Cloning and expression in Escherichia coli of the regulatory sacU gene from Bacillus subtilis.
The regulatory wild-type locus sacU, which has a pleiotropic effect in Bacillus subtilis, notably on the synthesis of secreted proteins, was obtained from a colony bank of Escherichia coli harboring recombinant cosmids representative of the B. subtilis genome. It was shown that the sacU gene is located on a 2.4-kilobase KpnI-EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. The wild-type phenotype was recovered after transformation of SacU-, SacUh, and SacU- Rec- strains with the recombinant cosmid, indicating that the sacU locus has been cloned in totality. The sacU gene was expressed in a minicell-producing E. coli strain, and it was shown that it coded for a 46-kilodalton protein. In addition to the hypersecretion of proteins, SacUh mutants were characterized by the presence of a 46-kilodalton protein in the membrane fraction in higher amounts than were found in the wild-type strain. These mutants were also devoid of a 36-kilodalton polypeptide corresponding to the flagellin subunit. Analysis of the mRNA content of a secreted protein (levansucrase) in SacU- and SacUh mutants strongly suggested that the pleiotropic action of the sacU gene on the synthesis of levansucrase is exerted at a posttranscriptional level in B. subtilis cells and is probably correlated with the mechanism of secretion of exoenzymes.
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005809:Genes, Regulator; D006602:Hexosyltransferases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008970:Molecular Weight; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D008565:Membrane Proteins; D006602:Hexosyltransferases; C021513:levansucrase