Expression of a pepT homologue from Bacillus subtilis

Abstract We isolated pepT from Bacillus subtilis , a gene with homology to various tripeptidases from different bacterial sources, pepT is preceded by genes encoding a two component regulatory system. Its expression is activated during stationary phase. In minimal medium this activation is boosted in the presence of phosphate. The response regulator is preceded by putative promoter consensus sequences recognized by the stationary phase specific sigma factors σ ^H, σ ^F, and σ ^G. This is in accordance with the initiation of expression at the beginning of stationary phase. Inactivation of pepT causes no obvious phenotype.     

SpoIVB has two distinct functions during spore formation in Bacillus subtilis

The Bacillus subtilis SpoIVB protein is a critical component of the intercompartmental signal-transduction pathway that activates the sigma factor, σ^K, in the mother cell of the sporulating cell. SpoIVB, synthesized in the forespore chamber, must act across two layers of phospholipid membrane to facilitate proteolytic processing of inactive pro-σ^K to active σ^K. We have used a genetic approach to dissect SpoIVB function and found that this protein has two distinct developmental functions. One function is that of intercompartmental signalling of pro-σ^K processing. The other role is essential to spore formation and is illustrated by mutations of SpoIVB which allow cell–cell signalling of pro-σ^K processing but prevent the formation of viable spores. Using localized and site-specific mutagenesis we have identified a functional domain of SpoIVB that is involved in its non-signalling role.     

A new RNA polymerase sigma factor, σF is required for the late stages of morphological differentiation in Streptomyces spp.

A gene ( sigF ) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial σ⁷⁰ family. Homologues were present in other Streptomyces spp., and that of the genetically well-studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 ( S. coelicolor ) and 295 ( S. aureofaciens ) amino acid residues, both showing greatest similarity to σ^B of Bacillus subtilis . However, while σ^B is involved in stationary-phase gene expression and in the general stress response in B. subtilis , σ^F affects morphological differentiation in Streptomyces , Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. σ^F is proposed to control the late stages of spore development in Streptomyces .     

Molecular genetic characterization of new bovine kappa-casein alleles CSN3F and CSN3G and genotyping by PCR-RFLP

In order to characterize the two new kappa-casein variants F and G (CSN3^F and CSN3^G) recently detected in Ayrshire and Pinzgauer cattle, exon IV of CSN3 from heterozygous animals was amplified by polymerase chain reaction (PCR), cloned and sequenced. The sequencing data revealed single point mutations at nucleotide positions 10530 (G→A) for CSN3^F and 10790 (C→T) for CSN3^G, corresponding to amino acid exchanges in positions 10 (Arg→His) and 97 (Arg→Cys) respectively. These mutations alter recognition sites for the restriction enzymes HhaI and MaeII , which were subsequently used to confirm these polymorphisms in cattle carrying CSN3^F or CSN3^G. A PCR-restriction fragment length polymorphism (RFLP) genotyping procedure for all currently known CSN3 alleles (CSN3^A, CSN3^B, CSN3^C, CSNJ^E, CSN^F, CSN3^G) was developed.