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-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.

FIG. 1

Map of plasmid pSWEET-bgaB. (a) Significant features of the xylose-based expression system. Plasmid pSWEET-bgaB is a derivative of pDG364 (3), which allows integration into the B. subtilis chromosome at amyE via double recombination and selection with CHL (10 μg/ml). The plasmid also has an E. coli origin of replication, denoted ori, and an ampicillin resistance cassette (50 μg/ml) for routine cloning steps. On the outside of the plasmid map, restriction sites of interest are highlighted, including two PstI sites for convenient plasmid linearization, a PacI site (eight-base recognition sequence TTAATTAA) upstream of bgaB, and a polylinker downstream of bgaB (HindIII is not unique). (b) Close-up of key elements of the xylose expression system (not to scale). Shown are xylR, encoding the xylose repressor; the xyl intergenic region, including promoters for xylR (P_xylR), xylA (P_xylA), and xyl operator sequences (xylO); translationally truncated xylA (first 58 nucleotides followed by an in-frame TAA), including an optimized CRE in xylA (see Materials and Methods and reference 18); PacI 5′ cloning site; ribosome binding site (SD) native to B. subtilis tagD; and gene bgaB, encoding a thermostable β-galactosidase from B. stearothermophilus.

FIG. 2

Detection of reporter gene expression by X-Gal hydrolysis on solid media. (a) Strains EB106 (pSWEET) and EB107 (pSWEET-bgaB) were plated on LB–CHL–X-Gal in the presence of 2% xylose. (b) Strains EB103 (pSPAC-bgaB), EB104 (pSPAC-bgaB ΔlacI), and EB105 (pSPAC) were plated on LB–CHL–X-Gal in the presence of 1 mM IPTG. (c) All five strains were plated on LB–CHL–X-Gal in the absence of an inducer. Strains were grown overnight at 37°C and then incubated at 55°C for color development (up to 36 h).

FIG. 3

Linearity of heat-stable β-galactosidase assay. Strain EB104 was grown (LB-CHL with 1 mM IPTG) to mid-log phase (OD₆₀₀ = 0.5) and assayed for BgaB activity (see Materials and Methods) at different sample volumes (○, ▿, and □ denote 0.1, 0.2 and 0.5 ml of culture, respectively) for up to 4 h. β-Galactosidase activity was defined as micromoles of o-nitrophenol released after 0, 10, 30, 60, 120, and 240 min. Slopes for 0.1, 0.2, and 0.5 ml of culture were 0.11, 0.20, and 0.47 nmol/min. Errors are standard deviations from three separate experiments.

FIG. 4

Modulation of the xyl and spac expression systems. Saturated cultures of EB107 (pSWEET-bgaB) and EB103 (pSPAC-bgaB) were inoculated (1/100) into fresh LB-CHL media supplemented with increasing concentrations of inducer, i.e., 0.00002 to 6.3% xylose (○) and 100 nM to 1 mM IPTG (□). Cultures were grown to mid-log phase and assayed for BgaB activity (see Materials and Methods). β-Galactosidase units are picomoles of o-nitrophenol released per minute per milliliter of culture at an OD₆₀₀ of 0.5. Errors are standard deviations from at least three separate experiments.

FIG. 5

Xylose-dependent complementation of a temperature-sensitive mutant in teichoic acid biosynthesis. Strains were plated on LB agar in the presence or absence of an inducer and incubated at 30°C (permissive temperature) or 47°C (nonpermissive temperature). Strains EB4 (tag-12), EB6 (tag⁺), EB123 (tag-12 pSWEET-tagD), and EB127 (tag-12 pSPAC-tagD) were plated at 30°C (a), plated at 47°C (b), supplemented with 1 mM IPTG at 47°C (c), and supplemented with 2% xylose at 47°C (d). Strains shown in panels b through d were plated as indicated in panel a.

FIG. 6

Growth profile of the B. subtilis tag-12 mutant and conditional complementation with tagD under xylose control. Overnight cultures (LB) of EB4 (tag-12), EB6 (tag⁺), and EB123 (tag-12 pSWEET-tagD) were inoculated (1/100) into fresh medium (LB) and grown for 25 h, with periodic monitoring of growth by OD₆₀₀. Growth curves for EB4 and EB6 are represented by the symbols ○ and ●, respectively. EB123 was grown in the presence (▾) and absence (▿) of 0.2% xylose. Growth was at 30°C (permissive temperature for tag-12) until mid-log phase, when cultures were shifted to 47°C (restrictive temperature for tag-12) at 300 min (denoted by the arrow).