Identification of a membrane protein involved in activation of the KinB pathway to sporulation in Bacillus subtilis

Abstract

The initiation of sporulation in Bacillus subtilis is dependent on the phosphorylation of the Spo0A transcription factor mediated by the phosphorelay and by two major kinases, KinA and KinB. Temporal expression of these kinases was analyzed, and an assessment of their respective contributions to the production of Spo0AϳP was undertaken. The results show that KinB is expressed and activated prior to KinA; i.e., the two kinases are solicited sequentially in the sporulation process and are thought to be activated by different signaling pathways. A strategy was developed to isolate mutations specifically affecting the KinB pathway, using the newly improved mini-Tn10 delivery vector pIC333. Several mutants were obtained, one of which carried a transposon in a gene coding for a small integral membrane protein, named KbaA. Inactivation of the kbaA gene appeared to affect KinB activity but not transcription of kinB. A Spo ؉ suppressor (kinB45) of the kbaA null mutation was isolated in the promoter region of kinB. An eightfold increase of kinB expression levels over wild-type levels was observed in the kinB45 mutant. Thus, overexpression of the kinB-kapB operon was sufficient to overcome the sporulation defect caused by inactivation of kbaA in a KinA ؊ strain. Transcription of kinB was found to be repressed by SinR, while the kinB45 mutant was no longer sensitive to SinR regulation. Implications of these observations on the transcriptional regulation of kinB and the role of KbaA in KinB activation are discussed.

Alkaline Serine Protease AprE Plays an Essential Role in Poly-γ-glutamate Production during Natto Fermentation

“…For vpr inactivation, the vpr-upstream and vpr-downstream fragments were amplified with two primer pairs, vpr-F1/F2 and vpr-B1/B2 (Table 2) respectively. A fragment of the spectinomycin resistance gene (aadII) cassette of plasmid pIC333 23) was prepared by PCR with primer pair aad-F/aad-B ( Table 2). The transformants were selected on plates containing spectinomycin (100 mg/mL).…”

KapB is a lipoprotein required for KinB signal transduction and activation of the phosphorelay to sporulation in Bacillus subtilis

“…Several lines of evidence indicate that KinA and KinB play distinct roles in the initiation of sporulation (Dartois et al, 1996) and are likely to be regulated by different input signals. Although KinA is a cytoplasmic kinase (Perego et al, 1989;Antoniewski et al, 1990), the KinB amino domain consists exclusively of six membrane-spanning helices with virtually no outside loop.…”

“…KinB displays unique properties in that it is expressed and activated before KinA, it responds to distinct input signals (Dartois et al, 1996; and requires the product of the co-transcribed kapB ORF for its function (Trach and Hoch, 1993). To specifically investigate the KinB signalling pathway, transposon mutagenesis was carried out using the spectinomycin mini-Tn10 delivery vector pIC333 (Steinmetz and Richter, 1994) in the ⌬kinA96 strain MB340, where sporulation is entirely dependent on KinB.…”

“…To specifically investigate the KinB signalling pathway, transposon mutagenesis was carried out using the spectinomycin mini-Tn10 delivery vector pIC333 (Steinmetz and Richter, 1994) in the ⌬kinA96 strain MB340, where sporulation is entirely dependent on KinB. The mini-Tn10 insertion library was screened for Spo0 mutants (see Dartois et al, 1996 for details). To discriminate between mutants with defects in spo0 genes from those affected in kinB or in KinB-signalling genes, the transposon insertions were transferred into the wild-type strain (KinA þ ) and those that did not alter the recipient Spo þ phenotype on plates were assumed to affect only the KinB pathway.…”

“…The insertion target sequence was rescued together with the mini-Tn10 transposon as described previously (Dartois et al, 1996), and sequenced. The transposon was found to be inserted in a lgt-like gene encoding the lipoprotein glyceryl transferase that participates in post-translational lipid modification of prolipoproteins before their processing by the signal peptidase II (Sankaran and Wu, 1994a).…”