Single multiplex polymerase chain reaction for environmental surveillance of toxigenic—Pathogenic O1 and Non-O1vibrio cholerae

A multiplex PCR assay was developed for the detection of toxigenic and pathogenic V. cholerae from direct water sources using specific primers targeting diverse genes, viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes, ompW acts as internal control for V. cholerae, the ctx gene as a marker for toxigenicity and tcp for pathogenicity. The sensitivity of multiplex PCR was 5 x 10(4) V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. Toxigenic V. cholerae were artificially spiked in different water samples, filtered through a 0.45 microm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8 V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence of V. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenic-pathogenic and nonpathogenic V. cholerae.     

The Hybrid Pre-CTXΦ-RS1 Prophage Genome and Its Regulatory Function in Environmental Vibrio cholerae O1 Strains

The cholera toxin genes of Vibrio cholerae are encoded by CTXΦ, a lysogenic bacteriophage. Infection with this phage plays a determinant role in toxigenicity conversion and the emergence of new clones of pathogenic V. cholerae. Multiple phage alleles, defined by sequence types of the repressor gene rstR, have been found, showing the divergence of phage genomes. Pre-CTXΦ, which is characterized by the absence of toxin genes, is predicted to be the precursor of CTXΦ. We have found a new pre-CTXΦ prophage genome (named pre-CTX^ZJΦ for its novel rstR allele) in nontoxigenic V. cholerae O1 isolates that were obtained during surveillance of the estuary water of the Zhujiang River. A novel hybrid genome of the helper phage RS1 was identified in an environmental strain carrying pre-CTX^ZJΦ in this study. The chromosomal integration and genomic arrangement of pre-CTX^ZJΦ and RS1 were determined. The RS2 of pre-CTX^ZJΦ was shown to have a function in replication, but it seemed to have lost its ability to integrate. The RstR of pre-CTX^ZJΦ exerted the highest repression of its own rstA promoter compared to other RstRs, suggesting rstR-specific phage superinfection immunity and potential coinfection with other pre-CTXΦ/CTXΦ alleles. The environmental strain carrying pre-CTX^ZJΦ could still be infected by CTX^ETΦ, the most common phage allele in the strains of the seventh cholera pandemic, suggesting that this nontoxigenic clone could potentially undergo toxigenicity conversion by CTXΦ infection and become a new toxigenic clone despite already containing the pre-CTXΦ prophage.     

Development of Stable Vibrio cholerae O1 Hikojima Type Vaccine Strains Co–Expressing the Inaba and Ogawa Lipopolysaccharide Antigens

We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co–express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba– and Ogawa–LPS antigens which are preserved after formalin–inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin–inactivated whole–cell vaccine preparations of these strains elicited strong intestinal IgA anti–LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole–cell vaccines.     

The rpoH gene encoding σ32 homolog of Vibrio cholerae

The Vibrio cholerae rpoH gene coding for the heat-shock sigma factor, σ³², has been cloned and shown to functionally complement Escherichia coli rpoH mutants. The nt sequence of the gene has been determined and the deduced aa sequence is more than 80% homologous to the E. coli rpoH gene product. Downstream of the V. cholerae rpoH gene, an unidentified dehydrogenase gene (udhA) is present on the opposite strand facing rpoH. The predicted secondary structure of the 5′-proximal region of V. cholerae rpoH mRNA is apparently different from the conserved secondary structures of the rpoH mRNA reported for several bacterial species. The `RpoH box', a stretch of 9 aa (QRKLFFNLR) unique to σ<sup>32</sup> factors, and the `downstream box' sequence complementary to a part of the 16S rRNA, have been detected.     

Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field?

The bacterium Vibrio cholerae is a natural inhabitant of aquatic ecosystems across the planet. V. cholerae serogroups O1 and O139 are responsible for cholera outbreaks in developing countries accounting for 3–5 million infections worldwide and 28.800–130.000 deaths per year according to the World Health Organization. In contrast, V. cholerae serogroups other than O1 and O139, also designated as V. cholerae non-O1/O139 (NOVC), are not associated with epidemic cholera but can cause other illnesses that may range in severity from mild (e.g. gastroenteritis, otitis, etc.) to life-threatening (e.g. necrotizing fasciitis). Although generally neglected, NOVC-related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. NOVC strains are also believed to potentially contribute to the emergence of new pathogenic strains including strains with epidemic potential as a direct consequence of genetic exchange mechanisms such as horizontal gene transfer and genetic recombination. Besides general features concerning the biology and ecology of NOVC strains and their associated diseases, this review aims to highlight the most relevant aspects related to the emergence and potential threat posed by NOVC strains under a rapidly changing environmental and climatic scenario.     

