Negative Regulation by the Bacillus subtilis GerE Protein*

Hiroshi Ichikawa, Richard HalbergDagger , and Lee Kroos§

From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

GerE is a transcription factor produced in the mother cell compartment of sporulating Bacillus subtilis. It is a critical regulator of cot genes encoding proteins that form the spore coat late in development. Most cot genes, and the gerE gene, are transcribed by sigma K RNA polymerase. Previously, it was shown that the GerE protein inhibits transcription in vitro of the sigK gene encoding sigma K. Here, we show that GerE binds near the sigK transcriptional start site, to act as a repressor. A sigK-lacZ fusion containing the GerE-binding site in the promoter region was expressed at a 2-fold lower level during sporulation of wild-type cells than gerE mutant cells. Likewise, the level of SigK protein (i.e. pro-sigma K and sigma K) was lower in sporulating wild-type cells than in a gerE mutant. These results demonstrate that sigma K-dependent transcription of gerE initiates a negative feedback loop in which GerE acts as a repressor to limit production of sigma K. In addition, GerE directly represses transcription of particular cot genes. We show that GerE binds to two sites that span the -35 region of the cotD promoter. A low level of GerE activated transcription of cotD by sigma K RNA polymerase in vitro, but a higher level of GerE repressed cotD transcription. The upstream GerE-binding site was required for activation but not for repression. These results suggest that a rising level of GerE in sporulating cells may first activate cotD transcription from the upstream site then repress transcription as the downstream site becomes occupied. Negative regulation by GerE, in addition to its positive effects on transcription, presumably ensures that sigma K and spore coat proteins are synthesized at optimal levels to produce a germination-competent spore.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Starvation induces the Gram-positive bacterium Bacillus subtilis to initiate a series of morphological changes that result in the formation of a dormant spore (1). Early in the sporulation process a septum forms that divides the cell into a larger mother cell compartment and a smaller forespore compartment. Each compartment contains a copy of the genome, and different genes are expressed in each compartment. Gene expression drives further morphogenesis, including migration of the septum to engulf the forespore in a double membrane, deposition of cell wall-like material called cortex between the membranes, and synthesis in the mother cell of proteins that assemble on the surface of the forespore to produce a tough shell known as the coat. The developmental process culminates with lysis of the mother cell to release a mature spore. When nutrients become available again, the spore germinates, producing a cell that resumes growth and division.

The program regulating transcription of sporulation genes is exceptionally well understood (2). It involves the synthesis and activation of four compartment-specific sigma  subunits of RNA polymerase (RNAP),1 each of which directs the enzyme to transcribe a particular set of genes. sigma F and sigma G control forespore-specific gene expression. In the mother cell, activation of sigma E is followed by the synthesis and activation of sigma K. In addition, two small, DNA-binding proteins, SpoIIID and GerE, activate or repress transcription of many mother cell-specific genes (3-6). The mother cell transcription factors form a hierarchical regulatory cascade in which the synthesis of each factor depends upon the activity of the prior factor, in the order sigma E, SpoIIID, sigma K, and finally GerE (7).

In addition to positive regulation between one transcription factor and the next in the mother cell cascade, there is evidence of negative regulation as well. sigma K RNAP initiates a feedback loop that inhibits transcription of the sigE gene encoding sigma E (8). Since sigma E RNAP transcribes the spoIIID gene (9-11), production of SpoIIID is also negatively regulated (8, 12). This facilitates the switch from the early sigma E- and SpoIIID-directed pattern of gene expression to the late sigma K- and GerE-directed pattern. GerE is not a component of this feedback loop (8); however, there was reason to believe that GerE might initiate a second feedback loop. GerE was shown previously to inhibit transcription in vitro of the sigK gene encoding sigma K (4). Whether GerE inhibits sigK transcription in vivo was in doubt, though, because a sigK-lacZ fusion was not overexpressed in gerE mutant cells (13). We have resolved this paradox by mapping a GerE-binding site in the sigK promoter region. This showed that the sigK-lacZ fusion examined previously did not contain the entire GerE-binding site. Here, we demonstrate that GerE represses sigK expression about 2-fold in vivo, and we discuss the implications of this negative feedback.

