INTRODUCTION

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The Spo0A protein of Bacillus subtilis is a member of the phosphorylation-activated response regulator family of two-component signal transduction proteins (3, 16, 35, 42) and is required in a signal transduction pathway that controls the initiation of sporulation in response to nutrient limitation (10, 47). Spo0A functions as both a repressor and activator of gene transcription during the transition from exponential growth to the stationary phase (43) and during the early stages of sporulation (39, 45, 47). Phosphorylated Spo0A represses the transcription of a key transition state regulator, AbrB, by binding to the promoter of the abrB gene (43, 45). In addition, phosphorylated Spo0A is required for the activation of transcription of key sporulation-specific genes, which include spoIIA (47), spoIIE (53), and spoIIG (6, 7). Spo0A has been shown to bind to specific sequences in the DNA upstream of the promoters it positively regulates and downstream of the promoters it negatively regulates (41). The Spo0A recognition sequence in DNA is referred to as the 0A box and consists of the 7-bp sequence 5'-TGTCGAA-3' (6, 43).

The response regulator family of two-component signal transduction proteins is characterized by a conserved amino-terminal phosphoacceptor domain and unique carboxyl-terminal effector domains (3, 35, 42) (see Fig. 1A). Response regulators that function as transcription factors, i.e., OmpR (38), UhpA (28), and others (3, 35, 42), generally consist of two domains: the phosphoacceptor domain and an effector domain containing DNA-binding and transcriptional activation functions. The phosphoacceptor domain functions as a receiver of a signal from a protein that senses the environment and is phosphorylated to activate the effector functions. The Spo0A protein consists of this two-domain structure, with DNA-binding and transcriptional activation functions residing in the carboxyl-terminal domain (18) (see Fig. 1A). Recent studies (9, 24) have identified residues of the carboxyl-terminal effector domain involved in transcriptional activation at Spo0A-dependent, ^A-dependent promoters. However, the residues of Spo0A involved in DNA binding have yet to be identified.

Brown et al. (8) have identified a region of the Spo0A effector domain that is a strong candidate to contain a DNA-binding function. An alignment of the amino acid sequences of multiple Spo0A homologs from diverse Bacillus and Clostridium species revealed three regions of high conservation in the effector domain of the Spo0A protein. One region consists of 15 amino acids perfectly conserved among four diverse species and contains features in common with a helix-turn-helix (HTH) DNA-binding domain. Analysis by the Dodd and Egan weight matrix (12) scored this region as positive for a putative HTH DNA-binding domain. This scoring matrix utilizes a reference set of proteins that includes 91 proteins with known HTH or suspected HTH DNA-binding domains for comparison (12). Therefore, the high conservation seen in the Spo0A homologs from bacteria that had been diverged for over a billion years and the predicted fit we saw by the Dodd and Egan analysis (8) argue strongly that this conserved region is of functional significance and possibly contains an HTH DNA-binding domain.

To test the functional significance of this putative HTH DNA-binding motif of Spo0A and to further define the structure and function of the effector domain of Spo0A, we used site-directed mutagenesis to make Gly and/or Ala changes in residues of this conserved stretch of Spo0A. We then analyzed the effects of the substitutions on sporulation, sporulation-specific transcriptional activation and repression, and DNA binding.

	MATERIALS AND METHODS

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Bacterial strains, culture media, genetic techniques, and in vitro manipulation of DNA. Bacterial strains used in this work are listed in Table 1. Oligonucleotides used in this work are listed in Table 2. Routine microbiological procedures and enzymatic manipulations of DNA were carried out using standard methods (5, 23). The concentrations of antibiotics used for selection on Luria-Bertani (LB) or Difco sporulation medium (DSM) agar and in culture were 5 µg/ml for chloramphenicol, 3 µg/ml for neomycin, 100 µg/ml for spectinomycin, and 100 µg/ml for ampicillin.