Sur les ferments protéolytiques duVibrio cholerae et duVibrio El Tor

Conclusions On peut conclure, en résumé, que l'hémodigestion et la liquéfaction de la gélatine sont des manifestations de 2 ferments.Au surplus, les comportements du pouvoir de scission de la gélatine et du pouvoir hémodigestif furent comparés chez 4 souches deV. El Tor cultivées sur gélose et conservées sur un milieu renfermant du blanc d'oeuf. A cette occasion on a eu recours au viscosimètre de Ostwald.Il fut observé que, chez les souches deV. El Tor cultivées sur gélose, la scission de la gélatine et l'hémodigestion furent plus intenses que chez des souches conservées sur milieu à l'oeuf. En cutre, chez ces dernières, le pouvoir hémodigestif avait entièrement disparu.     

Survival and proliferation of the lysogenic bacteriophage CTXΦ in Vibrio cholerae

The lysogenic phage CTXΦ of Vibrio cholerae can transfer the cholera toxin gene both horizontally(inter-strain) and vertically(cell proliferation). Due to its diversity in form and species, the complexity of regulatory mechanisms, and the important role of the infection mechanism in the production of new virulent strains of V.cholerae, the study of the lysogenic phage CTXΦ has attracted much attention. Based on the progress of current research, the genomic features and their arrangement, the host-dependent regulatory mechanisms of CTXΦ phage survival, proliferation and propagation were reviewed to further understand the phage's role in the evolutionary and epidemiological mechanisms of V. cholerae.     

Effects of Furazolidone on the Infection of Vibrio cholerae by Bacteriophage φ149

Furazolidone in concentrations which had little effect on the growth of host organisms greatly reduced the yield of phage 149 from the host Vibrio cholerae OGAWA 154. This phage was resistant to the in vitro action of the drug. The phage yield of infected bacteria depended significantly on the time of addition or withdrawal of the drug. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration. The latent period of phage multiplication and also the eclipse period did not change significantly from the control values. A concentration of 0.05 μg of furazolidone per ml inhibited DNA synthesis by about 50% in phage-infected cells and only by about 18% in noninfected ones, relative to the respective controls. RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. Quantitative deduction of the length of furazolidone-treated cells from their phage adsorption characteristics and its agreement with previous electron microscopy data indicated that furazolidone did not affect the phage receptors.     

Sulfonamide inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae

The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. VchCA, the α-CA from this species was investigated earlier, whereas the β-class enzyme, VchCAβ was recently cloned, characterized kinetically and its X-ray crystal structure reported by this group. Here we report an inhibition study with sulfonamides and one sulfamate of this enzyme. The best VchCAβ inhibitors were deacetylated acetazolamide and methazolamide and hydrochlorothiazide, which showed inhibition constants of 68.2–87.0 nM. Other compounds, with medium potency against VchCAβ, (K_Is in the range of 275–463 nM), were sulfanilamide, metanilamide, sulthiame and saccharin whereas the clinically used agents such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, zonisamide and celecoxib were micromolar inhibitors (K_Is in the range of 4.51–8.57 μM). Identification of potent and possibly selective inhibitors of VchCA and VchCAβ over the human CA isoforms, may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzymes.     

Anion inhibition studies of the α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae

An α-carbonic anhydrase (CA, EC 4.2.1.1) has been recently cloned and characterized in the human pathogenic bacterium Vibrio cholerae, denominated VchCA (Del Prete et al. J. Med. Chem. 2012, 55, 10742). This enzyme shows a good catalytic activity for the CO₂ hydration reaction, comparable to that of the human (h) isoform hCA I. Many inorganic anions and several small molecules were investigated as VchCA inhibitors. Inorganic anions such as cyanate, cyanide, hydrogen sulfide, hydrogen sulfite, and trithiocarbonate were effective VchCA inhibitors with inhibition constants in the range of 33–88 μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with K_Is of 7–43 μM. Halides (bromide, iodide), bicarbonate and carbonate were much less effective VchCA inhibitors, with K_Is in the range of 4.64–28.0 mM. The resistance of VchCA to bicarbonate inhibition may represent an evolutionary adaptation of this enzyme to living in an environment rich in this ion, such as the gastrointestinal tract, as bicarbonate is a virulence enhancer of this bacterium.     