Overexpression of sigK is not the only defect in gerE mutant cells. Expression of some cot genes encoding spore coat proteins is reduced or absent, whereas expression of other genes is increased. The resulting spores have defective coats and germinate inefficiently (14). Previously, it was shown that purified GerE binds to DNA sequences matching the consensus RWWTRGGY-YY (where R is purine; W is A or T; and Y is pyrimidine) and activates transcription in vitro from the cotB and cotC promoters (4) and from three promoters in the cotVWXYZ cluster (3). The position of binding ranges in the different promoters from well upstream of the -35 region to partially overlapping it. Here, we show that GerE binds to the cotD promoter region at a position typical for activation of transcription, and to a position downstream, which may cause repression of transcription. Our mapping of GerE-binding sites in the cotD and sigK promoters provides the first information about how GerE acts as a transcriptional repressor.

    EXPERIMENTAL PROCEDURES

DNase I Footprinting-- DNA fragments labeled at only one end were prepared as follows. For analysis of the sigK promoter region, a HindIII-XbaI fragment of pBK16 (15) was labeled at the 3' ends by the Klenow enzyme fill-in reaction and [alpha -32P]dCTP or at the 5' ends by treatment with alkaline phosphatase followed by T4 polynucleotide kinase and [gamma -32P]ATP. In both cases, the labeled DNA was digested with PstI, which cleaved off a small fragment containing the labeled HindIII end, so it did not interfere with subsequent DNase I footprinting. The labeled XbaI end is 164 bp downstream of the sigK transcriptional start site (TSS). For analysis of the cotD promoter region, pLRK100 (15) was digested with EcoRI, which cleaves 227 bp upstream of the cotD TSS, and labeled either at the 3' end by the Klenow enzyme fill-in reaction and [alpha -32P]dATP or at the 5' end by treatment with alkaline phosphatase followed by T4 polynucleotide kinase and [gamma -32P]ATP. In both cases, the labeled DNA was digested with HindIII, which cleaves downstream of the cotD promoter, to produce a 443-bp fragment that was purified by elution from an 8% polyacrylamide gel (16). Labeled DNA fragments were incubated with different amounts of GerE gel purified from Escherichia coli engineered to overproduce the protein as described previously (3) and then mildly digested with DNase I according to method 2 as described previously (4), except 3 pmol of probe was used and a 7-fold (w/w) excess of poly(dI-dC) as compared with probe was added as competitor. After DNase I treatment, the partially digested DNAs were electrophoresed in a 7% polyacrylamide gel containing 8 M urea alongside a sequencing ladder generated by subjecting the appropriate end-labeled DNA to the chemical cleavage reactions of Maxam and Gilbert as described previously (16).

Construction of a sigK-lacZ Fusion-- DNA between -115 and +28 relative to the sigK TSS was amplified by the polymerase chain reaction (PCR) using pBK16 (15) as the template. The upstream primer was 5' GGGAATTCGATGAAGAATATTTTTAAC 3' and the downstream primer was 5' GCGAAGCTTCCACAAAAGTATGTA 3'. EcoRI and HindIII restriction sites (underlined) were designed into the 5' ends of the upstream and downstream primers, respectively, to allow directional subcloning of the PCR product into EcoRI-HindIII-digested pTKlac (17). The resulting plasmid pHI16 was linearized by digestion with BsaI and transformed into B. subtilis ZB307, in which marker replacement-type recombination created an SPbeta ::sigK-lacZ specialized transducing phage as described previously (18). A phage lysate was prepared by heat induction and used to transduce B. subtilis SG38 (spo+ trpC2) and 522.2 (gerE36 trpC2) (19) with selection for resistance to chloramphenicol (5 µg/ml) on LB agar as described previously (20).

Measurement of beta -Galactosidase Activity-- Sporulation was induced by nutrient exhaustion in DSM at 37 °C as described previously (20). Samples (1 ml) were collected at hourly intervals during sporulation, cells were pelleted, and pellets were stored at -70 °C prior to the assay. The specific activity of beta -galactosidase was determined by the method of Miller (21), using o-nitrophenol-beta -D-galactopyranoside as the substrate. One unit of enzyme hydrolyzes 1 µmol of substrate per min per unit of initial cell absorbance at 595 nm.