Site-directed mutagenesis and mutant strain construction. Site-specific mutations in spo0A were made using the Unique Site Elimination mutagenesis kit (Pharmacia) based on the method of Deng and Nickoloff (11). Mutagenesis and selection were carried out according to the manufacturer's instructions. The desired base substitutions were made directly in a pKK223-3-0A clone using mutagenic primers S210G/A, E213G/A, and E214G/A and the selection primer STOM (Table 2). Each mutagenic primer was designed with degeneracy to allow the introduction of mutations that would result in both the glycine and alanine substitutions desired. The presence of the desired mutation was confirmed by sequencing the spo0A allele from positive clones using the fmol sequencing kit (Promega). The mutant spo0A alleles were then subcloned into the pSPC101 integrational vector (24) on a BglII-HindIII fragment containing a portion of the spo0A gene and the relevant mutations. The mutant alleles were subsequently introduced into B. subtilis JKH72 by transformation with selection for spectinomycin resistance. Spectinomycin-resistant transformants resulted from a single reciprocal recombination event between mutant spo0A sequences and the chromosomal spo0A locus generating a single intact copy of the spo0A gene. The presence of the mutation was verified by PCR amplification and sequencing of the intact copy of the spo0A gene from these strains.

Sporulation frequency assay. B. subtilis strains were grown with shaking at 37°C in DSM containing the appropriate antibiotics until 12 h after entry into stationary phase. The number of viable cells was then determined by dilution and plating onto LB agar containing the appropriate antibiotics. The number of chloroform-resistant spores was determined by a 10-min incubation of 0.45 ml of culture with 50 µl of chloroform at room temperature (RT) and dilution and plating. The sporulation frequency was defined as the number of chloroform-resistant spores compared to the total number of viable cells before chloroform treatment. The sporulation frequency for each mutant was calculated as the average of at least three independent assays.

-Galactosidase and -glucuronidase assays. -Galactosidase assays were performed by the fluorometric method of Youngman (54). Control and mutant spo0A strains containing both a spoIIA-lacZ transcriptional fusion and a spoIIE-gus transcriptional fusion were grown as previously described (24). Bacteria were cultured for assay at 37°C with shaking. At various intervals during growth and sporulation, 0.5-ml samples were collected and frozen in liquid nitrogen. Samples were stored at 70°C until assayed.

Immunoblot detection of Spo0A proteins. Polyclonal anti-Spo0A antibodies were isolated previously (6). Samples (10 to 25 ml) of B. subtilis cultures grown in DSM at 37°C with shaking were collected, the cells were harvested at various time points, and cell lysates were prepared as previously described (24). Total protein was quantitated using the Bio-Rad protein microassay procedure as described by the manufacturer. Protein samples (1.25 to 5.0 µg) were separated by electrophoresis through a sodium dodecyl sulfate-12% polyacrylamide gel. Proteins were electroblotted onto a PVDF-Plus membrane (Micron Separations, Inc.).

Spo0A purification. Wild-type and mutant spo0A alleles were amplified using either the 0ANdeI and pUCrev primers or the 0ANdeI and 0A4HindIII primers (Table 2) as forward and reverse primers, respectively. The products were then inserted in frame with the His-tag coding sequence between the NdeI and HindIII sites of the pET15b expression vector (Novagen). All proteins were isolated from Escherichia coli strain BL21(DE3)/pLysS containing the pET15b derivatives of the spo0A alleles. E. coli strains were grown at 37°C with shaking in LB medium (1 liter) containing 100 µg of ampicillin per ml and 35 µg of chloramphenicol per ml to an optical density at 600 nm of 0.6 and induced with 1 mM isopropyl--D-thiogalactopyranoside (IPTG) for 3 to 4 h to overproduce Spo0A proteins. After induction, cells were harvested by centrifugation at 6,000 rpm at 4°C for 10 min in a Beckman JA-10 rotor and then washed with 0.25 volume of cold 50 mM Tris-HCl (pH 8.0)-2 mM EDTA. Cells were repelleted, quick-frozen in liquid nitrogen, and stored at 70°C until needed.

