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Spo0A is a DNA binding protein in Bacillus subtilis that is essential for the initiation of endospore formation (reviewed in reference 6). Spo0A activates transcription from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, ^A, and RNA polymerase containing a secondary sigma factor, known as ^H (12, 15, 17). Three sporulation-specific promoters that are activated by Spo0A have been characterized extensively: spoIIG and spoIIE, which are ^A dependent, and spoIIA, which is ^H dependent (7, 18, 20). Spo0A binds to these promoters at sites overlapping the 35 region and may interact with the sigma subunit of RNA polymerase. Baldus et al. (1) found that transcription from the ^A-dependent, Spo0A-dependent promoters spoIIG and spoIIE was reduced in mutants of B. subtilis in which ^A contained either of two single-amino-acid substitutions, lysine at position 356 replaced by glutamate (K356E) or histidine at position 359 replaced by arginine (H359R). However, these substitutions had no effect on the use of a Spo0A-independent promoter, tms. Moreover, alanine substitutions at positions 356 and 359 had similar Spo0A-specific effects (14). A single-amino-acid substitution in Spo0A, serine at position 231 replaced by a phenylalanine (S231F), was recently shown to partially suppress the effect of the H359R mutation in ^A (2). These results support the hypothesis that spoIIG and spoIIE promoter activation by Spo0A requires the region of ^A near positions 356 to 359, possibly because Spo0A interacts with this region of ^A at these promoters.

To determine if the amino acids in the corresponding region of ^H are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in ^H, from 201 to 210 (Fig. 1). We expected that strains in which an alanine substitution in ^H resulted in loss of interaction with Spo0A would exhibit reduced sporulation efficiency because utilization of ^H-dependent, Spo0A-dependent promoters such as spoIIA is required for endospore formation. Moreover, if the alanine substitution in ^H specifically prevented its interaction with Spo0A, then the ^H mutant would retain the ability to direct transcription from promoters such as citG and spoVG, since use of these promoters does not require direct interaction with Spo0A (4). The sigH allele on plasmid pJB3 was mutagenized in vitro by a multiple-step PCR procedure (3) and used to replace the wild-type allele in the B. subtilis chromosome by transformation of strain JH642 (Table 1) to spectinomycin resistance as described previously (1). Spectinomycin-resistant transformants resulted from a single crossover event between the plasmid-encoded sigH allele and the chromosomal sigH allele. Four sigH mutant alleles, which resulted in the substitution of alanine for amino acids at positions 203, 207, 208, and 209 in ^H, produced transformants that appeared to sporulate as efficiently as the wild-type strain on DSM agar (13). Six alleles of sigH produced strains that appeared sporulation deficient (spo) on DSM agar (Fig. 1). Of these, four alleles (producing alanine substitutions at positions 202, 204, 206, and 210 in ^H [Fig. 1]) appeared to inactivate all ^H-dependent promoter activity (measured as described in reference 1; data not shown) and were not further studied. The remaining two alleles, which resulted in substitutions at 201 and 205 in ^H, each caused a specific decrease in the Spo0A-dependent, ^H-dependent spoIIA promoter activity, while the Spo0A-independent, ^H-dependent promoters spoVG and citGp2 appeared to be active (data not shown).

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Amino acid alignment of the 35 recognition regions in the carboxyl terminus of sigma factors in B. subtilis. Conserved regions are shaded according to Sun et al. (16). The position number for the last amino acid shown in each sigma factor is indicated. 70 of E. coli is not shown, but 70 and A are almost identical in this region. Amino acid substitutions are indicated by the arrow from the wild-type amino acid to the altered amino acid. The + and  signs below the H sequence indicate the phenotypes of alanine substitutions at that amino acid in H. A + sign indicates an allele which produced a functionally wild-type H protein, and a  sign designates a completely inactive H protein. The  indicates an amino acid substitution at 596 in 70, R596H, which specifically suppresses the D38N mutation in  cI (8, 9). The asterisk indicates amino acid substitutions in A (K356E and H359R) which specifically prevent transcription from Spo0A-dependent promoters (1).

In order to examine the quantitative effects of these amino acid substitutions in ^H, we isolated derivatives of our sigH mutants that had lost the integrated plasmid but retained the mutant sigH allele. To isolate these strains, we screened for sporulation-deficient (spo; determined as described in reference 1), spectinomycin-sensitive mutants after transformation of B. subtilis JH642 (trpC2 phe-1) to tryptophan prototrophy with a mixture of 40 ng of B. subtilis ZB307A chromosomal DNA (Table 1) and 2 µg of the pJB3sigHQ201A and pJB3sigHR205A plasmids. Transformants were selected on minimal plates for sporulation containing 50 µg of phenylalanine and screened for spectinomycin sensitivity and a spo phenotype. DNA sequence analysis demonstrated that the spo mutants, but none of the Spo⁺ isolates, contained the Q201A or R205A mutations in sigH. Two of these spo strains, EUC97Q1 and EUC97R1, which express ^H Q201A and R205A, respectively, produced only about 1% of the spores made by the strain containing wild-type sigH (Table 2).