Western Blot Analysis-- Cells were induced to sporulate by nutrient exhaustion in DSM at 37 °C as described previously (20). Samples (1 ml) were collected at hourly intervals during sporulation, and whole-cell extracts were prepared as described previously (22). Protein concentrations were determined by the method of Bradford (23). Proteins (5 µg) were separated by sodium dodecyl sulfate (SDS)-14% Prosieve (FMC BioProducts) polyacrylamide gel electrophoresis with Tris/Tricine electrode buffer (0.1 M Tris, 0.1 M Tricine, 0.1% SDS) and electroblotted onto Immobilon-P membranes (Millipore). Blots were incubated as described previously with polyclonal anti-pro-sigma K antibodies that detect both pro-sigma K and sigma K (22). The secondary antibody was horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Bio-Rad). Chemiluminescence detection (ECL) was performed according to the manufacturer's instructions (Amersham Pharmacia Biotech). The signal intensities were quantified using a GS 505 Molecular Imager System (Bio-Rad).

In Vitro Transcription-- sigma K RNAP was partially purified from gerE mutant cells as described previously (15). The enzyme was comparable in protein composition and in cotD- and sigK-transcribing activities to fraction 24 shown in Fig. 2 of Kroos et al. (15). Transcription reactions (45 µl) were performed as described previously (24), except that RNAP was allowed to bind to the DNA template for 10 min at 37 °C before the addition of nucleotides (the labeled nucleotide was [alpha -32P]CTP). Heparin (6 µg) was added 2 min after the addition of nucleotides to prevent reinitiation. After the reactions were stopped, 20 µl of each reaction mixture was subjected to electrophoresis in a 5% polyacrylamide gel containing 8 M urea, and transcripts were detected by autoradiography. The signal intensities were quantified using a Storm 820 PhosphorImager (Molecular Dynamics). To prepare a DNA template containing the cotD promoter and lacking the upstream GerE-binding site, DNA between -44 and +227 relative to the TSS was amplified by the PCR using pLRK100 (15) as the template. The upstream primer was 5' CGGTTTGCATCAGAACATGT 3' (the underlined portion corresponds to the nontranscribed strand of the cotD promoter region), and the downstream primer was 5' GGAAGCTTGCATGCCTGCA 3' (the underlined portion corresponds to a HindIII site downstream of the cotD promoter in pLRK100).

    RESULTS

Location of the GerE-binding Site in the sigK Promoter Region-- Purified GerE was shown previously to strongly inhibit transcription in vitro of the sigK gene by sigma K RNAP (4). However, expression of a sigK-lacZ fusion in vivo was unaffected by a gerE mutation (13). The discrepancy between the two results could be explained if the sigK-lacZ fusion did not contain a binding site for GerE that mediates repression. To see if GerE binds specifically in the sigK promoter region and, if so, to determine the position of binding, we performed DNase I footprinting experiments.

Fig. 1 shows that GerE protected a stretch of DNA from DNase I digestion that included the TSS of sigK and extended downstream. The protection spanned positions -4 to +19 on the nontranscribed strand (Fig. 1A) and positions +1 to +20 on the transcribed strand (Fig. 1B). Complete protection from DNase I digestion was observed at the lowest concentration of GerE tested, indicating that GerE binds with relatively high affinity to this site as compared with other GerE-binding sites mapped previously (3, 4). Fig. 1C shows the sequence of the sigK promoter in the region protected by GerE. Within the protected region are two sequences in inverted orientation that overlap slightly and match the consensus sequence for GerE binding (Fig. 1D).