Electrophoretic gel mobility shift assays. Oligonucleotides 0ABoxa and 0ABoxb (Table 2) were annealed to create a double-stranded 40-bp fragment corresponding to the spoIIE promoter sequence from position 59 to position 20 with a change at position 40 from a TA to a CG base pair to generate a consensus 0A box sequence (5'-TGTCGAA-3') . Oligonucleotides Boxa and Boxb (Table 2) were annealed to create a second double-stranded 40-bp fragment with six base pair substitutions in the 0A box to destroy the binding site. Double-stranded fragments were 5' end labeled with T4 polynucleotide kinase (Promega) and [-³²P]ATP (6,000 Ci mmol¹) as previously described (5).

	RESULTS

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Rationale and construction of mutations in the putative HTH DNA-binding domain of Spo0A. To determine the functional significance of a highly conserved stretch of amino acid sequence in the carboxyl-terminal effector domain of Spo0A from B. subtilis, we used site-directed mutagenesis to make specific amino acid substitutions in the coding sequence (Fig. 1). The residues targeted for mutagenesis were chosen on the basis of the polar nature of their side chains. Crystallographic and genetic studies have identified specificity-determining contacts between DNA and many HTH-containing proteins, which include CAP (13-15, 56),  cI Rep (26, 27), 434 Rep (4, 50), and TrpR (20, 32, 37). What is apparent from these studies is that while not all specificity-determining contacts occur in the second helix, or "recognition helix," of the HTH-binding motif, polar residues in this helix are important for sequence-specific recognition (22, 33, 34). Quite often, residues 1, 2, 5, and 6 have been shown to play a critical role in specificity determination, and it is thought that residues such as Gln, Asn, Ser, Tyr, Arg, Lys, Glu, and His are commonly used for DNA recognition (22, 33, 34). In addition, helical-wheel projections of the second helix of the putative Spo0A HTH motif show that this helix has an amphipathic character in which hydrophobic residues are clustered on one side of the helix and hydrophilic ones are clustered on the other (Fig. 1B). This structure would allow the hydrophobic side to face the protein core and the hydrophilic residues on the opposite face to contribute sequence-specific contacts to the DNA.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   (A) Domain structure of Spo0A and the mutations examined in this study. The N-terminal half of Spo0A to residue 127 forms the conserved phosphoacceptor domain, which contains the presumed site of phosphorylation, D56. The C-terminal 113 residues form the effector domain of Spo0A, which contains a putative HTH DNA-binding domain from residue 198 to residue 218 (shaded box) identified by Brown et al. (8) and a transcriptional activation domain (A AR) from residue 227 to residue 240 (solid box) (24). The sequence of the proposed HTH DNA-binding domain, which includes a stretch of 15 amino acids perfectly conserved among four diverse species of Bacillus and Clostridium, is shown. Residues in the recognition helix of the putative HTH domain examined in this study are indicated. (B) Helical-wheel projection of the putative HTH recognition helix from residue A209 to residue E221. Residues changed in this study are in bold. Alanine substitutions were made at all three of the changed residues, and glycine substitutions were made at both E213 and R214.

Sporulation phenotype of Spo0A mutants in B. subtilis. To determine the sporulation phenotype of the Spo0A mutants, mutant spo0A alleles were subcloned into the integrational vector pSPC101 and introduced into B. subtilis JKH72. When screening was performed with DSM plates, which allowed us to directly score the sporulation phenotype by colony morphology, all of the mutants except S210A exhibited a Spo phenotype and failed to sporulate efficiently.

