mother cell cytoplasm (Bagyan et al., 1996
). One possibility is that expression of the yraGDydhDEF cluster is required for spore coat assembly and is negatively regulated by SpoVT, an idea that is currently under test.
The K regulon.
Activation of K results from the activity of
G, and hence occurs following the completion of engulfment.
K directs the expression of at least one gene required for synthesis of the spore cortex (Piggot & Coote, 1976
), of most of the genes encoding coat structural components (Driks, 1999
; Henriques & Moran, 2000
), genes required for spore germination (Moir et al., 2002
; Setlow, 2003
), and mother-cell lysis (e.g. Nugroho et al., 1999
).
The decisive role of K in assembly of the spore coat is evidenced by the large number of genes encoding coat structural components found in this regulon (reviewed by Driks, 1999
; Henriques & Moran, 2000
). Several coat proteins are cysteine-rich, for example, those encoded by the cotVWXYZ cluster (Zhang et al., 1993
), and there is ample evidence for extensive disulphide bond formation within the coat (Driks, 1999
; Henriques & Moran, 2000
). Disulphide bonds are normally rare, and virtually absent from intracellular proteins. It is not known whether the mother-cell cytosol becomes oxidative at a late stage, allowing for extensive disulphide formation within the coat, or if cross-linking takes place following spore release into the surrounding medium. We found that genes encoding several enzymes of the cysteine biosynthesis pathway are induced under
K control. For example, the putative yrhAB operon encodes a presumptive cysteine synthase (yrhA) and a cystathionine gamma-synthase (yrhB), and the yubC gene encodes a putative cysteine dioxygenase (Fig. 4d
and Supplementary Table S4). Analysis of these genes will likely provide clues to the mechanisms by which disulphide bonds are incorporated into the spore coat lattice. The gene (tgl) for a spore-specific transglutaminase which appears to associate with the spore coat and which is presumed to promote formation of
-(
-glutamyl)lysine cross-links in coat proteins (Kobayashi et al., 1996
, 1998
; Ragkousi & Setlow, 2004
) is also found in the
K regulon (Supplementary Table S4). This confirms early expectations, based solely on inspection of the tgl promoter region, which also suggested its requirement for GerE (Kobayashi et al., 1998
). However, since tgl was found among class 2 genes (Fig. 3
and Supplementary Table S4), its requirement for GerE is questionable. In any event, synthesis of the enzyme takes place concomitantly with production of most of the coat structural components, suggesting that the Tgl-mediated post-translational modification of the coat components is a late event in spore morphogenesis.
Another notable feature of the K regulon is the proportion of genes that appear to encode enzymes involved in polysaccharide biosynthesis and/or glycosylation. In addition to the previously identified
K-controlled genes of the spsAK, cgeAB and cgeCDE operons whose expression affects the spore surface properties (Roels & Losick, 1995
; Stragier & Losick, 1996
), yfnE encodes a putative glycosyl transferase, and yodU and ypqP code for products with similarity to enzymes involved in capsular polysaccharide biosynthesis (Supplementary Table S4). Moreover, several other genes appear to be involved in sugar mobilization, often organized in operons (e.g. those in the putative ytlABC or ytcABC operons) (Supplementary Table S4). These observations are intriguing, because previous work has failed to reveal extensive glycosylation of the spore coat proteins (reviewed by Driks, 1999
; Henriques & Moran, 2000
). It may be that B. subtilis has the genetic potential for extensive glycosylation of the spore surface, but that this phenotype is not expressed under laboratory conditions or is under regulation by as yet unknown signals.
Our analysis also sheds some light on an intriguing aspect of sporulation. It has long been known that sporulating cultures of B. subtilis accumulate sulpholactic acid (reviewed by Piggot & Coote, 1976). We found that putative genes for sulpholactic acid synthesis are probably part of two putative operons induced under
K control. One (yitBAyisZ) encodes a putative sulphate adenylyltransferase (yitA), a phospho-adenylylsulphate sulphotransferase (yitB) and an adenylylsulphate kinase (yisZ); the other (yitCD) encodes a putative phosphosulpholactate phosphatase (yitC) and a presumptive phosphosulpholactate synthase (yitD) (Fig. 4d
and Supplementary Table S4). It is not known whether synthesis of sulpholactic acid is important for sporulation or not, and, if it is important, why it takes place late in the mother-cell compartment of the sporulating cell. However, some Bacillus species do not appear to accumulate sulpholactic acid (Piggot & Coote, 1976
). A detailed functional analysis of these two operons may provide insight into the biological relevance of the production of sulpholactic acid by sporulating B. subtilis.
Validation of the results.