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Fig. 1.   GerE footprints in the sigK promoter region. Radioactive DNA fragments separately end-labeled on the nontranscribed (A) or transcribed (B) strand were incubated in separate reactions with a carrier protein (bovine serum albumin, 310 pmol) only (lane 5) or with 6 pmol (lane 1), 12 pmol (lane 2), 60 pmol (lane 3), or 120 pmol (lane 4) of gel-purified GerE in addition to the carrier protein and then subjected to DNase I footprinting in a total volume of 45 µl. Hatched boxes indicate the region protected from DNase I digestion by GerE. Arrowheads denote the boundaries of protection, and numbers to the left refer to positions relative to the TSS, as deduced from sequencing ladders generated by chemical cleavage of the respective end-labeled DNA at purines (lane G over A) or guanines (lane G). C, position of the GerE-binding site in the sigK promoter region. The nucleotide sequence of the nontranscribed strand of the sigK promoter region is shown (13). Overlining and underlining indicate regions on the nontranscribed and transcribed strands, respectively, protected by GerE from DNase I digestion. The TSS is shown by an arrow. D, nucleotide sequences within the GerE-protected region of the sigK promoter are aligned with the consensus sequence for GerE binding (3). Matches to the consensus sequence are shown as capital letters, and numbers refer to positions relative to the TSS. Note that the sequence shown for the binding site between +18 and +7 is from the strand opposite that shown in C.

We conclude that GerE binds to a site in the sigK promoter region that overlaps the TSS and extends downstream. Presumably, GerE binding to this site represses sigK transcription in vitro (4) by interfering with RNA polymerase binding and/or a subsequent step in initiation. The location of the GerE-binding site provides a plausible explanation for the lack of an effect of a gerE mutation on sigK-lacZ expression reported previously (13). The sigK portion of the fusion extended only to +4, so it did not contain the entire GerE-binding site.

GerE Inhibits sigK Expression in Vivo-- To determine whether GerE affects sigK expression in vivo, we constructed a new sigK-lacZ transcriptional fusion that includes the GerE-binding site. DNA between -115 and +28 relative to the sigK TSS was fused to lacZ, and the fusion was recombined into phage SPbeta . The resulting SPbeta ::sigK-lacZ phage was transduced into wild-type and gerE mutant B. subtilis, creating lysogens. Fig. 2 shows the average beta -galactosidase activity during sporulation of three isolates of each type. The average maximum activity was 2-fold higher in gerE mutant cells than in wild-type cells, demonstrating that GerE inhibits sigK expression in vivo.


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Fig. 2.   sigK-lacZ expression in wild-type and gerE mutant cells. sigK-directed beta -galactosidase activity was measured at the indicated times during sporulation of congenic wild-type (SG38, ) and gerE mutant (522.2, triangle ) strains. Points on the graph are averages for isolates of each type, and error bars show 1 S.D. of the data.

We also compared the level of sigK gene products in developing wild-type and gerE mutant cells. The primary translation product of sigK is pro-sigma K, an inactive precursor that is proteolytically processed to active sigma K (22). We used anti-pro-sigma K antibodies to detect both pro-sigma K and sigma K in extracts of cells subjected to Western blot analysis. Fig. 3 shows that the levels of pro-sigma K and sigma K are higher in gerE mutant cells than in wild-type cells late in sporulation. Quantitation of the combined pro-sigma K plus sigma K signal for the experiment shown in Fig. 3 and three additional experiments showed that the maximum level of SigK gene products in wild-type cells during sporulation, on average, reached 57% (±6%, 1 S.D.) of the level in gerE mutant cells. In all four experiments, the level of both pro-sigma K and sigma K was elevated in T7 and T8 samples from the gerE mutant compared with wild type. These results show that GerE normally inhibits the accumulation of pro-sigma K and sigma K during the late stages of sporulation. It is likely that GerE represses transcription of the sigK gene, reducing the synthesis of pro-sigma K and sigma K. This would explain the similar 2-fold decrease of sigK-directed beta -galactosidase activity (Fig. 2) and pro-sigma K plus sigma K (Fig. 3) in wild-type cells compared with gerE mutant cells. The alternative explanation that GerE in wild-type cells causes increased turnover of pro-sigma K and sigma K and a similar increase in turnover of beta -galactosidase is unlikely because beta -galactosidase activity from a sigK-lacZ fusion lacking the GerE-binding site identified in Fig. 1 is similar during sporulation of wild-type and gerE mutant cells (13), as is beta -galactosidase activity from lacZ fusions to many other genes.


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Fig. 3.   Levels of pro-sigma K and sigma K during sporulation of wild-type and gerE mutant cells. Whole-cell extracts were prepared from wild-type (SG38) and gerE mutant (522.2) cells collected at the indicated number of hours after the onset of sporulation in DSM. Proteins were fractionated by SDS-PAGE and subjected to Western blot analysis with anti-pro-sigma K antibodies, which detect both pro-sigma K and sigma K.

