Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure.
High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis secretion mutant after nitrosoguanidine mutagenesis. At 42 degrees C, but not at 30 degrees C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and and was also defective in the secretion of subtilisin Carlsberg. The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non-permissive temperature. The secretion defect was not linked to the secA/div locus.
Jacobs
M F
MF
Department of Microbiology, Technical University of Denmark, Lyngby.
Andersen
J B
JB
Borchert
T V
TV
Kontinen
V P
VP
D001412:Bacillus subtilis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors; D014994:Xylose / Q000494:pharmacology; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C074322:XylR protein, Pseudomonas; D014994:Xylose; D001616:beta-Galactosidase; D013381:Subtilisins