Development and characterization of a xylose-dependent system for expression of cloned genes in Bacillus subtilis: conditional complementation of a teichoic acid mutant.
We have developed a xylose-dependent expression system for tight and modulated expression of cloned genes in Bacillus subtilis. The expression system is contained on plasmid pSWEET for integration at the amyE locus of B. subtilis and incorporates components of the well-characterized, divergently transcribed xylose utilization operon. The system contains the xylose repressor encoded by xylR, the promoter and 5' portion of xylA containing an optimized catabolite-responsive element, and intergenic xyl operator sequences. We have rigorously compared this expression system to the isopropyl-beta-D-thiogalactopyranoside-induced spac system using a thermostable beta-galactosidase reporter (BgaB) and found the xyl promoter-operator to have a greater capacity for modulated expression, a higher induction/repression ratio (279-fold for the xyl system versus 24-fold with the spac promoter), and lower levels of expression in the absence of an inducer. We have used this system to probe an essential function in wall teichoic acid biosynthesis in B. subtilis. Expression of the teichoic acid biosynthesis gene tagD, encoding glycerol-3-phosphate cytidylyltransferase, from the xylose-based expression system integrated at amyE exhibited xylose-dependent complementation of the temperature-sensitive mutant tag-12 when grown at the nonpermissive temperature. Plasmid pSWEET thus provides a robust new expression system for conditional complementation in B. subtilis.
Bhavsar
A P
AP
Antimicrobial Research Centre, Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
Zhao
X
X
Brown
E D
ED
D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D015870:Gene Expression; D005816:Genetic Complementation Test; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D009876:Operon; D010957:Plasmids / Q000235:genetics; D013682:Teichoic Acids / Q000378:metabolism*; D014994:Xylose / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D013682:Teichoic Acids; D014994:Xylose; D009713:Nucleotidyltransferases; C020675:glycerol-3-phosphate cytidylyltransferase; D001616:beta-Galactosidase