An A257V mutation in the bacillus subtilis response regulator Spo0A prevents regulated expression of promoters with low-consensus binding sites

Abstract

In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

FIG. 1.

Spo0A(A257V) is efficiently phosphorylated. Spo0A and Spo0A(A257V) were incubated with the phosphorelay components KinA, Spo0F and Spo0B, and [γ-³²P]ATP for various times, as indicated, before the reaction was terminated to determine steady-state phosphorylation (A and B) or the rate of phosphorylation (C and D). (A and C) The radiolabeled components were separated by 15% SDS-PAGE, and ³²P-labeled Spo0A or Spo0A(A257V) was detected by exposure to a phosphor screen. (B and D) The extent of phosphorylation was determined by PhosphorImager analysis. Symbols: Spo0A∼P, white bars in panel B, filled circles in panel D; Spo0A(A257V)∼P, gray bars in panel B, open circles in panel D. WT, wild type. Representative images are shown. The error bars indicate standard deviations.

FIG. 2.

Spo0A(A257V) represses transcription from the abrB promoter in vitro. Phosphorylated or unphosphorylated mutant and wild-type (WT) Spo0A proteins were incubated with the initiating nucleotides ATP, UTP, and GTP and a linear DNA fragment encoding both the P1 and P2 transcription initiation sites of the abrB promoter. CTP and heparin were added 3 minutes after the addition of RNAPσ^A to permit transcript elongation. The reactions were terminated after 5 minutes, and the transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel. ³²P-labeled abrB transcripts were detected by exposure to a phosphor screen (A), and the level of transcripts produced from the abrB promoter was determined by PhosphorImager analysis (B). Filled circles, Spo0A∼P; filled triangles, Spo0A; open circles, Spo0A(A257V)∼P; open triangles, Spo0A(A257V). Representative PhosphorImages are shown. The error bars indicate standard deviations.

FIG. 3.

Spo0A(A257V)∼P stimulates spoIIG transcription in vitro. Phosphorylated or unphosphorylated Spo0A or Spo0A(A257V) was incubated with a linear DNA fragment encoding the spoIIG operon promoter and the initiating nucleotides ATP and GTP. RNAPσ^A was added to the mixture and allowed to initiate transcription for 2 min prior to addition of the remaining nucleotides and heparin. Following a 5-minute incubation, the elongated transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel. ³²P-labeled spoIIG transcripts were detected by exposure to a phosphor screen (A), and the level of transcription was determined by PhosphorImager analysis (B). Filled circles, Spo0A∼P; filled triangles, Spo0A; open circles, Spo0A(A257V)∼P; open triangles, Spo0A(A257V). WT, wild type. A representative phosphorimage is shown. The values reflect the average of three independent experiments. The error bars indicate standard deviations.

FIG. 4.

Spo0A(A257V)∼P stimulates the rate of transcription initiation at the spoIIG promoter. Transcription was initiated by adding RNAPσ^A to a reaction mixture containing a linear DNA fragment encoding the spoIIG operon promoter, the initiating nucleotides (ATP and GTP), and either Spo0A or Spo0A(A257V) (phosphorylated or unphosphorylated). At various times, aliquots were removed and added to heparin plus CTP and UTP. After the reactions were terminated, ³²P-labeled spoIIG transcripts were separated by electrophoresis through an 8% denaturing polyacrylamide gel and detected by exposure to a phosphor screen (A), and their levels were determined by PhosphorImager analysis (B). The symbols indicate the addition of Spo0A∼P (filled circles), Spo0A (filled triangles), Spo0A(A257V)∼P (open circles), and Spo0A(A257V) (open triangles). A representative phosphorimage is shown. The values reflect the average of three independent experiments. The error bars indicate standard deviations.

FIG. 5.

Elevated amounts of Spo0A(A257V) cannot stimulate transcription of spoIIG-lacZ or spoIIA-lacZ promoter fusions in vivo. B. subtilis strains were grown in SSM, and samples were collected and harvested during the transition from an exponential to a stationary phase of growth; time zero indicates the end of exponential growth. (A) Whole-cell extracts were separated by electrophoresis through 12% SDS-PAGE, transferred onto nitrocellulose membranes, subjected to immunoblot analysis using an anti-Spo0A polyclonal antibody, and detected using a VersaDoc MP 4000 system. (B) Protein levels were quantified by densitometry using purified Spo0A as a standard. (C) Transcription of spoIIG-lacZ and spoIIA-lacZ promoter fusions in B. subtilis Spo0A overexpression strains was determined by assaying β-galactosidase activity in samples of cells grown in SSM. Closed circles, spo0A; closed triangles, spo0A crsA47; open circles, spo0A(A257V); open triangles, spo0A(A257V) crsA47.

FIG. 6.

Binding of wild-type and mutant Spo0A proteins to abrB 0A box spacing variants. Spo0A∼P and Spo0A(A257V)∼P were incubated with a mixture of double-stranded ³²P-labeled oligonucleotides that contained the consensus 0A boxes from the abrB promoter and different inputs of unlabeled competitor DNAs with consensus 0A boxes separated by spacers of various lengths. The proteins were allowed to bind DNA for 2 minutes prior to challenge with calf thymus DNA in glycerol and were separated on a running 8% nondenaturing polyacrylamide gel. (A) ³²P-labeled DNA was detected by exposure to a phosphor screen, and the levels of bound versus unbound DNA were determined by PhosphorImager analysis. (B) The data are plotted as (DNA_free + DNA_bound)/DNA_bound versus the normalized input for wild-type abrB, abrB+1, and abrB+3 competitor DNAs. Each data point represents the average from three separate experiments. The slopes of the curves are reported in Table 4. Filled circles, Spo0A∼P; open circles, Spo0A(A257V)∼P.

FIG. 7.

Spo0A(A257V) stimulates transcription from spoIIG promoters with high-consensus 0A boxes. B. subtilis strains JH642 (spo0A) and JH695 [spo0A(A257V)] were transformed with DNA encoding spoIIG-lacZ promoter mutants (spoIIG-1 through spoIIG-5) (Table 5). The strains were grown in SSM, and samples were collected and harvested during the transition from an exponential to a stationary phase of growth; time zero indicates the end of exponential growth. Filled squares, spoIIG1; open triangles, spoIIG2; filled triangles, spoIIG3; open circles, spoIIG4; filled circles, spoIIG5.