PCR confirmation of insertional disruption and deletion of six genes in four <i>Vibrio</i> species.

<p>(A) Lane M: DNA marker DL2000; lanes 1–3: PCRs using primers <i>hem</i>-TF/pLP-UR to test wild type, insertional disruption and negative control (water as a template) of <i>hem</i> in <i>V</i>. <i>alginolyticus</i> E0601; lanes 4–6: PCRs using primers <i>hem</i>-TF/<i>hem</i>-TR to test wild type, deletion mutant and negative control (water as a template) of <i>hem</i> in <i>V</i>. <i>alginolyticus</i> E0601 (PCR tests for insertional disruption and deletion of <i>hem</i> using pLP11 and pLP12 had the same results, so only the results from pLP11 were shown); lanes 7–9: PCRs using primers <i>degS</i>-TF/pLP-UR to test wild type, insertional disruption and negative control (water as a template) of <i>degS</i> in <i>V</i>. <i>cholerae</i> HN375; lanes 10–12: PCRs using primers <i>degS</i>-TF/<i>degS</i>-TR to test wild type, deletion mutant and negative control (water as a template) of <i>degS</i> in <i>V</i>. <i>cholerae</i> HN375; lanes 13–15: PCRs using primers <i>vasC</i>-TF/pLP-UR to test wild type, insertional disruption and negative control (water as a template) of <i>vasC</i> in <i>V</i>. <i>cholerae</i> HN375; lanes 16–18: PCRs using primers <i>vasC</i>-TF/<i>vasC</i>-TR to test wild type, deletion mutant and negative control (water as a template) of <i>vasC</i> in <i>V</i>. <i>cholerae</i> HN375. (B) Lane M: DNA marker DL2000; lanes 1–3: PCRs using primers <i>impB</i>-TF/pLP-UR to test wild type, insertional disruption and negative control (water as a template) of <i>impB</i> in <i>V</i>. <i>vulnificus</i> ATCC 27562; lanes 4–6: PCRs using primers <i>impB</i>-TF/<i>impB</i>-TR to test wild type, deletion mutant and negative control (water as a template) of <i>impB</i> in <i>V</i>. <i>vulnificus</i> ATCC 27562; lanes 7–9: PCRs using primers <i>ascS</i>-TF/pLP-UR to test wild type, insertional disruption and negative control (water as a template) of <i>ascS</i> in <i>V</i>. <i>parahaemolyticus</i> E06135; lanes 10–12: PCRs using primers <i>ascS</i>-TF/<i>ascS</i>-TR to test wild type, deletion mutant and negative control (water as a template) of <i>ascS</i> in <i>V</i>. <i>parahaemolyticus</i> E06135; lanes 13–15: PCRs using primers <i>pilO</i>-TF/pLP-UR to test wild type, insertional disruption and negative control (water as a template) of <i>pilO</i> in <i>V</i>. <i>parahaemolyticus</i> E0680; lanes 16–18: PCRs using primers <i>pilO</i>-TF/<i>pilO</i>-TR to test wild type, deletion mutant and negative control (water as a template) of <i>pilO</i> in <i>V</i>. <i>parahaemolyticus</i> E0680.</p

Schematic diagram of wild type, insertional mutation and deletion mutation of targeted genes.

<p>Targeted genes are shown with light gray arrows and their adjacent genes are shown with black arrows. Black triangles represent annealing sites of external or internal primers. The gene names prefixed with “T-” represent the names of the truncated genes.</p

An outline of evidences that <i>vmi480</i> and <i>vmi470</i> constitute a TA module.

<p>An outline of evidences that <i>vmi480</i> and <i>vmi470</i> constitute a TA module.</p

Features of <i>vmi480</i> and <i>vmi470</i>.

<p><b>(a)</b> Schematic diagram of a potential toxin-antitoxin system comprising <i>vmi480</i> and <i>vmi470</i>. <i>vmi480</i> and <i>vmi470</i> are located on the same strand and are separated from flanking genes, <i>mobI</i> and <i>res2</i>. There is a 5-bp intergenic region between <i>vmi480</i> and <i>vmi470</i>. Upstream of <i>vmi480</i>, there is a promoter region for <i>vmi480</i> and <i>vmi470</i>. These features suggest that <i>vmi480</i> and <i>vmi470</i> belong to one transcriptional unit and that they are functionally related. The bending arrow represents the position of the promoter, and the white letters on the black background represent the -10 and-35 regions of the promoter. <b>(b)</b> Conserved domain analysis of Vmi470. At the N-terminal of Vmi470, there is a DNA-binding helix-turn-helix domain, and at the C-terminal of Vmi470, there is a Zn-dependent peptidase domain.</p

Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in <i>Vibrio</i> Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, <i>vmi480</i>

<div><p>Despite that <i>Vibrio</i> spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in <i>Vibrio</i>. In some cases, deletion of target DNAs in <i>Vibrio</i> can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene <i>sacB</i> does not work well in <i>Vibrio</i> cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in <i>Vibrio</i> species based on suicide T-Vectors carrying a novel counterselectable marker, <i>vmi480</i>. We explored two uncharacterized genes, <i>vmi480</i> and <i>vmi470</i>, in a genomic island from <i>Vibrio mimicus</i> VM573 and confirmed that <i>vmi480</i> and <i>vmi470</i> constitute a two-component toxin-antitoxin system through deletion and expression of <i>vmi480</i> and <i>vmi470</i>. The product of <i>vmi480</i> exhibited strong toxicity to <i>Escherichia coli</i> cells. Based on <i>vmi480</i> and the P_BAD or P_TAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two <i>Ahd</i>I sites. Both vectors linearized by <i>Ahd</i>I digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by <i>E</i>. <i>coli</i> β2163, six genes from four representative <i>Vibrio</i> species were easily deleted. By using the counterselective marker <i>vmi480</i>, we obtained 3–12 positive colonies (deletion mutants) among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in <i>Vibrio</i> species.</p></div

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

The effect of the expression of <i>vmi480</i> and <i>vmi470</i> on the growth of <i>E</i>. <i>coli</i> cells.

<p>a, b, c: expression of <i>vmi480</i>-<i>470</i> (LP134), <i>vmi470</i> (LP135), and <i>vmi480</i> (LP192) were blocked by D-glucose (0.3%), respectively. d, e, f: expression of <i>vmi480</i>-<i>470</i> (LP134), <i>vmi470</i> (LP135), and <i>vmi480</i> (LP192) were activated by L-arabinose (0.2%), respectively.</p

Additional file 4: Table S4. of Comparative genomic analysis of six new-found integrative conjugative elements (ICEs) in Vibrio alginolyticus

ORFs in ICEValHN396 and their similarity with related ICEs. (DOCX 19 kb

Additional file 7: Figure S2. of Comparative genomic analysis of six new-found integrative conjugative elements (ICEs) in Vibrio alginolyticus

Alignment generated using WebACT of ICEValHN437, SXT, ICEVchBan9 and ICEVchBan8. Comparisons between the regions < 80 bp are filtered. Numbers show the identity values of the compared regions. Red areas indicate homologous regions; blue areas indicate inversions. (TIF 493 kb

Additional file 2: Table S2. of Comparative genomic analysis of six new-found integrative conjugative elements (ICEs) in Vibrio alginolyticus

ORFs in ICEValA056-2 and their similarity with related ICEs. (DOCX 22 kb