Journal:

Article Title: A Selective Medium and a Specific Probe for Detection of Vibrio vulnificus

doi:

Figure Lengend Snippet: List of the collection strains used to evaluate the selectivity of VVM and the specificity of the V3VV probe

Article Snippet: V. cholerae CECT 658, V. mimicus LMG7896 T , and V. vulnificus NCIMB 2046 T were used to determine the threshold of detection when mixed bacterial suspensions were analyzed. table ft1 table-wrap mode="anchored" t5 caption a7 Species strain a VVM c V3VV d Aeromonas hydrophila CECT 398 − − Edwarsiella ictaluri F87-88 b − − Pseudomonas aeruginosa CECT 116 G+ − Vibrio aestuarianus LMG 7909 T − − Vibrio alginolyticus LMG 4408 T − − Vibrio anguillarum ATCC 43305 G+ − Vibrio campbelli LMG 11216 T Y+ − Vibrio carchariae LMG 7890 T Y+ − Vibrio cholerae CECT 658 G+ − Vibrio cincinnatiensis LMG 7891 T − − Vibrio costicola CCM 2811 − − Vibrio damsella LMG 7892 T − − Vibrio diazotrophicus LMG 7893 T − − Vibrio fluvialis LMG 7894 T − − Vibrio furnissii LMG 7910 T − − Vibrio gazogenes NCIMB 2250 T − − Vibrio harveyi LMG 4404 T − − Vibrio mediterranei LMG 11258 T G+ − Vibrio metschnikovii LMG 11664 T − − Vibrio mimicus LMG 7896 T G+ − Vibrio mytili CECT 632 − − Vibrio natriegens LMG 10935 T − − Vibrio navarrensis LMG 15976 T Y+ − Vibrio nereis LMG 3895 T − − Vibrio ordalii ATCC 33509 T − − Vibrio parahaemolyticus ATCC 27969, NCIMB 2850, ATCC 17803, ATCC 33844 − − Vibrio pelagius ATCC 3897 T − − Vibrio proteolyticus LMG 3772 T G+ − Vibrio scophthalmi CECT 4638 T − − Vibrio splendidus LMG 4042 T − − Vibrio tubiashii LMG 10936 T − − Vibrio vulnificus CECT 898, CECT 897, NCIMB 2046 T Y+ + Yersinia ruckeri CECT 955 − − Open in a separate window a ATCC, American Type Culture Collection (United States); CECT, Colección Española de Cultivos Tipo (Spain); CCM, Czech Collection of Microorganisms (Czech Republic); LMG, Laboratorium voor Microbiologie (Belgium); NCIMB, National Collection of Industrial and Marine Bacteria (United Kingdom). b National Fisheries Research Laboratory (United States). c Growth on VVM (−, no growth; G, green; Y, yellow). d Hybridization using the V3VV probe.

Techniques:

Journal: PLoS ONE

Article Title: Vibrio vulnificus Type 6 Secretion System 1 Contains Anti-Bacterial Properties

doi: 10.1371/journal.pone.0165500

Figure Lengend Snippet: Bacterial strains.

Article Snippet: V . vulnificus DAL-79040 , Clinical isolate, T6SS1- T6SS2+ , 2 , CEFAS.

Techniques: Clone Assay

Journal: Frontiers in Microbiology

Article Title: Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor

doi: 10.3389/fmicb.2021.655845

Figure Lengend Snippet: CEM-VV-specific DNAzyme selection. Briefly, (1) biotin-containing tags were fixed to the original DNAzyme library by polymerase chain reaction (PCR), which could be attached to the streptavidin-coated magnetic beads. (2) The magnetic beads were washed with 0.2 M NaOH to remove the DNA chain, which was not connected. (3) DNA was bound to crude extracellular mixtures of Vibrio vulnificus (CEM-VV) to form a specific structure, resulting in a cleavage reaction. (4) Magnetic separation and alcohol precipitation were used to recover DNA as a library of next round selection. (5) Lib-3-P1 and Lib-3-P2 were added for polymerase chain reaction (PCR), and the next round of selection was performed.

Article Snippet: The supernatant was stored at −20°C as the CEM of V. vulnificus (CEM-VV) for downstream experiments.

