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.

Purified, Recombinant TagF Protein from Bacillus subtilis 168 Catalyzes the Polymerization of Glycerol Phosphate onto a Membrane Acceptor in Vitro

“…This study strongly suggested that tagF encodes the main chain polymerase (Scheme 2). In the work reported here, we have further characterized one such mutant, tagF1, by inserting a wild type copy of tagF under the control of a xylose-based expression system (15) at amyE. We have thus demonstrated an unequivocal role for tagF in the thermosensitivity of this strain through xylosedependent complementation of the mutant at the non-permissive temperature.…”

“…The resulting PCR product was cloned into PacI and BamHI restriction sites within pSWEET-bgaB (15), displacing the bgaB gene, and the resulting insert was sequenced using primers JS20 -JS25. The insertional plasmid pSWEET-tagF was used to engineer strain EB247 to contain a second complementing copy of tagF at the amyE locus resulting in the creation of strain EB311.…”

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

“…Complementation Assay-pSWEET-bgaB was kindly provided by Amit P. Bhavsar, McMaster University, Hamilton, Ontario, Canada (25). To create pSWEET-expressing Pdx1, a PacI site and nucleotides Ϫ24 to Ϫ1 of the B. subtilis tagD gene providing a ribosomal binding site (RBS) were introduced at the 5Ј-end and a stop codon and a BamHI at the 3Ј-end of Pdx1 and cloned into the PacI/BamHI sites of pSWEET.…”

“…All other variations are located on the surface of the molecule opposite to the synthase interaction site, e.g. in loops Pdx2 [25][26][27][28][29][30] Fig. 4, A and B).…”

“…Two different constructs of Pdx1 cloned into the expression vector pSWEET (25) were used for the complementation assays. One construct contains the coding sequence of Pdx1 with a ribosome binding site (RBS-Pdx1), whereas the other is lacking the ribosomal binding site (Pdx1) and thereby prohibiting translation of the parasite protein.…”