GerE Inhibits cotD Transcription in Vitro-- In addition to possible inhibitory effects of GerE on transcription of genes in the sigma K regulon (due to the inhibition of sigma K accumulation by GerE), GerE stimulates expression of certain genes in the sigma K regulon (3, 4, 7). Expression of a cotD-lacZ transcriptional fusion was 7-fold higher in developing wild-type cells than in gerE mutant cells (7). It was shown previously that purified GerE stimulates cotD transcription by sigma K RNAP 2-3-fold (4). We discovered that at a higher concentration GerE inhibits cotD transcription in vitro. Fig. 4 shows the result of an in vitro transcription experiment with a mixture of DNA templates containing the cotD or cotC promoter. The cotC promoter was included as an internal control because GerE has been shown to bind to a site centered at -68.5 relative to the TSS and to activate transcription by sigma K RNA polymerase (4). Lower levels of GerE stimulated cotD transcription about 2-fold (based on quantitation of signals in the experiment shown and one additional experiment), as reported previously (4), but higher concentrations of GerE inhibited cotD transcription. The inhibition was specific to cotD, since cotC transcription was activated at the highest concentration of GerE tested.


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Fig. 4.   Effect of GerE on cotD transcription in vitro. DNA templates (0.2 pmol each template) were transcribed with partially purified sigma K RNAP (0.2 µg) alone (lane 1) or with 50 (lane 2), 100 (lane 3), 200 (lane 4), or 400 pmol (lane 5) of gel-purified GerE added immediately after the sigma K RNAP. DNA templates were pLRK100 (15) linearized with HindIII (225-base cotD transcript) and a HaeIII-EcoRI restriction fragment from pHI1(4) (196-base cotC transcript). The positions of run-off transcripts of the expected sizes, as judged from the migration of end-labeled DNA fragments of MspI-digested pBR322, are indicated.

Location of the GerE-binding Site in the cotD Promoter Region-- To understand how GerE both positively and negatively affects cotD transcription, we tested whether GerE binds in the promoter region by performing DNase I footprinting experiments. Fig. 5 shows that GerE protected a stretch of DNA from DNase I digestion that included the -35 region of the cotD promoter and extended upstream. On the nontranscribed strand, the protection spanned positions -39 to -25 relative to the TSS (Fig. 5A), whereas on the transcribed strand positions -60 to -23 were protected. We could not be certain whether protection extends farther upstream on the nontranscribed strand due to the absence of DNase I cleavage in this region even in the absence of GerE. GerE appears to bind to the cotD promoter region with lower affinity than to the sigK promoter region, since a higher concentration of GerE was required to observe protection from DNase I digestion (compare Figs. 1 and 5). Fig. 5C shows the sequence of the cotD promoter in the region protected by GerE. Typically, GerE protects a stretch of about 20 bp from DNase I digestion (3, 4). The long region of protection (nearly 40 bp) observed on the transcribed strand in the cotD promoter region suggests that GerE binds to two sites. Within this region is a perfect match (positions -53 to -42) to the consensus sequence for GerE binding (Fig. 5D). A second match (7 out of 10) to the consensus sequence is present at positions -20 to -31, in inverted orientation with respect to the first match (Fig. 5D), which probably accounts for GerE binding in this region.


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Fig. 5.   GerE footprints in the cotD promoter region. Radioactive DNA fragments separately end-labeled on the nontranscribed (A) or transcribed (B) strand were incubated in separate reactions with a carrier protein (bovine serum albumin, 310 pmol) only (lane 1) or with 12 (lane 2), 25 (lane 3), 50 (lane 4), or 100 pmol (lane 5) of gel-purified GerE in addition to the carrier protein and then subjected to DNase I footprinting in a total volume of 45 µl. See Fig. 1 legend for explanation of boxes, arrowheads, and numbers. C, position of the GerE-binding site in the cotD promoter region. The nucleotide sequence of the nontranscribed strand of the cotD promoter region (7) is aligned with respect to conserved nucleotides found in the -35 regions of promoters transcribed by sigma K RNAP (34), shown above the sequence. Nucleotides in the cotD -35 region that match the consensus are shown as boldface capital letters. Overlining and underlining indicate regions on the nontranscribed and transcribed strands, respectively, protected by GerE from DNase I digestion. The dashed lines indicate regions of uncertain protection due to a lack of DNase I digestion in these regions. Numbers refer to positions relative to the TSS. M indicates A or C. D, nucleotide sequences within the GerE-protected region of the cotD promoter are aligned with the consensus sequence for GerE binding (3). Matches to the consensus sequence are shown as capital letters, and numbers refer to positions relative to the TSS. Note that the sequence shown for the binding site between -20 and -31 is from the strand opposite that shown in C.