Techniques: Selection, Polymerase Chain Reaction, Magnetic Beads

Journal: Frontiers in Microbiology

Article Title: Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor

doi: 10.3389/fmicb.2021.655845

Figure Lengend Snippet: (A) The biosensor-based assay responded to RFD-VV-M2 in a blank culture medium and seven bacteria. Inset: Cleaving gel image for RFD-VV-M2 specificity detection within 1 h. (B) Biosensor assay of RFD-VV-M2 using CEM-VV from different concentrations of V. vulnificus . Inset: Gel micrograph showing the activity for 1 h. (C) The RFD-VV-M2-based biosensor exhibited no signal in response to trypsin-treated CEM-VV. Inset: Gel micrograph showing the biosensor activity. (D) Assessment of the molecular weight of the target protein. Inset: Gel images of the response to different molecular weight samples; all the cleavage reactions were conducted in buffer B for 1 h.

Article Snippet: The supernatant was stored at −20°C as the CEM of V. vulnificus (CEM-VV) for downstream experiments.

Techniques: Biosensor Assay, Bacteria, Activity Assay, Molecular Weight

Journal: Frontiers in Microbiology

Article Title: Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor

doi: 10.3389/fmicb.2021.655845

Figure Lengend Snippet: (A) RFD-VV-M2 concentration optimization (100–300 mM) on the sensor. (B) Optimization of fluorescence reaction time (0–20 min). (C) Detection of V. vulnificus in crab, fish, shrimp, and oyster samples under the concentration of 10 7 CFU/ml. Red columns indicated different detection. Control: negative control. Inset: An active sensor exhibiting a positive fluorescent signal. (D) Detection of different concentrations of V. vulnificus in infected shrimp. Inset: The tissue fluid of infected shrimp rendered a fluorescent signal within 5 min. Control: negative control. (E) Corresponding agarose gel electrophoresis results of real-time PCR for V. vulnificus detection with primers L-vvh and R-vvh. M, D2000 Plus DNA marker; Control, negative control. (F) Corresponding agarose gel electrophoresis results of real-time PCR for shrimps spiked with V. vulnificus. M, D2000 Plus DNA marker; Control: negative control.

Article Snippet: The supernatant was stored at −20°C as the CEM of V. vulnificus (CEM-VV) for downstream experiments.

Techniques: Concentration Assay, Fluorescence, Control, Negative Control, Infection, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Marker

Journal: Frontiers in Microbiology

Article Title: Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor

doi: 10.3389/fmicb.2021.655845

Figure Lengend Snippet: Detection of V. vulnificus in spiked seafood samples.

Article Snippet: The supernatant was stored at −20°C as the CEM of V. vulnificus (CEM-VV) for downstream experiments.

Techniques: Control, Positive Control

Journal: Frontiers in Microbiology

Article Title: Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor

doi: 10.3389/fmicb.2021.655845

Figure Lengend Snippet: Detection of V. vulnificus in spiked seafood samples.

Article Snippet: The supernatant was stored at −20°C as the CEM of V. vulnificus (CEM-VV) for downstream experiments.