Effect of Spo0A mutants on transcriptional activation of sporulation-specific genes. Sporulation-specific gene expression was assessed by -galactosidase and -glucuronidase assays on strains containing mutant Spo0A derivatives and both a spoIIA-lacZ and a spoIIE-gus transcriptional fusion. These two promoters require Spo0A and different forms of RNA polymerase holoenzyme for transcription and therefore represent two different classes of Spo0A-dependent promoters (51-53). Since these two promoters contain multiple Spo0A binding sites and since transcription requires the spo0A gene product (6, 47, 53), we expected that amino acid substitutions in Spo0A that result in a DNA-binding defect would eliminate activation by Spo0A. The ability of mutant Spo0A to activate the transcription of the spoIIA and spoIIE genes was assayed at intervals through the end of the exponential phase and 6 h into the stationary phase (Fig. 2). A strain containing an insertional disruption of the spo0A gene (null) was included as a negative control.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Effects of spo0A mutants on transcriptional activation at Spo0A-dependent promoters. The effects of spo0A mutants on the expression of spoIIA-lacZ (A) and spoIIE-gus (B) transcriptional fusions are shown. Samples were collected and assayed as described in Materials and Methods. T0 marks the end of exponential growth. Samples were collected at half-hour time points from T2 until T1 and then hourly until T6. Solid circles, wild type; open squares, the Spo0A null strain; inverted solid triangles, S210A; open diamonds, E213G; solid diamonds, E213A; open triangles, R214G; solid triangles, R214A. Data were averaged from at least three independent trials. Error bars represent the standard errors of the means. One unit of activity is defined as 1 pmol of MUGluc or MUGal hydrolyzed per ml of culture sample per min, normalized for culture cell density (turbidity) (54).

Protein levels of Spo0A mutants in B. subtilis. To determine if the mutant Spo0A proteins were present in B. subtilis, we made total cell extracts from the mutant strains. Cell extracts were then analyzed by immunoblotting with rabbit polyclonal anti-Spo0A antiserum (Fig. 3). All five mutants produced stable full-length protein. However, all of the mutants had reduced levels of protein compared to the wild type. The two mutants with changes at E213 had levels of Spo0A protein four- to fivefold lower than wild-type levels. The S210A, R214G, and R214A mutants had two- to threefold lower Spo0A production. There is no direct correlation between the severity of the sporulation phenotype of the mutant and the level of Spo0A mutant protein.

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Immunoblot analysis of wild-type and mutant Spo0A proteins. B. subtilis strains containing wild-type or mutant spo0A alleles were grown, and culture samples were collected and harvested at T1. Samples containing 1.25, 2.5, or 5.0 µg of total cellular protein were subjected to electrophoresis through a 12% polyacrylamide gel. Samples were electroblotted on to a PVDF-Plus membrane, and the Spo0A protein was probed with anti-Spo0A antibody as described in Materials and Methods. The Spo0A protein is indicated by the arrowheads. wt, wild type.

Ability of the mutant Spo0A proteins to bind to DNA in vitro. In order to assess the DNA-binding ability of the mutant Spo0A proteins in vitro, we first subcloned the mutant spo0A genes into the pET15b expression vector (Novagen) and isolated His-tagged variants of wild-type and mutant Spo0A proteins. Proteins were isolated using affinity purification and were determined to be 50 to 95% pure by Coomassie blue staining (data not shown). We then used these partially purified proteins in electrophoretic gel mobility shift assays. Two complexes were formed when His-Spo0A was added to a radiolabeled 40-bp DNA fragment corresponding to the 59-to-20 region of the spoIIE promoter containing a single base pair mismatch (TA to CG) at position 40 to create a consensus 0A box (0ABox fragment) (Fig. 4A and data not shown). At low concentrations of protein (0.069 to 2.3 µM), a single shifted complex was detected (Fig. 4A). At high concentrations (6.9 to 69 µM), the lower complex disappeared and was replaced by smearing or a second slowly migrating complex very high in the gel (data not shown). We determined that the second complex was due to a nonspecific interaction, as we detected the same complex in an assay with a DNA fragment in which the 0A box had multiple substitutions (Box fragment), and because a vast excess of salmon sperm DNA (10 µg) could effectively compete for the formation of this complex but not the lower complex (data not shown). Formation of the Spo0A-DNA complex was sequence specific, as the addition of the unlabeled 0ABox fragment effectively competed for the formation of this complex, but the Box fragment, which contains a multiple-substitution derivative of the 0ABox fragment, did not (Fig. 4B).

