No abstract

The phosphorelay is the signal-transduction system recognizing and integrating environmental signals to initiate sporulation. The major signal input to the phosphorelay is an ATP-dependent kinase, KinA, responsible for phosphorylating the SpoOF protein. Mutants lacking KinA, however, still sporulate, suggesting that other kinases can fulfil its role. In order to identify these kinases, genes for kinases were isolated by hybridization using a degenerate oligonucleotide probe designed for common regions of this class of kinases. A gene for a second kinase, KinB, was isolated which gave a sporulation negative phenotype when inactivated in a kinA background. The kinB locus was sequenced and found to be a small operon consisting of the kinB gene and another gene, kapB, transcribed from a single.sigma A.-dependent promoter. Inactivation of either kinB or kapB in a kinA strain led to severe sporulation deficiency. The kinB gene coded for a 47774 M(r) protein with the carboxyl half of this protein highly homologous to the same domain of KinA. The amino-terminal domain of KinB was hydrophobic with six recognizable membrane-spanning regions. The kapB gene coded for a moderately charged, probably soluble, protein of 14,668 M(r) with no homology to any known protein. Genetic evidence suggests that KapB is required either for the function of KinB or for its expression. Although double mutants kinA kinB cannot sporulate and assume a stage 0 phenotype, the SpoA approximately P-dependent regulation of the abrB gene is normal in these strains, suggesting that low levels of SpoA approximately P accumulate even in the absence of both kinases. This accumulation is dependent on functional spo0F and spo0B genes and its source is unknown. The KinA and KinB pathways are the only pathways capable of producing sufficient Spo0A approximately P to allow initiation and completion of sporulation under laboratory conditions.

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 SpoOA and SPOOF sporulation regulatory proteins. SpoOF protein was a much better phosphoreceptor for this kinase than SpoOA 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 SpoOA protein.Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spoOB, spoOE, and spoOF mutations but not of spoOA mutations. The results suggest that the spoOB-, spoOE-, and spoOF-dependent pathway of activation (phosphorylation) of the SpoOA 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 SpoOA protein.

Transcription of the spoOB gene and genes downstream of it was investigated by S1 nuclease protection experiments. The MATERIALS AND METHODS Bacterial strains, media, and transformation. Escherichia coli DH5a (F-endAl hsdRJ7 supE44 thi-l X-relAl gyrA96 recAl 480dlacZzAM15) competent cells were purchased from Bethesda Research Laboratories, Inc., and transformed as specified in the accompanying procedure. E. coli JM109[endAl hsdRJ7 supE44 thi-l X-recAl gyrA96 relAl A(lacproAB) (F' traD36 proAB lacI4, lacZAM15)] were made competent and transformed by the method of Dagert and Ehrlich (9). E. coli strains were maintained on L broth, and, when appropriate, ampicillin, chloramphenicol, and X-Gal (5-bromo-4-chloro-3-indoyl-3-D-galactopyranoside; Bethes-