The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis.

The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis. We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spoIIA promoter. Core RNA polymerase or holoenzyme containing sigma A was reconstituted with sigma H protein and used to transcribe the spoIIA promoter. Formation of heparin resistant transcription initiation complexes required that the spoIIA template be supercoiled. Topoisomers of the spoIIA template were created and transcribed at various temperatures. Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spoIIA promoter.

Control of the initiation of sporulation in Bacillus subtilis by a phosphorelay.

Sporulation in Bacillus subtilis is a developmental process induced as a response to nutritional stress. Activation of sporulation-specific gene transcription is under the control of the spoOA gene product. The SpoOA protein and the SpoOF protein are both homologous to response regulator proteins of two-component regulatory systems which control bacterial responses to a variety of environmental challenges. Response regulators are activated by specific kinases which phosphorylate them. In this study, it was shown that phosphorylation of SpoOA occurs via a phosphotransferase which is the product of the spoOB locus. The phosphodonor in this reaction is the phosphorylated form of SpoOF. It is postulated that SpoOF acts as a secondary messenger that can be phosphorylated by a variety of kinases depending on the particular environmental stress. The series of phosphate transfer reactions in this system is called a phosphorelay. The end product of this series of reactions is SpoOA approximately P which is shown to have greater affinity for the DNA target, the OA box, of SpoOA on the abrB promoter than the unphosphorylated form. SpoOA approximately P, but not SpoOA, was shown to be an activator of transcription of the spoIIA operon which codes for the sporulation-specific sigma factor sigma F. Thus, the initiation of sporulation is dependent on SpoOA approximately P which arises through the phosphorelay and which acts as a transcription factor to repress certain genes, e.g. abrB, and activate others, e.g. spoIIA.

Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay.

Stage 0 sporulation (spo0) mutants of Bacillus subtilis are defective in the signal transduction system initiating sporulation. Two of the products of these genes, Spo0A and Spo0F, are related to response regulator components of two-component regulatory systems used to control environmental responses in bacteria. The Spo0F response regulator was found to be the primary substrate for phosphorylation by the sporulation-specific protein kinase, KinA. Phosphorylated Spo0F was the phosphodonor for a phosphotransferase, Spo0B, which transferred the phosphate group to the second response regulator, the transcription regulatory protein Spo0A. This phosphorelay provides a mechanism for signal gathering from several protein kinases using Spo0F as a secondary messenger. These divergent signals are integrated through Spo0B phosphotransferase to activate the Spo0A transcription factor. This system provides for many levels of control to prevent capricious induction of sporulation.

The transition state transcription regulator AbrB of Bacillus subtilis is autoregulated during vegetative growth.

The DNA-binding AbrB protein of Bacillus subtilis is an ambiactive transcriptional regulator of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Studies on the transcriptional control of AbrB synthesis using abrB-lacZ fusions indicated that the abrB gene was autoregulated. This was consistent with the observation that purified AbrB protein bound specifically to the promoter region of its own gene in DNase I protection experiments. The structural gene mutation abrB4 abolished the autoregulation and purified AbrB4 protein did not have the promoter binding properties associated with the wild-type protein. Both AbrB and AbrB4 proteins were shown to be hexamers of 10,500 Dalton subunits and subunit exchange occurred between the proteins in vitro. However, the presence of only one or two mutant subunits dramaticaly altered the DNA-binding ability of the multimeric protein. The results support a model in which autoregulation of the abrB gene is an important factor in preventing sporulation-associated genes from being expressed during vegetative growth.

Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis.

The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins. Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro. Mutants with deletion mutations in the kinA gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein. Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations. The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the Spo0A protein.

The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein.

The product of the abrB gene of Bacillus subtilis is an ambiactive repressor and activator of the transcription of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Purified AbrB protein binds specifically in a highly co-operative fashion to fragments of DNA containing the promoters it affects. DNase I footprints of the binding regions in these promoters revealed large protected areas of 50-120 nucleotides or more depending on the promoter. Methylation protection experiments gave protected guanine residues on only one face of the DNA helix. A consensus sequence could be deduced around these guanine residues that was not found around non-protected guanine residues in the footprint region. The results suggested that stationary phase functions and sporulation are repressed during active growth by AbrB and other transition state regulators by binding to the affected promoters in a concentration-dependent manner.

Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis.

The four individual phosphotransfer steps in the multicomponent phosphorelay system controlling sporulation in Bacillus subtilis have been characterized kinetically using highly purified samples of the individual protein components in vitro. The autophosphorylation of KinA is the initial occurrence, and a divalent metal ion is required. KinA-mediated phosphotransfer, which displays a 57,000-fold preference (kcat/Km) for catalysis of Spo0F-P formation relative to Spo0A-P formation, is shown to proceed via a hybrid ping-pong/sequential mechanism with pronounced (> or = 40-fold) substrate synergism by Spo0F of KinA autophosphorylation. In addition, evidence is presented for formation of an abortive KinA.Spo0F complex. Kinetic parameters derived for Spo0F-P and Spo0A as substrates for Spo0B, the second phosphotransferase in the phosphorelay chain, indicate that Spo0B-mediated production of Spo0A-P is 1.1-million-fold more efficient (kcat/KSpo0A) than the direct KinA-mediated process. A rationale is presented for a four component cascade as the means for controlling sporulation, which focuses on the utility of synergistic interactions among the phosphorelay components that may be modulated by environmental stimuli.