Figure 1.
Schematic representation of the experimental setup.
The rational behind this study was to engineer V. cholerae strains so that large gene clusters can be artificially expressed independent of growth condition restraints. The idea was to integrate the lacUV5-promoter controlled T7 RNA polymerase-encoded gene together with its repressor gene (lacI; both derived from E. coli BL21(DE3) [12]) into the V. cholerae chromosome. Using this strain, specific genes could be put under control of the T7 RNA polymerase solely by integrating the T7 RNA polymerase-dependent promoter sequence (indicated in black box; PT7) at the respective locus on the chromosome using natural transformation. Alternatively, the PT7 sequence was integrated on a plasmid as is commonly done in E. coli overexpression systems.
Table 1.
Bacterial strains and plasmids.
Figure 2.
Functionality of T7 RNA polymerase dependent reporter plasmid.
Plasmid pBR-[PT7]-GFP (panel A) was transferred into chemically competent E. coli BL21(DE3) cells. Transformed bacteria were grown in the absence or presence of 1 mM IPTG as indicated and tested for GFP expression using epifluorescence microscopy (panel B). Panel B upper row: phase contrast images showing all cells; middle row: green fluorescence channel with short exposure time (40 msec); lower row: green fluorescence channel with longer exposure time (1000 msec).
Figure 3.
Testing for T7 RNA polymerase-dependent expression of gfp reporter construct in V. cholerae.
Plasmid pBR-[PT7]-GFP was transferred into V. cholerae strain A1552 (WT; on the left) or its T7 RNA polymerase derivative AT7RNAP (on the right). Plasmid-containing bacteria were grown in rich medium either in the absence (−) or in the presence (+) of the inducer IPTG. Expression of gfp driven by the T7 RNA polymerase-dependent promoter was visualized by epifluorescence microscopy (green channel; lower row; same exposure time was applied to all samples). The corresponding phase contrast images are shown above.
Figure 4.
Insertion of the T7 RNA polymerase-dependent promoter sequence by TransFLP.
A: Schematic representation depicting the strategy to integrate the T7 RNA polymerase-dependent promoter sequence into the V. cholerae chromosome. Upper row: the Vibrio pathogenicity island (VPI-1 or tcp island) is indicated for the WT strain of V. cholerae (freely adapted from [46] and based on [47]; not to scale). Middle row: the transforming PCR-derived DNA fragment included parts of the genes tcpH and tcpA as flanking regions (in blue) to allow homologous recombination with the chromosome. In addition the PCR fragment carried the FRT-site (red rectangles) flanked kanamycin resistant cassette (aph; green arrow) and the T7 RNA polymerase-dependent promoter sequence (black box; according to Fig. 1). Lower row: the structure of the VPI-1 island after natural transformation and FLP recombination of the WT strain using the PCR fragment indicated in the middle row as transforming DNA material. B: PCR-based verification of site-directed insertion. The T7 RNA polymerase-dependent promoter sequence was inserted into the V. cholerae genome by chitin-induced natural transformation followed by FLP-mediated excision of the antibiotics resistance cassette (TransFLP; [3], [4]). The correctness of the resulting strain was tested by PCR using primer pair T7tcp-chk-up & T7tcp-chk-down and genomic DNA as template. The expected fragment sizes for the wild type (A1552; lane 1) and the ctx minus parental strain (AΔctxAB-T7RNAP; lane 2) (both 1′671 bp in length) as well as for the newly created T7 RNA polymerase-dependent promoter-containing strain AΔctxAB-[PT7]-tcp-T7RNAP (lane 3; 1′784 bp) are indicated by arrows. L, 1 kb ladder (Invitrogen; sizes indicated on the left).
Figure 5.
Expression of VPI-1 genes in a T7 RNA polymerase-dependent manner.
Expression of genes upstream and downstream the T7 RNA polymerase dependent promoter sequence was tested using qRT-PCR. A: Schematic representation of the genomic region of interest in the final strain AΔctxAB-[PT7]-tcp-T7RNAP. Genes whose expression was tested by qRT-PCR in panels B–C are indicated in bold and underlined. The PT7 promoter is indicated as a black rectangle with a black arrow on top. B–D: Comparison of gene expression between the engineered V. cholerae strain and its parental strains. Gene expression was measured in the engineered V. cholerae strain containing both the T7 RNA polymerase gene and the PT7 promoter sequence and compared to its two parental strains each containing only one of both elements. Bacteria were grown in rich medium in the presence of 1 mM IPTG. Genes indicated on the X-axis were tested for their expression level using qRT-PCR (normalized to the housekeeping gene gyrA). The values shown on the Y-axis depict the relative expression difference between strains AΔctxAB-[PT7]-tcp-T7RNAP ([PT7]+, T7RNAP+) and AΔctxAB-[PT7]-tcp ([PT7]+, T7RNAP−)(panel B), between strains AΔctxAB-[PT7]-tcp-T7RNAP ([PT7]+, T7RNAP+) and AΔctxAB-T7RNAP ([PT7]−, T7RNAP+)(panel C), and between the two control strains AΔctxAB-T7RNAP ([PT7]−, T7RNAP+) and AΔctxAB-[PT7]-tcp ([PT7]+, T7RNAP−) (panel C), respectively. Relative expression levels of recA are shown for comparison reason. Average of two independent biological replicates.
Figure 6.
Phenotypes of T7 RNA polymerase-mediated expression of the tcp cluster.
The V. cholerae strains AΔctxAB-[PT7]-tcp (lane 1), AΔctxAB-T7RNAP (lane 2), and AΔctxAB-[PT7]-tcp-T7RNAP (lane 3) were grown under shaking conditions in LB medium without or with supplementation of 1 mM IPTG. A: Macroscopic observation of an autoagglutination phenotype. Bacterial cultures were allowed to settle before pictures were taken. Agglutinated bacteria are indicated by a black arrow in the rightmost image. B: Microscopic observation of a microcolony formation phenotype. Microcolony formation of the cells was visualized using light microscope. Pictures were taken using phase contrast (upper panel) or DIC (lower panel).
Figure 7.
Visualization of TCP fibers by scanning electron microscopy.
V. cholerae cells were grown in rich medium as described for Fig. 6. At that stage bacteria were transferred to silicon wafers and processed for SEM. A representative image of V. cholerae strain AΔctxAB-[PT7]-tcp-T7RNAP containing both the T7 RNA polymerase gene and PT7 promoter preceding the tcp cluster is shown in panel A (EHT = 2.00 kV, WD = 3.7 mm, Mag = 13.48 k X). The control strain lacking the PT7 promoter sequence upstream the tcp cluster is shown in panel C (EHT = 2.00 kV, WD = 3.8 mm, Mag = 24.91 k X). The white rectangles in panel A and C indicate the regions that are magnified in panels B and D, respectively. Scale bar = 1 μm.