Crystal structure of periplasmic catecholate-siderophore binding protein VctP from Vibrio cholerae at 1.7 Å resolution

VctP, one of the two essential siderophore-binding PBPs from the pathogen Vibrio cholerae, plays an important role in the transport of enterobactin and vibriobactin, which have quite different configurations of iron coordination, from the periplasm to the inner membrane. The current study reports the crystal structure of VctP from V. cholerae N16961 at 1.7 Å resolution. A structural comparison of VctP with its homologues and the results of molecular docking indicate that enterobactin and vibriobactin share the same binding pocket. Significantly, a basic triad consisting of Arg137, Arg226 and Arg270 is used to balance the three negative charges of ferric-enterobactin, while a basic dyad consisting of Arg137 and Arg270 is used to balance the two negative charges of ferric-vibriobactin.     

Quinazoline–sulfonamides with potent inhibitory activity against the α-carbonic anhydrase from Vibrio cholerae

Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were synthesized by simple, eco-friendly procedures. These compounds were tested for their ability to inhibit the α-carbonic anhydrases (CA, EC 4.2.1.1) from Vibrio cholerae (VchCA) as well as the human α-CA isoforms, hCA I and hCA II. Nine compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme VchCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic isoform II. The best VchCA inhibitor had a K_I of 2.7 nM. Many of these developed compounds showed high selectivity for inhibition of the bacterial over the mammalian CA isoforms, with one compound possessing selectivity ratios as high as 97.9 against hCA I and 9.7 against hCA II. Compound 9d was another highly effective VchCA inhibitor presenting a selectivity ratio of 99.1 and 8.1 against hCA I and hCA II, respectively. These results suggest that sulfonamides with quinazoline backbone could be considered suitable tools to better understand the role of bacterial CAs in pathogenesis.     

Molecular Determinants of Mechanical Properties of V.?cholerae Biofilms at?the Air-Liquid Interface

Biofilm formation increases both the survival and infectivity of Vibrio cholerae, the causative agent of cholera. V. cholerae is capable of forming biofilms on solid surfaces and at the air-liquid interface, termed pellicles. Known components of the extracellular matrix include the matrix proteins Bap1, RbmA, and RbmC, an exopolysaccharide termed Vibrio polysaccharide, and DNA. In this work, we examined a rugose strain of V. cholerae and its mutants unable to produce matrix proteins by interfacial rheology to compare the evolution of pellicle elasticity in real time to understand the molecular basis of matrix protein contributions to pellicle integrity and elasticity. Together with electron micrographs, visual inspection, and contact angle measurements of the pellicles, we defined distinct contributions of the matrix proteins to pellicle morphology, microscale architecture, and mechanical properties. Furthermore, we discovered that Bap1 is uniquely required for the maintenance of the mechanical strength of the pellicle over time and contributes to the hydrophobicity of the pellicle. Thus, Bap1 presents an important matrix component to target in the prevention and dispersal of V. cholerae biofilms.     

DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of Vibrio cholerae

The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB''s requirement for single-stranded-DNA chromosomal integration of CTXφ phage dependent on XerCD recombinases.Vibrio cholerae, the etiologic agent of cholera, is a gram-negative bacterium which hosts several specific filamentous phages (1, 7, 8, 9, 10, 11, 13). CTXφ phage has been the most studied due to its role in pathogenicity and horizontal gene transfer (6). This phage is usually integrated into the genomes of toxigenic strains of V. cholerae, but it is also able to replicate directly from the bacterial chromosome (6) and to produce infective phage particles with potential for transducing the cholera toxin genes into nonpathogenic environmental strains (6, 13). Another filamentous phage important for its role in horizontal gene transfer is VGJφ, which is able to recombine with the CTXφ genome to originate a hybrid phage endowed with the full potential for virulence conversion. The hybrid phage shows an increased infectivity due to its specificity for the receptor mannose-sensitive hemagglutinin (receptor mannose-sensitive hemagglutinin pilus), which is ubiquitous among environmental strains (1, 2). Therefore, elucidating the biology of these phages is crucial for understanding the evolution of bacterial pathogenesis.The genomes of CTXφ and VGJφ carry the putative homologous rstB and open reading frame 112 (orf112) genes, respectively. The requirement of rstB for the integration of CTXφ into the bacterial chromosome has been described (14). However, the biochemical function of the gene product has not been elucidated. Genes rstB and orf112 are positional and size homologues of genes encoding single-stranded DNA (ssDNA)-binding proteins (SSB) in other filamentous phages (1). It is expected that the proteins encoded by rstB and orf112 exert similar functions in the biology of their respective phages (1). Thus, we wanted to evaluate the ssDNA-binding activity of these ORF products.To asses whether the Orf112 product and RstB have SSB activity, sufficient amounts of pure proteins are required. This paper describes quick purification protocols used to obtain both protein species and the evaluation of their DNA binding activities. The Orf112 protein was obtained from V. cholerae strain 569B (serogroup O1, Inaba classical biotype) infected with VGJφ, which expresses high levels of the protein. The infected bacteria were inoculated into 300 ml of LB broth and were cultured with shaking overnight at 200 rpm and 37°C. Parallel uninfected batches of 569B were also processed. Cells were collected by centrifugation for 15 min at 9,000 × g and at 4°C and stored at −20°C until processed.A recombinant rstB gene with a hexahistidine tag coding region fused to the C terminus of the respective protein product (RstB-His) was constructed by cloning the gene into the expression vector pBAD/Myc-HisC (Invitrogen). The rstB gene was PCR amplified from the CTXφ genome using the oligonucleotides CNC06-171 (5′-AGTTCCATGGGGAAATTATGGGTGATAAT-3′) and CNC06-173 (5′-CATCAAGCTTTAATGGGT-3′), which introduce restriction sites for NcoI and HindIII at the amplicon ends. The amplified fragment was digested with both enzymes and cloned into the same sites of pBAD/Myc-HisC. In the resultant construction, named pBAD/Myc-HisC-rstB 9, expression of the recombinant protein is inducible by arabinose.Plasmid pBAD/Myc-HisC-rstB 9 was electroporated into Escherichia coli Top 10. A 1-ml sample of an overnight, 5-ml, ampicillin-supplemented LB broth culture of transformed Top 10 was inoculated into 300 ml of fresh broth. The culture was incubated with orbital shaking at 200 rpm and 37°C until it reached an optical density at 600 nm of 0.5. To induce expression of the RstB-His protein, 0.002% (wt/vol) arabinose was added and the culture was reincubated for three additional hours. Parallel batches of pBAD/Myc-HisC-transformed E. coli Top 10 were processed as a negative control. Cells were sedimented by centrifugation for 15 min at 9,000 × g and 4°C and stored at −20°C until processed.The expression of the Orf112 and RstB-His proteins was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cell extracts of VGJφ-infected 569B and E. coli Top 10 transformed with pBAD/Myc-HisC-rstB 9 contained proteins with apparent molecular sizes of 12.7 kDa (Fig. ​(Fig.1)1) and 16 kDa (Fig. ​(Fig.2B),2B), respectively, which are not observed in cells from control cultures. The sizes match those predicted from orf112 (12.72 kDa) (see reference 1) and recombinant rstB-his (16.8 kDa).Open in a separate windowFIG. 