A region in Bacillus subtilis sigmaH required for Spo0A-dependent promoter activity

Abstract

Spo0A activates transcription in Bacillus subtilis from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, sigmaA, and RNA polymerase containing a secondary sigma factor, known as sigmaH. The region of sigmaA near positions 356 to 359 is required for Spo0A-dependent promoter activation, possibly because Spo0A interacts with this region of sigmaA at these promoters. To determine if the amino acids in the corresponding region of sigmaH are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in sigmaH (201 to 210). Two alanine substitutions in sigmaH, at glutamine 201 (Q201A) and at arginine 205 (R205A), significantly decreased activity from the Spo0A-dependent, sigmaH-dependent promoter spoIIA but did not affect expression from the sigmaH-dependent, Spo0A-independent promoters citGp2 and spoVG. Therefore, promoter activation by Spo0A requires homologous regions in sigmaA and sigmaH. A mutant form of Spo0A, S231F, that suppresses the sporulation defect caused by several amino acid substitutions in sigmaA did not suppress the sporulation defects caused by the Q201A and R205A substitutions in sigmaH. This result and others indicate that different surfaces of Spo0A probably interact with sigmaA and sigmaH RNA polymerases.

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. ς⁷⁰ of E. coli is not shown, but ς⁷⁰ and ς^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 ς^H sequence indicate the phenotypes of alanine substitutions at that amino acid in ς^H. A + sign indicates an allele which produced a functionally wild-type ς^H protein, and a − sign designates a completely inactive ς^H protein. The λ indicates an amino acid substitution at 596 in ς⁷⁰, R596H, which specifically suppresses the D38N mutation in λ cI (8, 9). The asterisk indicates amino acid substitutions in ς^A (K356E and H359R) which specifically prevent transcription from Spo0A-dependent promoters (1).

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 β-galactosidase (βgal) activity (11). An independent transductant from each strain was assayed for β-galactosidase activity and found to express essentially the same levels of activity as those shown (data not shown).

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 β-galactosidase (βgal) activity (11). An independent transductant from each strain was assayed for β-galactosidase activity and found to express essentially the same levels of activity as those shown (data not shown).

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 β-galactosidase (βgal) activity (11). An independent transductant from each strain was assayed for β-galactosidase activity and found to express essentially the same levels of activity as those shown (data not shown).