An Immunological Assay for the Sigma Subunit of RNA Polymerase in Extracts of Vegetative and Sporulating Bacillus subtilis

Abstract

The activity of the a subunit of Bacillus subtilis RNA polymerase decreases markedly during the first hours of sporulation [T. The onset of sporulation by Bacillus subtilis is associated with a change in the template specificity of RNA polymerase (1). Enzyme isolated from sporulating bacteria is unable to transcribe certain phage DNA templates as actively in vitro as RNA polymerase from vegetative B. subtilis. However, RNA polymerase in certain mutants blocked at an early stage of spore formation retains, throughout stationaryphase, the template recognition properties characteristic of enzyme from vegetative bacteria (2, 3). The change in template specificity is apparently caused by a marked decrease in the activity of the a subunit of RNA polymerase rather than an alteration of the core subunits, ,B, f', and a (4, 5).However, it has previously not been possible to determine whether sporulating bacteria actually lack a polypeptide or whether the a polypeptide is present but its activity inhibited during spore formation. To distinguish between these alternatives, we report an immunological assay that provides a direct test for the presence of the a polypeptide in extracts of vegetative and sporulating B. subtilis. METHODSCells. Wild-type B. subtilis strain NCTC 3610 (ATCC 6051), a Marburg strain, was used for all experiments except where otherwise indicated. The asporogenous mutants (6) were kindly provided by P. Schaeffer.Media and Sporulation. Growth and sporulation of B. subtilis 3610 was in 121B medium (7) and sporulating cells were harvested 4.5 hr after the end of a logarithmic growth (T4.5) unless otherwise noted. For radioactive labeling of cells, the radioactive precursor was added during early logarithmic growth. Strain SMY and the asporogenous mutants were grown (8) in Difco sporulation medium (DSAI); cells were harvested either at mid-logarithmic phase or allowed to enter stationary phase by continued growth in DS'M or by resuspension of logarithmically growing cells in Sterlini-MNandelstam (SM) medium (9). In DSM and SAM media, sporulating or stationary-phase cells were harvested either 3 hr after the end of logarithmic growth (T3) or 3 hr after resuspending in SM medium (T3). After harvesting, cells were washed with buffer G (0.05 M Tris-HCl, pH 7.5, 0.01 MI MNlgCI2, 0.1 mMNi ethylenediaminetetraacetate, 0.1 mMI dithiothreitol, 10% v/v glycerol) containing 1.0 AM KC1 (10) and 5%lphenyl-methylsulfonyl fluoride solution (6 mg/ml of 95% ethanol) to remove extracellular proteases and then rapidly frozen.Cells Extracts Were Prepared from about 2 g of cells by disruption in a Braun homogenizer and high speed centrifugation as previously described (5)

Binding of σAand σBto Core RNA Polymerase after Environmental Stress inBacillus subtilis

“…There are by now several lines of evidence in favor of the view that, in some circumstances, different factors compete for a limiting pool of the core enzyme, both in E. coli and in B. subtilis (13,18,26,36). The experiments with a sigB mutant reported here support this view, since they show that Adependent transcription is much more strongly induced by stress in the mutant cells than in the wild type (Fig.…”

“…However, this conclusion, which was based on the finding that there is twofold more E than A in the cell, is open to question, as twothirds of the molecules of E are known to be involved in transcription elongation (9) and are therefore not in a state in which they can bind any factor. Furthermore, other measurements of the intracellular concentration of E and A have suggested that the two proteins are present at approximately the same molar concentration in sporulating cells (26,36). In addition, expression studies have suggested that A and H in B. subtilis compete for binding to the core RNA polymerase, as do 70 and S in Escherichia coli, since in both systems overexpression of one factor leads to a decrease in the gene expression that is dependent on the other factor (13, 18).…”

Chloramphenicol Restores Sigma Factor Activity to Sporulating Bacillus subtilis

“…4A) while the ratio for sporulation polymerase, 1.1 (Fig. 4B), was similar to that for purified core RNA polymerase lacking a- (3). In contrast, phasepartitioned RNA polymerase from sporulating cells that had been treated with chloramphenicol for 20 min before harvesting exhibited a ratio of 4.0 (Fig.…”

“…The increase in transcription ratio caused by chloramphenicol was also observed for polymerase from sporulating cells that had been infected by He after drug treatment (Table 2). RNA polymerase was prepared either by ammonium sulfate fractionation (3) or phase partitioning (3). Enzyme activities were plotted against protein concentration as illustrated in Fig.…”

“…Although sporulating bacteria contain as much a polypeptide as vegetative bacteria, a from sporulating B. subtilis is functionally inhibited and only weakly associated with RNA polymerase (3). It was previously observed that B. subtilis phage ye is unable to grow in sporulating bacteria (4) and that the time course of decrease in phage burst size during the first hours of spore formation closely parallels the decrease in a activity, as indicated by the reduced ability of RNA polymerase to transcribe he DNA in vitro (5).…”