1.SDS-PAGE monitoring of the purification process of Orf112 protein. Lane 1, broad-range protein molecular mass markers (Promega); lane 2, cell extract of non-VGJφ-infected V. cholerae 569B; lane 3, cell extract of VGJφ-infected V. cholerae 569B; lane 4, soluble fraction of the sonicate; lane 5, insoluble fraction of the sonicate; lane 6, precipitate at 30% (NH₃)₂SO₄; lane 7, supernatant at 30% (NH₃)₂SO_4; lane 8, precipitate at 50% (NH₃)₂SO₄; lane 9, supernatant at 50% (NH₃)₂SO₄; lane 10, Orf112 protein electro-eluted after preparative SDS-PAGE.Open in a separate windowFIG. 2.Isolation and purification of RstB-His. (A) Chromatogram on a Ni-CAM HC His tag affinity column. (B) SDS-PAGE monitoring of the purification process of RstB-His. Lane 1, broad-range protein molecular mass markers (Promega); lane 2, cell extract of uninduced cultures; lane 3, cell extract of expression-induced cultures; lane 4, soluble fraction of the sonicate from expression-induced cultures; lane 5, insoluble fraction of the sonicate from expression-induced cultures; lane 6, soluble fraction of the 8 M urea extract; lane 7, RstB-His eluted from the column.These proteins are not secreted into the growth medium (not shown); thus, they were released from the cells by ultrasonic disruption as previously described (4). V. cholerae was suspended in 15 ml of 20 mM Tris-HCl buffer, pH 7.5, while E. coli cells were suspended in 15 ml of 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0. Cell lysates were cleared by centrifugation for 40 min at 9,000 × g and 4°C. SDS-PAGE detected Orf112 protein in the soluble fraction, while RstB-His remained in the insoluble fractions of cell extracts (Fig. ​(Fig.1).1). Subsequently, 569B cell lysate supernatants containing Orf112 protein were fractionated with ammonium sulfate. At 30% ammonium sulfate, several contaminants precipitated but Orf112 protein remained in solution, while at 50% ammonium sulfate, Orf112 precipitated and was recovered by centrifugation. The pellet was washed twice with 50% ammonium sulfate and finally resuspended into 3 ml of 20 mM Tris-HCl buffer, pH 7.5. Removal of excess salt was achieved by gel filtration before the extract was applied to a preparative SDS-PAGE gel. Briefly, a 15% polyacrylamide gel (17 by 19 by 0.5 cm) was run at a constant current intensity of 100 mA and with free voltage at 4°C, until the bromophenol blue dye migrated off the gel. The gels were negatively stained with imidazole-zinc (3), and the Orf112 protein band was identified by comparing bands with the bands of a negative control applied in a neighboring lane of the same gel, where this protein was not visible. The ORF112 protein band was cut from the gel, and the slice was fragmented and introduced into a dialysis bag with a 6- to 8-kDa molecular mass cutoff in 10 ml of 24 mM Tris-HCl-250 mM glycine-0.5% (wt/vol) SDS buffer. The protein was electro-eluted for 5 h at a current intensity of 70 mA and with free voltage at 4°C. Reverse current was applied for 5 min to release membrane-bound proteins, and gel fragments were discarded. The same sample was dialyzed against 1 liter of 0.5 M Tris-HCl, 0.25 M glycine buffer, pH 7.5, for 24 h at 4°C with constant stirring. The dialysis was repeated with 20 mM Tris-HCl, 0.5 M NaCl buffer, pH 7.5, for 24 h. No contaminants were seen when 25 μg of this Orf112 protein-dialyzed extract was checked by SDS-PAGE and Coomassie brilliant blue staining (Fig. ​(Fig.1,1, lane 10).RstB-His protein was recovered from the insoluble fraction of the E. coli lysate by dissolving the lysate in 15 ml of a buffer containing 8 M urea, 20 mM sodium phosphate, 0.5 M NaCl, and 10 mM imidazole, pH 8.0. The mixture was stirred overnight at 4°C and cleared by centrifugation at 9,000 × g for 40 min. The supernatant was applied to a Ni-CAM HC matrix (Sigma), and urea was removed using a linear gradient from 8 to 0 M urea as previously described (5). The presence of 10 mM imidazole in the sample and binding buffer was intended to reduce the level of contaminants bound to the column. Protein was eluted using a gradient of imidazole (10 to 250 mM) in 20 mM sodium phosphate, 0.5 M NaCl buffer, pH 8.0. Fractions were assayed by SDS-PAGE, and those containing the RstB-His protein were pooled according to purity rather than yield. RstB-His protein was