The Upstream GerE-binding Site in the cotD Promoter Is Necessary for Activation of Transcription in Vitro but Not for Repression-- We hypothesized that GerE bound at the upstream site in the cotD promoter (i.e. recognizing the perfect match to the consensus centered at -47.5) activates transcription, because GerE binds at a similar position in the cotB (4) and cotVWX (3) promoters and activates transcription. GerE bound at the cotD promoter downstream site (centered at -25.5) might repress transcription, explaining the inhibition of cotD transcription we observed (Fig. 4). To test these ideas, we repeated the in vitro transcription experiments with a cotD DNA template lacking the upstream GerE-binding site. As shown in Fig. 6, GerE failed to activate transcription of this template. However, GerE still repressed transcription of this template, under conditions that permitted GerE to activate cotC transcription. We conclude that GerE binding to the upstream site in the cotD promoter is required to activate transcription. GerE binding to the downstream site probably represses transcription, although we cannot rule out the possibility that GerE binding farther downstream is also necessary and was not detected by DNase I footprinting.


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Fig. 6.   Effect of GerE on transcription in vitro of a cotD template lacking the upstream GerE-binding site. The amounts of DNA templates, sigma K RNAP, and GerE were the same as in the Fig. 4 legend. The cotD template was a 275-bp PCR fragment containing 44 bp upstream of the TSS (228-base transcript) prepared as described under "Experimental Procedures." The cotC template was described in the Fig. 4 legend. The positions of run-off transcripts of the expected sizes, as judged from the migration of end-labeled DNA fragments of MspI-digested pBR322, are indicated.


    DISCUSSION

Our results strongly support the model that GerE is a repressor of sigK transcription which lowers the level of pro-sigma K and sigma K in cells during the late stages of sporulation. The previous finding that GerE inhibits sigK transcription in vitro (4) can be explained by GerE binding near the TSS (Fig. 1) and acting as a repressor. The previous observation that expression of a sigK-lacZ fusion lacking the GerE-binding site in the promoter region is unaffected by a gerE mutation (13), together with our finding that expression of a sigK-lacZ fusion containing the GerE-binding site is 2-fold lower in wild-type cells than in gerE mutant cells (Fig. 2), demonstrates the importance of the GerE-binding site for sigK regulation in vivo. The lowering of sigK expression by GerE in wild-type cells appears to result in a comparable decrease in the level of SigK gene products (Fig. 3). The simplest interpretation of these results is that GerE represses sigK transcription, limiting the synthesis of pro-sigma K and sigma K during sporulation.

The negative effect of GerE on the sigma K level during sporulation would lower expression of genes for which sigma K is the limiting factor for transcription. Four genes in the sigma K regulon show a lower level of expression in wild-type cells than in gerE mutant cells. These are cotA (25), spoVF (26), csk22 (27), and cotM (28). In the case of cotA, there is evidence of a direct effect of GerE on transcription. Purified GerE inhibits cotA transcription in vitro (4). It is unknown how much of the increased cotA expression observed in a gerE mutant (25) results from loss of direct inhibition by GerE and how much results from an elevated level of sigma K. Also unknown is whether GerE represses spoVF, csk22, or cotM directly, or whether these genes are overexpressed in a gerE mutant because GerE fails to repress sigK. It should be possible to answer some of these questions and, more generally, to assess the importance of GerE repression of sigK during sporulation and germination, by deleting the GerE-binding site in the sigK promoter.

