Development and Characterization of a Xylose-Dependent System for Expression of Cloned Genes in Bacillus subtilis : Conditional Complementation of a Teichoic Acid Mutant

Abstract

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-␤-D-thiogalactopyranoside-induced spac system using a thermostable ␤-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.

Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis

“…Arabinose (1% w/v) was used as a carbon source in place of glucose. Xylose (1% w/v) was added to induce expression of Pxyl-rapI (Bhavsar et al, 2001). Antibiotics were used at the following concentrations for B. subtilis: kanamycin (5 μg ml −1 ), spectinomycin (100 μg ml −1 ), chloramphenicol (5 μg ml −1 ), tetracycline (10 μg ml −1 ), and a combination of erythromycin (0.5 μg ml −1 ) and lincomycin (12.5 μg ml −1 ) to select for macrolide-lincosamide-streptogramin (MLS) resistance.…”

Identification of Two Phosphate Starvation-induced Wall Teichoic Acid Hydrolases Provides First Insights into the Degradative Pathway of a Key Bacterial Cell Wall Component

“…We endeavored to complement the phosphate starvation growth phenotype in mutant strains by introducing a copy of glpQ or phoD at the amyE locus. To do this, we used a xyloseinducible promoter (33), but these attempts were frustrated by leakiness in the induction system that seemed to mask the effect of complementing genes during phosphate-limited growth. More compelling results were achieved with experiments that showed exogenous WTA stimulated phosphate-starved growth in complemented strains (supplemental Fig.…”

Characterization of the Bacillus subtilis GTPase YloQ and its role in ribosome function

“…A strain containing an additional copy of yloQ at the amyE locus on the chromosome was created using the plasmid pSWEET as described previously [15]. Primers yloQ-F and yloQ-R were used to PCR-amplify the gene and clone into pSWEET.…”

“…We set out to characterize the biological function of YloQ with the construction of a B. subtilis strain that conditionally expressed yloQ. Using methods developed in our laboratory [15], a copy of yloQ was placed under the control of a tightly regulated xyloseinducible promoter at the amyE locus. Subsequently, the wildtype copy of yloQ was replaced with a spectinomycin resistance cassette.…”