View larger version (64K): [in this window] [in a new window]   FIG. 4.   Ability of His-Spo0A and His-Spo0A mutants to bind and shift a 40-bp spoIIE promoter fragment in an electrophoretic gel mobility shift assay. (A) DNA binding by His-tagged wild-type and mutant Spo0A proteins. One nanogram (0.038 pmol) of a radiolabeled double-stranded oligonucleotide (0ABox) corresponding to the spoIIE promoter from position 59 to position 20 containing a single base pair substitution (from TA to CG) at position 40 to generate a consensus 0A box sequence was incubated with 1 µg of partially purified protein. All reaction mixtures also included 1 µg of salmon sperm DNA. Proteins were purified as described in Materials and Methods. The Spo0A-DNA complex is indicated by the arrowhead. P, unbound probe (B) Competition for Spo0A-DNA complex formation by a 40-bp spoIIE promoter DNA fragment containing either a consensus (0ABox) or mutated (Box) 0A box sequence. Assays were performed as described for panel A, except that 0.5 µg of protein was used and 400 ng of the indicated unlabeled oligonucleotide was added to the binding reactions. The Box oligonucleotide sequence consists of six base pair substitutions in the 0A box sequence contained within the 0ABox oligonucleotide. WT, wild type.

Ability of the Spo0A mutants to repress abrB transcription. To assess the ability of the mutant Spo0A proteins to repress abrB transcription, we introduced wild-type and mutant spo0A genes by chromosomal transformation into a strain containing an abrB-lacZ transcriptional fusion carried on a specialized SP transducing phage (9). Transcription of the abrB gene is repressed by the spo0A gene product during the transition from exponential to stationary phase at the onset of sporulation and requires that Spo0A bind to the abrB promoter (36). Therefore, the ability of the Spo0A mutants to bind in vivo and to repress abrB transcription was assayed by monitoring -galactosidase activity in these strains (Fig. 5). The S210A mutant repressed transcription of abrB in a manner similar to that of the wild type. The E213G and E213A mutants showed some derepression of abrB transcription but not to the same level as the null strain. abrB transcription was derepressed to the same level as in the null strain by the mutant R214A. However, unexpectedly, the R214G mutant showed higher levels of derepression of abrB transcription than the null strain. The same assay results were obtained with subsequent reconstruction of the abrB-lacZ strain containing the R214G mutant.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Ability of spo0A mutants to repress abrB transcription. The ability of Spo0A and mutants to repress abrB transcription was assayed using an abrB-lacZ transcriptional fusion carried on a specialized SP-transducing phage. Strains were cultured and assayed as described in the legend for Fig. 2. Samples were collected at half-hour time points from T2 until T1 and then hourly until T4. Each panel contains the mutant indicated: S210A (inverted solid triangles), E213G (open diamonds), E213A (solid diamonds), R214G (open triangles), or R214A (solid triangles). Data were averaged from at least three independent trials. Error bars represent the standard errors of the means. One unit of activity is defined as described in the legend for Fig. 2. Solid circles, wild type; open squares, Spo0A null strain.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Spo0A is a key transcriptional regulator involved in controlling the expression of genes required during the entry into stationary phase and the onset of endospore formation. In this study, we have made amino acid substitutions in a highly conserved region of the C-terminal domain of Spo0A that contains a possible HTH DNA-binding domain with the hope of isolating DNA-binding mutants of Spo0A. The results reported here give support for the proposed involvement of residue E213 in DNA binding and support a functional role for the conserved residues in this region.

The S210A mutant contains an alanine in the second position of the putative recognition helix and shows little effect on DNA binding. As serine and alanine side chains differ only by a hydroxyl group, it is unlikely that replacement with alanine would greatly perturb the structure of the mutant protein. Therefore, these results indicate that this residue is probably not involved in making specific contacts for DNA binding. However, the transcriptional activity of the spoIIA and spoIIE promoters positively regulated by Spo0A was decreased by this substitution. This result is consistent with the S210 residue being part of the HTH DNA-binding domain, as many mutations within and around the HTH DNA-binding domain in other proteins are known to affect transcriptional activation without necessarily affecting binding (25, 30).

