Activation of spo0A transcription by sigma H is necessary for sporulation but not for competence in Bacillus subtilis.

spo0A and spo0H are needed for the initiation of sporulation and for the development of genetic competence in Bacillus subtilis. Transcription of spo0A initiates from two promoters, Pv and Ps. Pv is active during vegetative growth and is recognized by RNA polymerase containing sigma A. Expression from Ps increases during sporulation and depends on sigma H, the spo0H gene product. A deletion mutation, spo0A delta Ps, that removes the promoter controlled by sigma H blocked sporulation but had no detectable effect on competence. These results indicate that expression of spo0A from Ps is necessary for sporulation and that the requirement for spo0H in competence development is not due to its role in expression of spo0A.

Alanine dehydrogenase (ald) is required for normal sporulation in Bacillus subtilis.

The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). The ski22::Tn917lac insertion mutation was in ald, the gene encoding alanine dehydrogenase. Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source. The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation. The spoVN::Tn917 mutation was also found to be an allele of ald. Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth. Expression of ald was normal in all of the regulatory mutants tested, including spo0A, spo0K, comA, sigB, and sigD mutants. The only gene in which mutations affected expression of ald was ald itself. This regulation is probably related to the metabolism of alanine.

Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis.

The development of competence in Bacillus subtilis is regulated by growth conditions and several regulatory genes. In complex media competence development is poor, and there is little or no expression of late competence genes. mec mutations permit competence development and late competence gene expression in complex media, and bypass the requirements for many of the competence regulatory genes. In this paper we describe the cloning and characterization of mecA. The mecA gene product acts negatively in the development of competence. Null mutations in mecA allowed expression of a late competence gene comG, under conditions where it is not normally expressed, including in complex media and in cells mutant for several competence regulatory genes. Overexpression of MecA from a multicopy plasmid resulted in inhibition of comG transcription. The DNA sequence of mecA was determined and the predicted gene product showed no significant similarity to any protein in the database. Expression of a mecA-lacZ translational fusion was constitutive during growth and did not vary significantly in the different media tested. The role of mecA in competence development and other stationary phase phenomena is discussed.

Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor.

Multiple physiological and environmental signals are needed to initiate endospore formation in Bacillus subtilis. One key event controlling sporulation is activation of the Spo0A transcription factor. Spo0A is a member of a large family of conserved regulatory proteins whose activity is controlled by phosphorylation. We have isolated deletion mutations that remove part of the conserved amino terminus of Spo0A and make the transcription factor constitutively active, indicating that the amino terminus normally functions to keep the protein in an inactive state. Expression of an activated gene product is sufficient to activate expression of several sporulation genes in the absence of signals normally needed for initiation of sporulation. Our results indicate that nutritional, cell density, and cell-cycle signals are integrated through the phosphorylation pathway that controls activation of Spo0A.

Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.