A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus

Abstract

Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

Figure 1.

Vanillate degradation by Caulobacter. (A) Conversion of vanillate into protocatechuate, as catalyzed by the vanillate demethylase (VanAB) complex. (B) Arrangement of the vanR, vanA and vanB genes in C. crescentus, Acinetobacter sp. ADP1 and Pseudomonas sp. HR199. (C) Cell densities achieved with vanillate or glucose as the sole carbon source. Minimal media composed of 0.5 mM vanillate (light bars) or glucose (dark bars), respectively, in M2 salts (6) were inoculated with washed cells of wild-type strain CB15N. The cultures were grown until the carbon source was depleted, and their optical densities were determined (growth cycle 1). Subsequently, the cells were subjected to another five growth cycles, each of which started with replenishment of the carbon source (0.5 mM final concentration), followed by growth to stationary phase and determination of the respective cell densities. Note: Stepwise addition of the carbon source was necessary due to a negative effect of vanillate on growth at concentrations higher than 0.5 mM. (D) Involvement of CC2393 (VanA) in vanillate degradation. M2G minimal medium containing no (−) or 0.5 mM (+) vanillate, respectively, was inoculated with wild-type strain CB15N (WT) or its ▵vanA-derivative MT231 and incubated for 24 h. Subsequently, the cells were pelleted, and the supernatant was analyzed spectrophotometrically at 286 nm (the absorption maximum of vanillate) to determine the amount of vanillate left in the medium. Sterile medium (w/o) was used as a control. Data represent the mean of three independent experiments (±SD). (E) Induction kinetics of vanillate degradation. Cells of wild-type strain CB15N were grown to exponential phase in M2G minimal medium, washed, and resuspended in M2G medium containing 0.5 mM vanillate to an OD₆₀₀ of 0.11. Samples were taken at one-hour intervals and analyzed for cell density (OD₆₀₀, open triangle) and vanillate content (E₂₈₆, filled circle).

Figure 2.

Characteristics of the vanAB promoter. (A) Overview of the vanAB promoter region. The vanR-vanAB intergenic region is shown in normal print, whereas the 5′ ends of the flanking vanR and vanA genes are given in boldface, with their orientations indicated by arrows. The transcriptional start site of vanA (see Figure 2B) and the putative −10 and −35 motifs of the vanA promoter are labeled. Diverging arrows and italic letters mark two copies of a perfectly palindromic sequence, which is likely to represent the VanR target site. (B) Determination of the vanAB transcriptional start site. Primer extension analysis was conducted on RNA extracted from cells of wild-type strain CB15N which had been grown in M2G medium in the presence (lane 5) or absence (lane 6) of 0.5 mM vanillate. In parallel, sequencing reactions were performed (lanes 1–4). The two reaction products and the corresponding +1 sites are indicated by arrows.

Figure 3.

Response of the vanAB promoter to different inducers and inducer concentrations. (A) Reporter constructs used to determine P_van promoter activity. The 5′ region of vanA, which was fused in frame to the lacZ gene, is shown in black. (B) Activity of P_van in the presence of different inducers. Wild-type strain CB15N was transformed with reporter plasmid pMT122 and grown in M2G minimal medium. In mid-exponential phase, vanillate, vanillin or vanillyl alcohol (vanOH) were added to a final concentration of 0.5 mM, respectively, and the activity of P_van was determined. The inset shows the structural formulas of the inducers used. (C and D) Response of P_van to different concentrations of vanillate. Cells of strain MT231 (▵vanA) transformed with reporter plasmid pMT122 were grown in M2G minimal medium (C) or PYE rich medium (D), exposed for 3 h to different concentrations of vanillate, and used to determine the activity of P_van. The inset in (C) shows P_van promoter activity at vanillate concentrations of 0–5 µM. Data depicted in panels (B), (C) and (D) represent the average of two independent experiments, each performed in triplicate. Standard deviations were smaller than 11% throughout.

Figure 4.

Influence of sugars on the activity of the vanAB promoter. Cells of wild-type strain CB15N transformed with reporter plasmid pMT122 (Figure 3A) were grown in PYE medium and exposed to 0.5 mM vanillate (w/o), a mixture of 0.5 mM vanillate and glucose or xylose at the indicated concentrations, or a mixture of 0.5 mM vanillate, 20 mM glucose and 20 mM xylose. Subsequently, the cell were subjected to β-galactosidase activity assays to determine the response of P_van under the different inducing conditions. Data represent the mean of two independent experiments (± SD), each performed in triplicate.

Figure 5.