The cotD gene, like many other genes in the sigma K regulon, is positively regulated by GerE. For several genes, GerE has been shown to bind upstream of the promoter -35 region and stimulate transcription by sigma K RNAP in vitro (3, 4). GerE can increase cotD transcription in vitro as shown previously (4), but we discovered that a higher concentration of GerE causes repression (Fig. 4). DNase I footprinting revealed that GerE protects not only DNA upstream of the -35 region in the cotD promoter, but the protection extends downstream through the -35 region (Fig. 5). Binding that extends through the -35 region has not been observed in other promoters activated by GerE (3, 4). We reasoned that GerE binding in the cotD -35 region might repress transcription, and we showed that truncation of cotD promoter DNA at -44 prevented activation, but allowed repression, by GerE (Fig. 6). These in vitro results suggest the model that a rising level of GerE in sporulating cells first activates cotD transcription by binding upstream of the -35 region and then represses transcription by binding to a second site just downstream. It should be straightforward to test the requirement for the upstream GerE-binding site for activation in vivo, by making the appropriate 5' deletion (e.g. to -44) and measuring expression of a fusion to a reporter gene. However, testing the role of the downstream GerE-binding site in repression in vivo will be more difficult. As noted above, one must be careful to distinguish between direct repressive effects of GerE and indirect effects due to GerE repression of sigK.

Fig. 7 illustrates the regulatory interactions between the four mother cell-specific transcription factors (circled) and our model for regulation of cotD transcription at different times during sporulation. Early in sporulation, sigma E and SpoIIID are active in the mother cell (Fig. 7A). At this time, sigK is transcribed by sigma E RNAP, with SpoIIID serving as an essential activator (6, 13). As we have shown for GerE (Figs. 5 and 6), it was shown previously that SpoIIID binds in the -35 region of the cotD promoter and represses transcription in vitro (6). Fig. 7B shows the regulatory interactions in the mother cell slightly later, at about 4 h into the sporulation process, when the primary product of the sigK gene, pro-sigma K, is processed to active sigma K in response to a signal from the forespore (22, 29). sigma K RNAP initiates a negative feedback loop that inhibits transcription of the sigE gene encoding sigma E, which in turn lowers expression of spoIIID (8, 12). As the levels of sigma E RNAP and SpoIIID fall, cotD transcription would no longer be repressed by SpoIIID. At the same time, sigma K RNAP transcribes gerE (4, 30), and, according to our model, the GerE produced initially activates cotD transcription and represses sigK transcription. For a period, all four mother cell-specific transcription factors probably affect transcription of sigK, because SpoIIID activates both sigma E RNAP and sigma K RNAP to transcribe sigK (6, 13), and GerE represses sigK transcription (4) (Figs. 1-3). The positive autoregulatory loop created by sigma K RNAP transcription of sigK is kept in check by two negative feedback loops (Fig. 7B). One inhibits transcription of sigE and therefore inhibits production of sigma E and SpoIIID (8). The other leads to synthesis of GerE, which represses sigK transcription directly (4) (Figs. 1-3). As the level of GerE rises later in sporulation, GerE may also repress cotD (Fig. 7C). Presumably, the complex regulatory interactions depicted in Fig. 7 ensure that the four transcription factors, as well as structural proteins like CotD under their control, are made at optimal levels for formation of the spore cortex and coat.


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Fig. 7.   Regulatory interactions between mother cell-specific transcription factors and a model for regulation of cotD transcription at different times during sporulation. Dashed arrows show gene (italicized) to product (proteins are circled) relationships. Solid arrows represent positive regulation of transcription. Lines with a barred end represent negative regulation of transcription. A, B, and C represent early, intermediate, and late stages of sporulation, respectively, as explained in the text.