obtained with 90% purity (Fig. ​(Fig.2B,2B, lane 7), according to a densitometry scan of Coomassie brilliant blue-stained gels, using a Gene Genius gel documentation system (Syngene Synoptics Ltd., Cambridge, United Kingdom). The gradient-based removal of urea allowed effective solubilization of RstB-His without significant precipitation of protein in the column, as described before (5).Biological activity was assayed by retardation assays of VGJφ genomic ssDNA by 0.5% agarose gel electrophoresis conducted with 20 mM EDTA, 40 mM Tris-acetate buffer. Various amounts of each protein and ssDNA from VGJφ (0.5 μg) mixed in 20% glycerol, 0.25 mM EDTA, 0.3 μM bovine serum albumin, 20 mM Tris-HCl up to a total volume of 40 μl were incubated at room temperature for 30 min and loaded into the gel for analysis. The electrophoresis was run at 100 V and 4°C until colorant exit. Ethidium bromide (1 μg/ml) was used for 30 min to stain DNA bands, which were documented in a Gene Genius gel system (Syngene Synoptics Ltd., Cambridge, United Kingdom).Orf112 protein exhibited DNA retardation activity, showing a high specificity of binding for the circular ssDNA of VGJφ, but was unable to bind double-stranded DNA (dsDNA) (Fig. ​(Fig.3A).3A). However, RstB was able to bind ssDNA as well as dsDNA substrates (Fig. ​(Fig.3B).3B). No retardation was observed with protein preparations from negative controls or when the DNA-protein mixture was inactivated with 1:1 (vol/vol) phenol-chloroform, indicating that the binding activity is intrinsic to the purified proteins.Open in a separate windowFIG. 3.Gel retardation assays of VGJφ-ssDNA by Orf112 protein or RstB-His binding, as measured by 0.5% agarose gel electrophoresis. (A) Binding of Orf112 protein. Lane 1, control of 500 ng of genomic ssDNA of VGJφ; lanes 2 to 6; same as for lane 1 plus 1.25, 2.50, 5.00, 10.0, and 20.0 μg of Orf112 protein, respectively; lane 7, same as for lane 6 but treated with phenol-chloroform; lane 8, linearized replicative-form dsDNA of VGJφ; lane 9, same as for lane 8 plus 20.0 μg of Orf112; lane 10, linearized pUC19; lane 11, same as for lane 10 plus 20.0 μg of Orf112. (B) Binding of RstB-His. Lane 1, control of 500 ng of genomic ssDNA of VGJφ; lane 2, same as for lane 1 plus 20 μg of RstB treated with phenol-chloroform; lanes 3 to 8, same as for lane 1 plus 0.62, 1.25, 2.50, 5.00, 10.0, and 20.0 μg of RstB-His, respectively; lane 9, linearized replicative-form dsDNA of VGJφ; lane 10, same as for lane 9 plus 20.0 μg of RstB-His; lane 11, linearized pUC19; lane 12, same as for lane 11 plus 20.0 μg of RstB-His.In the case of RstB, which binds to ssDNA and dsDNA substrates, we wanted to rule out the possibility that this effect was caused by the His tail fused to the recombinant protein. For this, we recloned RstB in the same vector as RstB-His but without the His tail. We used the same procedure described above for RstB-His but used oligonucleotides CNC06-171 (see above) and CNC06-172 (5′-TACTGCAGTCAAGATTTAATGGGTTG-3′) for RstB amplification. In this case, CNC06-172 introduced a restriction site for PstI, which was used for cloning into pBAD/Myc-HisC.For purification of this RstB variant, E. coli growth and protein expression induction was done as described for RstB-His. Again, RstB was recovered in the insoluble fraction after cell disruption by sonication. Inclusion bodies were resuspended in 15 ml of 50 mM phosphate buffer, pH 7.7, containing 8 M urea, and after overnight stirring at 4°C, the suspension was cleared by centrifugation (9,000 × g, 40 min). The supernatant was applied to an SP Sepharose fast-flow column (Amersham, United Kingdom), and urea was removed as described above for RstB-His. The RstB that bound to the matrix was eluted using a gradient of 0 to 2 M NaCl and was obtained with about 92% purity (data not shown). RstB without the His tail also showed binding activity toward ssDNA and dsDNA substrates (data not shown), ruling out the possibility that the His hexamer is responsible for the nonspecific binding of RstB-His.Since RstB has affinity for both ssDNA and dsDNA, the possibility exists that this protein simply binds any DNA nonspecifically due to an effect of a charge interaction with the phosphate backbone of DNA. In order to study the effect of the charge in the DNA-binding activity of RstB, NaCl was included in the reaction mixture at a concentration from 0 to 500 mM (Fig. ​(Fig.4).4). As can be seen in Fig. ​Fig.4,4, the retardation activity of ssDNA was only partially inhibited from a starting concentration of 400 mM (Fig. ​(Fig.4A),4A), while the retardation of dsDNA started to be partially inhibited at 300 mM NaCl (Fig. ​(Fig.4B).4B). Since RstB continues to bind at high salt concentrations, which should equilibrate the charge effect, these results indicate that the DNA binding activity is not due to the presence of positively charged amino acids in the protein backbone but rather to the presence of domains that specifically recognize the ssDNA or dsDNA.Open in a separate windowFIG. 