Open AccessDevelopment and Characterization of a Xylose-Dependent System for Expression of Cloned Genes in Bacillus subtilis : Conditional Complementation of a Teichoic Acid Mutant
Author(s) -
Amit P. Bhavsar,
Xumei Zhao,
Eric D. Brown
Publication year - 2001
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.67.1.403-410.2001
Subject(s) - complementation , operon , bacillus subtilis , repressor , biology , xylose , biochemistry , regulon , lac operon , xylose isomerase , mutant , microbiology and biotechnology , catabolite repression , gene , transcription (linguistics) , plasmid , gene expression , genetics , bacteria , fermentation , linguistics , philosophy
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.