Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A). Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.     

Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (sigma 43) operon.

The gene coding for Bacillus subtilis RNA polymerase major sigma 43, rpoD, was cloned together with its neighboring genes in a 7 kb EcoRI fragment. The complete nucleotide sequence of a 5 kb fragment including the entire rpoD gene revealed the presence of two other genes preceding rpoD in the order P23-dnaE-rpoD. The dnaE codes for DNA primase while the function of P23 remains unknown. The three genes reside in an operon that is similar in organization to the E. coli RNA polymerase major sigma 70 operon, which is composed of genes encoding small ribosome protein S21 (rpsU), DNA primase (dnaG), and RNA polymerase sigma 70 (rpoD). There is a relatively high degree of base and amino acid homology between the DNA primase and sigma genes. The most significant differences between the two operons are observed in the molecular size of the first genes (P23 and rpsU), the complete lack of amino acid homology between P23 and S21, the molecular weights of the two rpoD genes, the size of the intercistronic region between the first two genes, and the regulatory elements of the operon.     

Control of the expression and compartmentalization of (sigma)G activity during sporulation of Bacillus subtilis by regulators of (sigma)F and (sigma)E

During formation of spores by Bacillus subtilis the RNA polymerase factor sigma(G) ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of sigma(G) ordinarily requires prior activity of sigma(F) in the prespore and sigma(E) in the mother cell. Here we report that in spoIIA mutants lacking both sigma(F) and the anti-sigma factor SpoIIAB and in which sigma(E) is not active, sigma(G) nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of sigma(G) activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for sigma(G), from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to sigma(G). When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, sigma(G) became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature sigma(G) activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of sigma(G) activity, and pro-sigma(E) (but not sigma(E)) can counteract this effect of SpoIIGA. We find that in strains lacking sigma(F) and SpoIIAB and engineered to produce active sigma(E) in the mother cell without the need for SpoIIGA, sigma(G) also becomes active in the mother cell.     

A mutation in P23, the first gene in the RNA polymerase sigma A (sigma 43) operon, affects sporulation in Bacillus subtilis.

Mutations within P23, the first gene of the Bacillus subtilis sigma A operon, were not detrimental to vegetative growth or sporulation. One deletion of P23 resulted in a strain that sporulated earlier than the wild type. This aberrant phenotype may be due to the simultaneous deletion of a sigma H promoter from the sigma A operon.     

AbrB is a regulator of the sigma(W) regulon in Bacillus subtilis

The Bacillus subtilis global regulator AbrB was found to negatively control expression of sigW and genes of the sigma(W) regulon. AbrB bound to DNA regions in the autoregulatory sigW promoter and to some, but not all, of the other sigma(W)-dependent promoters in B. subtilis. Defects in antibiotic resistance properties caused by spo0A mutations are at least partially correlated with AbrB repression of the sigma(W) regulon.     

Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K

The bacterial endospore cortex peptidoglycan is synthesized between the double membranes of the developing forespore and is required for attainment of spore dehydration and dormancy. The Bacillus subtilis spoVB, spoVD and spoVE gene products are expressed in the mother cell compartment early during sporulation and play roles in cortex synthesis. Here we show that mutations in these genes block synthesis of cortex peptidoglycan and cause accumulation of peptidoglycan precursors, indicating a defect at the earliest steps of peptidoglycan polymerization. Loss of spoIV gene products involved in activation of later, sigma(K)-dependent mother cell gene expression results in decreased synthesis of cortex peptidoglycan, even in the presence of the SpoV proteins that were synthesized earlier, apparently due to decreased precursor production. Data show that activation of sigma(K) is required for increased synthesis of the soluble peptidoglycan precursors, and Western blot analyses show that increases in the precursor synthesis enzymes MurAA, MurB, MurC and MurF are dependent on sigma(K) activation. Overall, our results indicate that a decrease in peptidoglycan precursor synthesis during early sporulation, followed by renewed precursor synthesis upon sigma(K) activation, serves as a regulatory mechanism for the timing of spore cortex synthesis.     

Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor.

By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.     

FosB, a cysteine-dependent fosfomycin resistance protein under the control of sigma(W), an extracytoplasmic-function sigma factor in Bacillus subtilis

We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires sigma(W), and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn(2+)-dependent glutathione transferases but with a preference for Mg(2+) and L-cysteine as cofactors.     

Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates sigma(B) by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of sigma(B) from an anti-sigma factor inhibitor. By using the yeast dihybrid selection system to identify B. subtilis proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX). B. subtilis depleted of Obg no longer activated sigma(B) in response to environmental stress, but it retained the ability to activate sigma(B) by the ATP responsive pathway. Stress pathway components activated sigma(B) in the absence of Obg if the pathway's most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of sigma(B) and suggest that Obg may directly communicate with one or more sigma(B) regulators.