4.Effect of salt concentration and nonrelated dsDNA competition on the binding of RstB. (A) RstB binding to phage ssDNA in the presence of 0 to 500 mM NaCl as detected by 0.5% agarose gel electrophoresis. Lane 1, 400 ng of genomic ssDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 to 7, same as for lane 2 plus 100, 200, 300, 400, and 500 mM NaCl, respectively. (B) RstB binding to replicative-form phage dsDNA in the presence of 0 to 500 mM NaCl. Lane 1, 400 ng of dsDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 to 7, same as for lane 2 plus 100, 200, 300, 400, and 500 mM NaCl. (C) DNA-binding competition by RstB. Lane 1, 400 ng of genomic ssDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 and 4, same as for lane 2 plus 500 and 1,000 ng of sheared calf thymus dsDNA, respectively; lane 5, 500 ng of calf thymus DNA (control).We also investigated whether RstB-His has more affinity for the phage ssDNA than for a nonrelated dsDNA; a DNA-binding competition experiment in which the protein was incubated with a constant amount of ssDNA of VGJφ phage and increasing amounts of calf thymus DNA was performed (Fig. ​(Fig.4C).4C). The ssDNA of VGJφ was retarded by RstB-His even in the presence of 500 and 1,000 ng of calf thymus dsDNA (Fig. ​(Fig.4C,4C, lanes 3 and 4). These results indicate that RstB protein has more affinity for the phage ssDNA than for a non-phage-related dsDNA.Until we know more, RstB is the first protein of a filamentous phage which shows affinity for both ss- and dsDNA, at least in vitro. It is possible that RstB needs another protein from the host or the phage itself to recognize the ssDNA in a specific manner, or perhaps the affinity of RstB for both ss- and dsDNA is an intrinsic property of the protein, which is needed on one hand for binding to genomic ssDNA during the rolling circle replication of the phage and on the other hand for holding the hairpin dsDNA secondary structure formed by the phage genome that functions as the site for integration into the bacterial chromosome (12). This hairpin structure is used by XerCD recombinases as a substrate for recombining the phage genome with the bacterial chromosomal dif site (12), and RstB may act jointly with XerCD to achieve integration. This could explain the requirement of RstB for the integration of CTXφ.It is concluded that Orf112 and RstB proteins purified by the protocols described in this paper were biologically active and obtained at a high degree of purity, which paves the way for further characterization of these proteins. The SSB activities of these two proteins are shown for the first time. Consequently, we propose to rename their respective genes gV^VGJφ and gV^CTXφ and their proteins pV^VGJφ and pV^CTXφ, to follow the denomination of genes of canonical phages of the Inovirus genus. A biochemical and chemical-physical characterization of both proteins is in progress and will be published elsewhere. Should the in vitro role demonstrated for these proteins operate in vivo as well, it might explain their role for rolling circle replication and why rstB is required for CTXφ integration.     

TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes^1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons^1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene⁴. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)⁵ (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism⁶ and other representative of the genus Vibrio such as V. fischeri⁷. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region⁸. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences⁵. Several optimization steps concerning the initial protocol of chitin-induced natural transformation⁶ are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes⁸, the donation of PCR-derived DNA as transforming material⁹, and the addition of FLP-recombination target sites (FRT)⁵. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase¹⁰.