To monitor the effects of each amino acid substitution on the activation of specific promoters, we isolated an isogenic set of strains containing operon fusions of lacZ to spoIIG, spoIIA, or spoVG or to citG as described previously (1) (Table 1). The ^H Q201A and R205A mutations decreased the expression from the ^H-dependent, Spo0A-dependent promoter, spoIIA, more than 10-fold (Fig. 2A). The amount of activity in these strains is similar to that measured in strains expressing a Spo0A nonsense allele, which retains only 5% of wild-type spoIIA activity (2). However, the strains expressing the sigHQ201A and sigHR205A alleles displayed wild-type levels of the ^H-dependent, Spo0A-independent promoters citGp2 and spoVG (Fig. 2B and C). To determine if spoIIA promoter activity was indirectly reduced in the sigH mutant strains because of a decreased level of Spo0A, we measured the activity of the ^A-dependent, Spo0A-dependent promoter spoIIG and found it to be about 25% of the activity measured in the isogenic wild-type strain (data not shown). These levels are significantly higher than those in an otherwise isogenic strain expressing a spo0A nonsense allele (7.4% of wild-type spoIIG activity [2]). These results suggest that the side chains of Q201 and R205 of ^H are important for wild-type levels of Spo0A-dependent, ^H-dependent spoIIA promoter activity. On the other hand, these amino acid residues are not required for activity of the Spo0A-independent, ^H-dependent promoters citGp2 or spoVG.

View larger version (14K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   FIG. 2.   Effects of amino acid substitutions in sigH on spoIIA, citGp2, and spoVG promoter activities. B. subtilis JH642 (wild-type sigH) (circles), EUC97Q1 (sigHQ201A) (squares), and EUC97R1 (sigHR205A) (triangles) containing the spoIIA (A), citGp2 (B), or spoVG (C) promoter-lacZ fusions were grown in DSM liquid medium. Samples were taken from cultures growing at mid-log phase (T1), at the end of exponential growth (T0), and at 1-h intervals after the onset of stationary phase (T1 to T4) and were then assayed for -galactosidase (gal) activity (11). An independent transductant from each strain was assayed for -galactosidase activity and found to express essentially the same levels of activity as those shown (data not shown).

The sporulation-defective phenotype caused by an amino acid substitution in ^A (H359R) is suppressed by a single-amino-acid substitution (S231F) in Spo0A (2). We wished to determine whether the S231F form of Spo0A suppressed the spo phenotype of the ^H mutant strains. We transformed strains EUC97Q1, EUC97R1, and the wild-type parent, JH642, with plasmids pCB2spo0Awt as a control and pCB2spo0AS231F as described previously (2). Analysis of the sporulation efficiency of the resultant strains (Table 2) indicated that the spo0AS231F allele failed to suppress the spo phenotype caused by the amino acid substitutions in ^H. However, the spo0AS231F allele slightly increased the sporulation efficiency of an otherwise isogenic strain containing a wild-type sigH allele (Table 2).

Our results support the model in which spoIIA promoter activation requires an interaction between Spo0A and ^H. Two single alanine substitutions in ^H, at glutamine 201 (Q201A) and at arginine 205 (R205A), which significantly decrease activity from the Spo0A-dependent, ^H-dependent promoter spoIIA, do not affect expression from the ^H-dependent, Spo0A-independent promoters citGp2 and spoVG. These two amino acids lie in the region of ^H corresponding to the region in ^A that has been implicated in an interaction with Spo0A at the spoIIG and spoIIE promoters (1, 2, 14) (Fig. 1). Therefore, Spo0A appears to activate transcription by interacting with homologous regions in ^A and ^H. A homologous region in ⁷⁰ of Escherichia coli is also suspected to be involved in interaction with cI from phage lambda (8, 9) (Fig. 1).

It is not known if the same surface of Spo0A or different surfaces of Spo0A are important in its activation of ^A- and ^H-dependent promoters. An amino acid substitution in Spo0A (S231F) that was previously identified as a suppressor mutation of the sporulation defect caused by an amino acid substitution in ^A, H359R (2), displayed little or no suppression of the sporulation defects of the ^H mutants Q201A and R205A. Alanine substitutions in the 229 to 233 region of Spo0A did not reduce spoIIA transcription (2), and Hatt and Youngman (5) have isolated five additional mutant forms of Spo0A in the region of 227 to 240 that prevented spoIIG and spoIIE transcription, without affecting transcription from spoIIA. Therefore, another region of Spo0A may interact with ^H RNA polymerase to stimulate its activity. An amino acid substitution in Spo0A, A257T, has been found to prevent spoIIA transcription but not abrB repression (10). These results are consistent with the model that different amino acids in Spo0A are involved in the activation of ^H and ^A RNA polymerases. The ability of response regulators to interact with multiple sigma factors may increase the variety of responses made by bacteria with a limited number of transcription factors.