Our mapping of GerE-binding sites in the sigK and cotD promoters and our in vitro transcription experiments with cotD promoter fragments differing at the 5' end provide the first insight into how GerE acts as a repressor. In both the sigK and cotD promoters, GerE binds within the region likely to be bound by sigma K RNAP, although the footprints of sigma K RNAP on these promoters have not yet been determined. The positions of GerE binding in these promoters suggest that GerE may repress transcription by interfering with the initial binding of sigma K RNAP or a subsequent step in transcription initiation. It may be possible to distinguish between these possibilities by measuring the ability of sigma K RNAP to bind to these promoters in the presence or absence of GerE. In the case of cotD, it is unlikely that GerE inhibits elongation of transcripts or inhibits transcription by any other mechanism that does not depend on sequence-specific DNA binding, because GerE stimulated cotC transcription in the same reaction mixtures in which it inhibited cotD transcription (Figs. 4 and 6). These same in vitro transcription experiments, with cotD promoter fragments differing at the 5' ends, suggest that at a low GerE concentration, binding to a site upstream of the -35 region activates transcription, and at a higher GerE concentration, binding to a second site just downstream represses transcription. In the DNase I footprint experiment shown in Fig. 5, there is no indication that GerE binds with higher affinity to the upstream site (a perfect match to the GerE binding consensus sequence) than the downstream site (a 7 out of 10 match to the consensus). Although we cannot rule out a slight difference in affinities, another possible explanation is that sigma K RNAP competes with GerE for binding to the downstream site but not the upstream site.

GerE appeared to bind with higher affinity to the sigK promoter region than to the cotD promoter region (compare Figs. 1 and 5) or many other GerE-binding sites mapped previously (3, 4). Within the region of the sigK promoter protected by GerE from DNase I digestion are two sequences in inverted orientation that overlap by 4 bases and match the consensus sequence for GerE binding perfectly or in 7 out of 10 positions. An identical arrangement of sequences matching the GerE consensus was observed previously just upstream of the -35 region in the cotYZ promoter, where GerE appeared to bind with high affinity and activated transcription (3). It is unknown whether more than one molecule of GerE at a time binds the sigK and cotYZ sites. Also unknown is whether GerE is monomeric in solution. GerE is predicted to possess a helix-turn-helix DNA-binding motif similar to that in several proteins whose three-dimensional structure has been determined (31, 32). Some of these well characterized proteins are dimeric and recognize inverted repeats in the DNA-binding sites (33). The consensus GerE-binding sequence is not palindromic, so perhaps GerE can bind as a monomer. This does not exclude the possibility of interaction between monomers at sites that exhibit palindromic character, such as the sigK and cotYZ sites.

GerE appears to be similar to SpoIIID in terms of its DNA sequence recognition characteristics. The SpoIIID-binding site consensus sequence (WWRRACAR-Y) is of similar length and degeneracy as that recognized by GerE (RWWTRGGY-YY) and also is not palindromic (5, 6). Although SpoIIID appears to be monomeric in solution,2 many strong SpoIIID-binding sites exhibit a second good match to the consensus in inverted orientation relative to the best match (6). Mutational analysis will be required to assess the contribution of each consensus match to binding of GerE and SpoIIID at sites with palindromic character and to determine whether two monomers interact (e.g. bind cooperatively) at these sites.

In summary, we have investigated the biochemical basis for negative regulation by the GerE protein. It appears to act like a classical repressor, binding in promoter regions at sites that overlap the position of RNAP binding. GerE repression at the sigK promoter lowers sigma K production during sporulation about 2-fold, potentially regulating the expression of many genes in the sigma K regulon. In addition, GerE binds in the promoter regions of certain genes in the sigma K regulon and directly represses or activates transcription by sigma K RNAP. In the case of cotD, it is likely that GerE binds to a site upstream of the promoter -35 region and first activates transcription, then, as its level rises in sporulating cells, GerE binds to a site slightly farther downstream and represses transcription.

    ACKNOWLEDGEMENTS

We thank B. Kunkel, R. Losick, P. Zuber, and C. Moran for providing bacterial strains, phages, and plasmids. We thank S. Lu for providing anti-pro-sigma K antibodies and B. Zhang for helpful discussions. We are grateful to J. Kaguni for comments on the manuscript.

    FOOTNOTES

* This research was supported by Grant GM43585 from the National Institutes of Health and by the Michigan Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706.

§ To whom correspondence should be addressed. Tel.: 517-355-9726; Fax: 517-353-9334; E-mail: kroos{at}pilot.msu.edu.

2 B. Zhang and L. Kroos, unpublished work.

    ABBREVIATIONS

The abbreviations used are: RNAP, RNA polymerase; TSS, transcriptional start site; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; bp, base pair.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
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