Involvement of VanR in the regulation of vanAB expression. Cells of wild-type strain CB15N (WT) and its mutant derivative MT219 (ΔvanR) were transformed with reporter plasmids pMT122 (vanR) or pMT129 (ΔvanR) and grown to exponential phase in PYE medium. Subsequently, they were exposed to 0 mM (−) or 0.5 mM (+) vanillate, and used to determine the activity of P_van. Data represent the mean of two independent experiments (± SD), each performed at least in triplicate.

Figure 6.

Integration Plasmids. This schematic shows different sets of vectors designed for (A) integrating constructs at a chromosomal site of interest, (B) inserting a minimal P_xyl or P_van fragment upstream of a defined chromosomal locus, (C) creating C-terminal protein fusions encoded at the native locus of the gene of interest, and generating inducible (D) C-terminal or (E) N–terminal protein fusions encoded at the xylX or vanA locus. Plasmid names are determined as follows: each construct carries the prefix ‘p’, followed by a string of terms that describes its characteristic features and resistance gene. The individual terms are shown in red print and are to be lined up from left to right to yield the correct designation. For each category of plasmids, the name of one construct is given as an example. The different MCS (MCS, A-E) are detailed in Figure 7. The following gene names were used: RK2 origin of transfer (oriT), E. coli rrnB T1T2 transcriptional terminator (ter), pMB1 origin of replication (oriV), aminoglycoside adenylyltransferase (Spc/Str^R, aadA), neomycin phosphotransferase I (Kan^R, nptI), rifampicin ADP-ribosyltransferase (Rif^R, parr-2), AAC(3)-I aminoglycoside acetyltransferase (Gent^R, aacC1), tetracycline efflux permease (Tet^R, tetA) and repressor (tetR), chloramphenicol acteyltransferase (Cam^R, cat), AAC(3)-IV aminoglycoside acetyltransferase (Apr/Gent^R, aacC4), enhanced green fluorescent protein (egfp), enhanced yellow fluorescent protein (eyfp), enhanced cyan fluorescent protein (ecfp), Venus (venus), Cerulean (cerulean), mCherry (mCherry), FLAG-tag (flag), Strep-tag II (strep), tetracysteine tag (tcys). Plasmids are shown to scale, except where indicated by breaks or dotted lines.

Figure 7.

Multiple cloning sites. The sequences of the different MCS (MCS A–E) are shown in boldface. Flanking Shine-Dalgarno motifs (RBS, as present in P_van-containing constructs) and fusion tags (exemplified by egfp) are indicated. Restriction sites that are unique in all plasmids carrying the respective MCS are highlighted in boldface, whereas those that are unique in a subset of constructs only are shown in normal print.

Figure 8.

Application examples. (A) Expression of a mipZ-eyfp fusion, obtained by insertion of the mipZ gene into plasmid pVYFPC-1, under the control of P_van. Cells of strain MT232 (P_van-mipZ-eyfp) were examined by DIC and fluorescence microscopy before and 2h after induction with 0.5 mM vanillate. Note: MipZ is a spatial regulator mediating proper positioning of the cell division apparatus in Caulobacter (10). It forms a complex with the DNA partitioning protein ParB at the chromosomal origin of replication. Before initiation of chromosome replication, the MipZ·ParB·origin complex localizes to the flagellated pole of the cell. After entry into S phase, MipZ and ParB re-associate with the newly synthesized origin regions, generating two nucleoprotein complexes which are positioned at the two opposite cell poles. (B) Expression of an egfp-mipZ fusion, created using plasmid pXGFPN-2, under the control of P_xyl. The images show cells of strain MT236 (P_xyl-egfp-mipZ) before and two hours after induction with 0.03% xylose. (C) Expression of an ftsZ-eyfp fusion, constructed with the help of plasmid pVCHYC-3, under the control of P_van. Cells were withdrawn from a culture of strain MT240 (P_van-ftsZ-eyfp) before and 1h after induction with 0.5 mM vanillate and analyzed by microscopy. Note: The tubulin homologue FtsZ is a fundamental component of the bacterial cell division machinery. Upon initiation of chromosome replication and bipolar positioning of MipZ, it forms a ring-like structure at the cell center which recruits the other constituents of the cytokinetic ring and plays an essential role in the constriction process (10,60).

Figure 9.

Replicating plasmids. Shown are four sets of replicating shuttle vectors designed for the inducible expression of genes from (A) a high-copy number or (B) a low-copy number plasmid and for the construction of (C) C-terminal and (D) N-terminal fluorescent protein fusions, expressed under the control of P_van from a low-copy number plasmid. The nomenclature of these plasmids follows the same rules as described in Figure 6, but the prefix is ‘pB’ for high-copy number plasmids and ‘pR’ for low-copy number plasmids. The gene names used are as follows: pBBR1 replication protein (rep), pBBR1 mobilization protein (mob), RK2 replication initiatior protein (trfA), β-lactamase (Amp^R, bla). All other gene designations are detailed in Figure 6.