The E213 mutants, however, appear to be involved with DNA binding, as both the glycine and alanine substitution mutants showed decreased binding in vitro compared to that of the wild type. In addition, reductions in the ability to repress the transcription of abrB and to activate transcription of the spoIIA and spoIIE promoters are consistent with a loss of DNA binding. The more drastic effect seen at the promoters positively regulated by Spo0A may be due to the fact that these promoters have multiple 0A boxes (6, 53), and degenerate 0A boxes in these promoters may result in different levels of binding defects in the mutant proteins. Alternatively, since Spo0A functions require phosphorylation for activity in vivo (6, 17, 45), we cannot rule out the possibility that substitutions in E213 somehow affect phosphorylation. However, since a D56N mutation of Spo0A that cannot be phosphorylated cannot repress abrB transcription in vivo (45), our results argue that phosphorylation is not abolished in the E213 mutant strains. Whether there is decreased phosphorylation of our mutants is still in question, since the abrB promoter is most sensitive to low levels of phosphorylation (48), so repression may still occur even with decreased phosphorylation of Spo0A. Moreover, the effects we saw on transcriptional activation could be due to the decreased levels of protein in these strains. However, since DNA binding in vitro does not require phosphorylation (41, 47) and protein levels are standardized, it is clear that there are defects in binding when the E213 residue of Spo0A is replaced.

The substitutions at R214 completely abolished the activity of these proteins. This result is consistent with either an alteration in a sequence-specific contact that is required for DNA binding, a gross conformational change that destroys protein function, or a defect in the ability of the protein to be phosphorylated. There are a few points that argue against the last two cases. First, these proteins retained their ability to bind to DNA nonspecifically, as indicated by the slowly migrating complex that was formed at high concentrations of protein in the mobility shift assays (data not shown). Moreover, they were able to bind to heparin-agarose during affinity chromatography, although with a lower affinity than that of the wild-type protein (data not shown). Therefore, although nonspecific binding of the mutant proteins was not identical to that of the wild type, this binding was not abolished. These results are similar to those observed for mutations in the recognition helix of the  cII protein, which eliminate sequence-specific binding but do not eliminate nonspecific binding (25).

In addition, the R214G mutant appears to have a gain-of-function alteration that results in higher levels of -galactosidase expression in a strain containing an abrB-lacZ transcriptional fusion. We have shown that the higher expression from this strain is not due to a second site mutation at abrB. Therefore, either this mutant form of Spo0A either activates abrB transcription or interacts aberrantly at an additional locus. It seems more likely that the mutations we made could result in an altered specificity of Spo0A binding producing an indirect effect on abrB expression. In support of this alternative is the fact that protease expression in the position 214 mutants was intermediate to that of the wild-type and Spo0A null strains on milk plates. In the null strain, protease expression was abolished. Therefore, the results observed for the R214G mutant are not simply due to the destruction of all protein functions. Moreover, this result suggests that the regulation of abrB or repression by Spo0A may be more complicated than previously suspected.

Our studies support the hypothesis that the conserved region of Spo0A identified by Brown et al. (8) is functionally important. The S210A and E213 mutant proteins do maintain some binding activity while decreasing or destroying the ability to activate transcription, so these could be classified as positive-control mutants (1). Mutations within or in regions surrounding the binding motifs of the proteins  cI (30) and OmpR (2, 29) are positive-control mutations. Therefore, it is possible that these residues are not directly involved in contacting DNA but are still part of the DNA-binding domain. However, the results clearly indicate a role for E213 in DNA binding and implicate R214 as a key functional residue.

Ultimate determination of the functional significance of the conserved region of Spo0A requires determination of its molecular structure. Recently, it has been reported that the full-length protein and the C-terminal domain of Spo0A have both been crystallized (31). Together with genetic and biochemical studies such as the present study, the crystal structure of Spo0A will help us elucidate the functional domains of Spo0A so we can better understand how this protein functions compared to other members of the response regulator family of proteins.