Sigma factor displacement from RNA polymerase during Bacillus subtilis sporulation - PubMed (original) (raw)

Sigma factor displacement from RNA polymerase during Bacillus subtilis sporulation

J Ju et al. J Bacteriol. 1999 Aug.

Abstract

As Bacillus subtilis proceeds through sporulation, the principal vegetative cell sigma subunit (sigma(A)) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific sigma factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with sigma(A) and two mother cell sigma factors, sigma(E) and sigma(K), which successively replace sigma(A) on RNAP. Although the relative abundance of sigma(A) with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable sigma(A) which cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T(0)) to 2 to 8% by 3 h into the process (T(3)). In a strain that failed to synthesize sigma(E), the first of the mother cell-specific sigma factors, approximately 40% of the sigma(A) remained associated with RNAP at T(3). The level of sigma(A)-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a sigma(E) variant (sigma(ECR119)) that could bind to RNAP but was unable to direct sigma(E)-dependent transcription. The E-sigma(E)-to-E-sigma(K) changeover was characterized by both the displacement of sigma(E) from RNAP and the disappearance of sigma(E) from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the sigma(K) protein is required for the displacement of sigma(E) from RNAP and also confirmed that sigma(K) is needed for the loss of the sigma(E) protein. The results indicate that the successive appearance of mother cell sigma factors, but not necessarily their activities, is an important element in the displacement of preexisting sigma factors from RNAP. It suggests that competition for RNAP by consecutive sporulation sigma factors may be an important feature of the holoenzyme changeovers that occur during sporulation.

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Figures

FIG. 1

FIG. 1

Fractionation of extracts from sporulating and Spo0A− B. subtilis. Wild-type B. subtilis SMY (A) and its congenic Spo0A− variant S0A514 (B) were grown in DSM. Samples were harvested at the end of exponential growth (_T_0) or at 1.5-h intervals thereafter (_T_1.5, _T_3, and _T_4.5), the cells were disrupted, and the resulting crude extracts were fractionated by centrifugation through a linear gradient of 15 to 30% glycerol, as described in Materials and Methods. Fractions were collected and analyzed by Western blotting with anti-β′, anti-ςA, and anti-ςE antibodies as probes. Fraction 1 represents the bottom of the centrifuge tube. The two bands detected by the anti-ςE antibody in panel A at _T_1.5 represent ςE and its slightly larger precursor (pro-ςE).

FIG. 2

FIG. 2

Fractionation of SigE− and SigF− B. subtilis. Wild-type B. subtilis (SMY) (A) and its congenic SigF− (SF9030) (B), and SigE− (SEF500) (C), and SigE− SigF− (SEF500) (D) variants were grown to _T_3 in DSM and analyzed as described for Fig. 1. The protein band reacting with the anti-ςE antibody is ςE in wild-type B. subtilis (A) and pro-ςE in the SigF− strain (B).

FIG. 3

FIG. 3

Effect of chloramphenicol treatment on E-ςA persistence. Wild-type B. subtilis (SMY) was grown in DSM. At 1.5 and 3 h after the end of exponential growth, chloramphenicol was added to portions of the culture, which were harvested 0.5 h later. _T_2.0 + CM and _T_2.0 represent portions of the culture harvested at 2 h with and without chloramphenicol treatment, respectively. _T_3.5 + CM and _T_3.5 are similar cultures harvested at _T_3.5. The culture samples were analyzed as described for Fig. 1. The anti-ςE antibody detected pro-ςE and ςE at _T_2 and only ςE at _T_3.5.

FIG. 4

FIG. 4

Fractionation of crude extracts from SigK− B. subtilis. SigK− B. subtilis (SK1027) was grown in DSM, and samples were harvested at the end of growth (_T_0) and at 1.5-h intervals thereafter (_T_1.5, _T_3.0, and _T_4.5). The samples were analyzed as described for Fig. 1. The anti-ςE antibody detects pro-ςE and ςE at _T_1.5 and _T_3 and primarily ςE at _T_4.5.

FIG. 5

FIG. 5

Fractionation of crude extracts from sigK109CR B. subtilis. B. subtilis S410 (sigK109CR) was grown in DSM. Samples harvested at 4.5 h (_T_4.5) and 6 h (_T_6) after the end of growth were analyzed as described for Fig. 1 with antibodies against β′, ςA, ςE, and ςK as probes. The anti-ςE antibody detected ςE. The anti-ςK antibody detected ςK and its slower-migrating precursor, pro-ςK.

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