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: Expression of vvhBA upon exposure to murine blood and RAW 264.7 cells. A, the genes induced in V. vulnificus upon exposure to murine blood were identified by RNA-seq analysis. vvhBA was selected as the most highly induced extracellular toxin-encoding gene, and its induction was confirmed by qRT-PCR. Each column represents the vvhBA transcript level in V. vulnificus exposed to murine blood relative to M9G (negative control). Error bars represent the S.E. calculated using DeSeq2 for RNA-seq and the S.D. for qRT-PCR. B, V. vulnificus was exposed to DMEM (negative control) or RAW 264.7 cells in the presence or absence of l-NMMA. The vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript level in the cells exposed to DMEM without l-NMMA was set to 1. Error bars represent the S.D. *, p < 0.05; ***, p < 0.0005; ns, not significant.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: The effect of iscR mutation on vvhBA expression. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. **, p < 0.005; ***, p < 0.0005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR or IscR3CA and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT (pJH0311) and WT, a WT; ΔiscR (pJH0311) and ΔiscR, an iscR-deletion mutant; ΔiscR (pJH0311::iscR), an iscR-complemented strain with pKK1531; iscR3CA, a strain expressing apo-locked IscR.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Mutagenesis, Expressing, Isolation, Quantitative RT-PCR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: The effect of nitrosative stress and iron starvation on vvhBA and IscR expression. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3 and then exposed to 25 μm DEA NONOate for 20 min (A and B) or 50 μm DP for 10 min (C and D). A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT unexposed to DEA NONOate (A) or DP (C) were set to 1. Error bars represent the S.D. *, p < 0.05; ***, p < 0.0005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT, a WT; ΔiscR, an iscR-deletion mutant.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: Specific binding of IscR to PvvhBA and sequences of the PvvhBA regulatory region. A, a 501-bp DNA of the PvvhBA regulatory region (5 nm) was radiolabeled and then incubated with increasing amounts of IscR as indicated. For competition analysis, various amounts of the unlabeled DNA fragment were used as a self-competitor and added to a reaction mixture containing 5 nm radiolabeled DNA and 30 nm IscR. B1, a DNA-IscR complex; F, free DNA. B, the same DNA of the PvvhBA regulatory region (32.3 nm) was labeled with 6-FAM, incubated with increasing amounts of IscR as indicated, and then digested with DNase I. The regions protected by IscR are indicated by black boxes (ISCRB1, ISCRB2, ISCRB3). Nucleotide numbers shown are relative to the transcription start site of vvhBA, which was determined previously (17). C, sequence analysis of the PvvhBA regulatory region. The transcription start site of vvhBA and the putative translational initiation codon of VvhB are indicated by solid and dashed bent arrows, respectively. The putative −10 and −35 regions are underlined and the putative ribosome-binding site (AGGA) is boldface. The binding sequences of IscR are shown with the black boxes as described above. The binding sequences of HlyU (HLYUB; a white box) and H-NS (HNSB1, HNSB2, HNSB3, HNSB4, HNSB5, HNSB6; gray boxes) were determined later in this study (Fig. 6, C and D). The consensus sequences of the IscR-binding Type 2 DNA motif are indicated above the V. vulnificus DNA sequences. W, A or T; Y, C or T; R, A or G; x, any nucleotide.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Binding Assay, Incubation, Labeling, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: IscR and HlyU activate, but H-NS represses the vvhBA transcription. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. *, p < 0.05 relative to the WT; **, p < 0.005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT (pJH0311), a WT; ΔiscR (pJH0311), an iscR-deletion mutant; ΔhlyU (pJH0311), a hlyU-deletion mutant; Δhns (pJH0311), an hns-deletion mutant; ΔiscR (pJH0311::iscR), an iscR-complemented strain with pKK1531; ΔhlyU (pJH0311::hlyU), a hlyU-complemented strain with pZW1510; Δhns (pJH0311::hns), an hns-complemented strain with pGR1713.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Isolation, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: IscR relieves H-NS repression of vvhBA in cooperation with HlyU in vivo. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. *, p < 0.05; **, p < 0.005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR or IscR3CA, H-NS, HlyU, and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT, a WT; Δhns, an hns-deletion mutant; ΔiscRΔhns, an iscR hns double-deletion mutant; iscR3CAΔhns, an hns-deletion mutant expressing apo-locked IscR; ΔiscR, an iscR-deletion mutant; ΔhlyU, a hlyU-deletion mutant; ΔiscRΔhlyU, an iscR hlyU double-deletion mutant; iscR3CAΔhlyU, a hlyU-deletion mutant expressing apo-locked IscR.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: In Vivo, Isolation, Quantitative RT-PCR, Western Blot, Mutagenesis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: A proposed model for the regulation of vvhBA by multiple transcriptional regulators during host infection. Upon entering the host, V. vulnificus induces vvhBA expression in response to drastic environmental changes. IscR, along with HlyU, which is preferentially produced in the host (23), activates vvhBA by relieving H-NS repression by sensing nitrosative stress and iron starvation. Additionally, CRP activates vvhBA expression via Class I activation under certain nutrient-depleted conditions (17, 21). Meanwhile, a repressive interaction of H-NS and Fur at PvvhBA would be relieved in response to the increase in temperature and iron starvation in the host, respectively (32, 48). Taken together, the transcriptional regulators integrate diverse host-derived signals to collaboratively regulate vvhBA transcription during infection. Solid lines indicate activation of vvhBA by positive regulators, whereas dashed lines show relieved repression of vvhBA by negative regulators in the host. The transcription start site of vvhBA and the putative translational initiation codon of VvhB are indicated by solid bent arrows. The putative −10 and −35 regions, and ribosome-binding site (RBS) are underlined. ISCRB, an IscR-binding site; HLYUB, a HlyU-binding site; HNSB, an H-NS-binding site; CRPB, a CRP-binding site; FURB, a Fur-binding site.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Infection, Expressing, Produced, Activation Assay, Derivative Assay, Binding Assay