In this work, we analysed data from several time points during sporulation, in combination with mutants for the four compartment-specific sigma factors (including a spoIIIG bofA double mutant to better separate the late regulons). We also incorporated into our analysis data generated upon expression of active forms of the four sporulation-specific sigma factors in vegetative cells. The combination of these approaches forced us to use stringent criteria for the assignment of genes to the various regulons, thereby decreasing the occurrence of false positives. We also base this conviction on several lines of evidence. First, the fact that we were able to find most of the characterized sporulation genes in the correct regulon, and that we in no case assigned a known gene only to the incorrect regulon (see above). Note however that, with the exception of the ctpB gene (see above), certain genes previously assigned to a regulon were confirmed, but additionally assigned to a different regulon of the same cellular compartment. Examples are the spsJ, spsK and csk22 genes previously included in the K regulon (Henriques et al., 1997
), which we have also found to be expressed under the control of
E (Supplementary Table S2). However, the spsJ and spsK genes, as well as the related spsC, spsG and spsI genes, were also found to be under
E control in the study of Eichenberger et al. (2003)
. Second, we constructed fusions of the putative promoters of genes newly assigned to each of the regulons of the gfp gene and examined the time and compartment of expression, as well as their dependency on the putative cognate sigma factor. The promoters tested were randomly selected among genes which were not predicted to be embedded in operons, but for which no other information was available. Fig. 5
shows the results of this analysis. The yuiC gene was included in the
F regulon (Supplementary Table S1), and a fusion of its promoter to the gfp gene showed prespore-specific expression in a wild-type strain at the time when the activity of
F reaches a peak (Fig. 5a
), but not in cells of a
F mutant (Fig. 5b
). A fusion of the yhaX promoter to gfp was expressed at 150 min in the mother cell compartment of a wild-type (Fig. 5a
) but not in a
E mutant (Fig. 5b
), supporting its inclusion in the
E regulon (Supplementary Table S2). The yhcV promoter, predicted to be utilized by
G (Supplementary Table S3), was expressed later (210 min) in the prespore, and in a
G-dependent manner (Fig. 5a, b
), and transcription from the yxeE promoter in the mother cell occurred late and depended on
K (Fig. 5
), as predicted by our analysis (Supplementary Table S4). Lastly, we note that, since this study began, expression data, or in some cases detailed studies, of several genes of each regulon have been reported, and in all cases there was no discrepancy between our assignment and those studies [with the partial exception of the
E-controlled yvjB (ctpB) gene, as noted; see above]. These genes are marked in Supplementary Tables S1S4 and references are given.
|
On the other hand, expression of genes performing functions in the synthesis of biotin, histidine, phenylalanine and serine, as well as expression of A-dependent chemotaxis and motility genes (Aizawa et al., 2002
), was severely reduced immediately after the initiation of sporulation. With respect to repression of gene expression, it has to be considered that sporulating cells do not divide, and thus, provided that they are stable, components present during nutrient shift-down are not diluted by growth and can still perform their function.
Summarizing this part, there is evidence for the continued activity of A during sporulation, but the actual expression level of particular genes is subject to additional regulation by other regulatory loops.
Concluding remarks
Our approach to look at sporulation-specific gene expression at different times and to attempt to define the four compartment-specific sigma regulons using a combination of criteria clearly differentiates our work from other studies, namely those aimed at the definition of the E regulon (Eichenberger et al., 2003
; Feucht et al., 2003
). Our analysis provides a dynamic view of the pattern of utilization of genome information during the early and late stages of sporulation in both the prespore and mother cell chambers of the developing cell. Direct comparison with the results obtained by other groups for the
E regulon clearly suggests that we have here applied more selective criteria for gene identification, and that we may have missed several genes in the various regulons. However, we have minimized the chances of incorrect assignment. A significant proportion of genes in each regulon have no known function (60 % of
F, 55 % of
E, 62 % of
G, 65 % of
K), but the data also suggest that, at least for some regulons (or cellular compartments), there appears to be redundancy at the level of certain functions. For example, no obvious phenotype was found for mutants deficient in the production of the
E-governed CotJC Mn2+-type catalase (Henriques et al., 1995
), but at least one other putative Mn2+-dependent catalase (encoded by yjqC) was found in the
K regulon (Supplementary Table S4). The present list of sporulation-specific genes will also assist proteomics-based studies aimed at the identification of spore proteins, proteins associated with certain spore structures (Kuwana et al., 2002
; Lai et al., 2003
), or proteins that may be present as complexes at specific stages in morphogenesis. It will serve as a basis for studies of sporulation-specific gene expression under different nutritional conditions or in undomesticated strains of B. subtilis (Duc et al., 2004
). It will also allow comparisons of the changes in gene expression that take place during sporulation in other species, including an analysis of the relative contributions of the various sporulation-specific regulatory factors involved, or the role of vegetative factor
A in sporulation (Lai et al., 2003
).
The results also suggest that transcription of at least one group of genes in the two early F- or
E-dependent regulons is switched off prior to activation of the next sigma factor that operates in the same cellular compartment (Figs 2 and 3
) (Li & Piggot, 2001
). It could be that the switching off of earlier classes of gene expression responds to a morphological cue during the morphogenetic process, in much the same way as activation of the four compartment-specific sigma factors is coupled to the completion of key intermediate structures during sporulation (Errington, 2003
; Hilbert & Piggot, 2004
; Li & Piggot, 2001
). In any case, our analysis reinforces the view that sporulation gene expression is largely compartmentalized, both spatially and temporally.
![]() |
ACKNOWLEDGEMENTS |
---|
While this work was in revision, Eichenberger et al. (2004) published a comprehensive analysis of the mother-cell-specific programme of gene transcription during sporulation. Their analysis was not restricted to the role of the two mother-cell-specific sigma factors
E and
K, but also defined the contributions of the DNA-binding proteins GerE, GerR and SpoIIID. These authors assigned a total of 383 genes to the mother-cell-specific gene-expression programme, and they provide evidence for a hierarchical regulatory cascade of the five regulatory proteins mentioned above. In an extension of their previous work (Eichenberger et al., 2003
), these authors now analysed the impact of SpoIIID and GerR on the expression of the 272 members of the
E regulon. Eichenberger et al. (2004)
also provide detailed analyses of the
K-controlled genes and the modulation of their expression by GerE. Of the 144 genes assigned to the
K-regulon by Eichenberger et al. (2004)
, 89 were also recognized as
K-dependent in this study.
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Received 19 July 2004;
revised 4 October 2004;
accepted 7 October 2004.
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