Overexpression of the tcp Gene Cluster Using the T7 RNA Polymerase/Promoter System and Natural Transformation-Mediated Genetic Engineering of Vibrio cholerae

The human pathogen and aquatic bacterium Vibrio cholerae belongs to the group of naturally competent bacteria. This developmental program allows the bacterium to take up free DNA from its surrounding followed by a homologous recombination event, which allows integration of the transforming DNA into the chromosome. Taking advantage of this phenomenon we genetically engineered V. cholerae using natural transformation and FLP recombination. More precisely, we adapted the T7 RNA polymerase/promoter system in this organism allowing expression of genes in a T7 RNA polymerase-dependent manner. We naturally transformed V. cholerae by adding a T7-specific promoter sequence upstream the toxin-coregulated pilus (tcp) gene cluster. In a V. cholerae strain, which concomitantly produced the T7 RNA polymerase, this genetic manipulation resulted in the overexpression of downstream genes. The phenotypes of the strain were also in line with the successful production of TCP pili. This provides a proof-of-principle that the T7 RNA polymerase/promoter system is functional in V. cholerae and that genetic engineering of this organism by natural transformation is a straightforward and efficient approach

Regulatory elements involved in the expression of competence genes in naturally transformable Vibrio cholerae

BackgroundThe human pathogen Vibrio cholerae normally enters the developmental program of natural competence for transformation after colonizing chitinous surfaces. Natural competence is regulated by at least three pathways in this organism: chitin sensing/degradation, quorum sensing and carbon catabolite repression (CCR). The cyclic adenosine monophosphate (cAMP) receptor protein CRP, which is the global regulator of CCR, binds to regulatory DNA elements called CRP sites when in complex with cAMP. Previous studies in Haemophilus influenzae suggested that the CRP protein binds competence-specific CRP-S sites under competence-inducing conditions, most likely in concert with the master regulator of transformation Sxy/TfoX.ResultsIn this study, we investigated the regulation of the competence genes qstR and comEA as an example of the complex process that controls competence gene activation in V. cholerae. We identified previously unrecognized putative CRP-S sites upstream of both genes. Deletion of these motifs significantly impaired natural transformability. Moreover, site-directed mutagenesis of these sites resulted in altered gene expression. This altered gene expression also correlated directly with protein levels, bacterial capacity for DNA uptake, and natural transformability.ConclusionsBased on the data provided in this study we suggest that the identified sites are important for the expression of the competence genes qstR and comEA and therefore for natural transformability of V. cholerae even though the motifs might not reflect bona fide CRP-S sites

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNAbinding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and sitedirected modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria

Glucose- but Not Rice-Based Oral Rehydration Therapy Enhances the Production of Virulence Determinants in the Human Pathogen Vibrio cholerae

Despite major attempts to prevent cholera transmission, millions of people worldwide still must address this devastating disease. Cholera research has so far mainly focused on the causative agent, the bacterium Vibrio cholerae, or on disease treatment, but rarely were results from both fields interconnected. Indeed, the treatment of this severe diarrheal disease is mostly accomplished by oral rehydration therapy (ORT), whereby water and electrolytes are replenished. Commonly distributed oral rehydration salts also contain glucose. Here, we analyzed the effects of glucose and alternative carbon sources on the production of virulence determinants in the causative agent of cholera, the bacterium Vibrio cholerae during in vitro experimentation. We demonstrate that virulence gene expression and the production of cholera toxin are enhanced in the presence of glucose or similarly transported sugars in a ToxR-, TcpP- and ToxT-dependent manner. The virulence genes were significantly less expressed if alternative non-PTS carbon sources, including rice-based starch, were utilized. Notably, even though glucose-based ORT is commonly used, field studies indicated that rice-based ORT performs better. We therefore used a spatially explicit epidemiological model to demonstrate that the better performing rice-based ORT could have a significant impact on epidemic progression based on the recent outbreak of cholera in Haiti. Our results strongly support a change of carbon source for the treatment of cholera, especially in epidemic settings

Insertion of the T7 RNA polymerase-dependent promoter sequence by TransFLP.

<p>A: Schematic representation depicting the strategy to integrate the T7 RNA polymerase-dependent promoter sequence into the <i>V. cholerae</i> chromosome. Upper row: the <i>Vibrio</i> pathogenicity island (VPI-1 or <i>tcp</i> island) is indicated for the WT strain of <i>V. cholerae</i> (freely adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053952#pone.0053952-Faruque1" target="_blank">[46]</a> and based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053952#pone.0053952-Heidelberg1" target="_blank">[47]</a>; not to scale). Middle row: the transforming PCR-derived DNA fragment included parts of the genes <i>tcpH</i> and <i>tcpA</i> 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 (<i>aph</i>; 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>B: PCR-based verification of site-directed insertion.</b> The T7 RNA polymerase-dependent promoter sequence was inserted into the <i>V. cholerae</i> genome by chitin-induced natural transformation followed by FLP-mediated excision of the antibiotics resistance cassette (TransFLP; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053952#pone.0053952-DeSouzaSilva1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053952#pone.0053952-Blokesch1" target="_blank">[4]</a>). 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 <i>ctx</i> 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).</p

Visualization of TCP fibers by scanning electron microscopy.

<p><i>V. cholerae</i> 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 <i>V. cholerae</i> strain AΔctxAB-[P_T7]-tcp-T7RNAP containing both the T7 RNA polymerase gene and P_T7 promoter preceding the <i>tcp</i> cluster is shown in panel A (EHT = 2.00 kV, WD  = 3.7 mm, Mag  = 13.48 k X). The control strain lacking the P_T7 promoter sequence upstream the <i>tcp</i> 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.</p

Testing for T7 RNA polymerase-dependent expression of <i>gfp</i> reporter construct in <i>V. cholerae</i>.

<p>Plasmid pBR-[P_T7]-GFP was transferred into <i>V. cholerae</i> 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 <i>gfp</i> 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.</p

Functionality of T7 RNA polymerase dependent reporter plasmid.

<p>Plasmid pBR-[P_T7]-GFP (panel A) was transferred into chemically competent <i>E. coli</i> 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).</p

Phenotypes of T7 RNA polymerase-mediated expression of the <i>tcp</i> cluster.

<p>The <i>V. cholerae</i> strains AΔctxAB-[P_T7]-tcp (lane 1), AΔctxAB-T7RNAP (lane 2), and AΔctxAB-[P_T7]-tcp-T7RNAP (lane 3) were grown under shaking conditions in LB medium without or with supplementation of 1 mM IPTG. <b>A: Macroscopic observation of an autoagglutination phenotype.</b> Bacterial cultures were allowed to settle before pictures were taken. Agglutinated bacteria are indicated by a black arrow in the rightmost image. <b>B: Microscopic observation of a microcolony formation phenotype.</b> Microcolony formation of the cells was visualized using light microscope. Pictures were taken using phase contrast (upper panel) or DIC (lower panel).</p

Bacterial strains and plasmids.

*<p>VC numbers according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053952#pone.0053952-Heidelberg1" target="_blank">[47]</a>.</p