Journal: MicrobiologyOpen

Article Title: The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid-encoded system of Vibrio anguillarum?

doi: 10.1002/mbo3.65

Figure Lengend Snippet: Figure 2. Anguibactin production from Vibrio harveyi. (A) Bioassay to test anguibactin production. CC9-16ΔfvtAΔfetA was used as an anguibactin indicator strain. EDDA (40 lmol/L) and Cm (10 lg/mL) were added into AB media with the indicator strain. Five microliters of overnight culture of V. harveyi HY01 (pMMB208), V. harveyi BAA-1116 (pMMB208), and Vibrio anguillarum 96F (pMMB208) grown in AB broth with Cm was spotted on the plates, and incubated at 25°C. Presence of growth halos around spots was checked every 24 h. Vibrio anguillarum 96F (pMMB208) is a vanchrobactin producer and was used as a negative control. (B) Structure of anguibactin from V. anguillarum 775 (pJM1) (Jalal et al. 1989). (C) Confirmation of anguibactin biosynthesis in the V. harveyi HY01 strain. Positive mode electrospray ionization-mass spectra of the purified siderophores without (above) and with (below) 30% collision energy. The nominal masses (m/z) of the parental ion species and different fragmentation products are indicated in the spectra.

Article Snippet: Strains and plasmids Characteristics Reference or source Vibrio anguillarum strains 775 (pJM1) Wild type, Washington (serotype O1, pJM1) Crosa (1980) 775 (pJM1)-pMMB 775 (pJM1) harboring pMMB208 Naka et al. (2008) 96F-pMMB Vanchrobactin producer (serotype O1, plasmidless) harboring pMMB208 Naka et al. (2008) HNVA-8 CC9-16ΔfvtAΔfetA (anguibactin indicator strain) Naka and Crosa (2012) Vibrio harveyi strains ATCC BAA-1116 Marine (Ocean) isolate Lin et al. (2010) HY01 Dead, luminescing shrimp isolate Lin et al. (2010) HNVH-1 HY01ΔangR This study HNVH-2 HY01ΔangRΔfatA This study CAIM 148 Diseased shrimp (Penaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 513T Dead, luminescing amphipod (Talorchestia sp.) isolate V. harveyi type strain ATCC 14126 Lin et al. (2010) CAIM 1075 Oyster (Crassostrea gigas) isolate Lin et al. (2010) CAIM 1766 Sea horse (Hippocampus ingens) liver isolate Lin et al. (2010) CAIM 1792 Diseased shrimp (Litopenaeus vannamei) lesion isolate Lin et al. (2010) Vibrio campbellii strains 42A Healthy coral (Mussismilia hispida) isolate Lin et al. (2010) CAIM 115 Shrimp (Litopenaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 198 Shrimp (Litopenaeus sp.) hepatopancreas isolate Lin et al. (2010) CAIM 519T Seawater isolate V. campbellii type strain ATCC 25920 Lin et al. (2010) CAIM 1500 Snapper (Lutjanus guttatus) liver isolate Lin et al. (2010) Escherichia coli strains DH5a F , /80lacZDM15, endA1, recA1, hsdR17, (rK mK +), supE44, thi-1, gyrA96, relA1, D(lacZYA-argF)U169, k Laboratory stock S17-1kpir k-pir lysogen; thi pro hsdR hsdM+recA RP4 2-Tc::Mu-Km::Tn7 (Tpr Smr) Simon et al. (1983) Plasmids pGEM-T Easy A vector for the cloning of PCR products with blue/white screening, Apr Promega pBluescript II Cloning vector, Ampr Stratagene pBluescript-Km Cloning vector, Kmr This study pDM4 Suicide plasmid sacB gene, R6K origin, Cmr Milton et al. (1996) pHN11 pDM4 harboring ΔangR of V. harveyi HY01 This study pHN12 pDM4 harboring ΔfatA of V. harveyi HY01 This study pMMB208 A broad-host-range expression vector; Cmr IncQ lacIq Ptac; polylinker from M 13mp19 Morales et al. (1991) pHN13 pMMB208 harboring V. harveyi angR This study pHN14 pMMB208 harboring V. harveyi fatA This study ATCC and CAIM strains were obtained from American Type Culture Collection (http://www.atcc.org) and Collection of Aquatic Important Microorganisms (http://www.ciad.mx/caim), respectively.

Techniques: Bioassay, Incubation, Negative Control

Journal: MicrobiologyOpen

Article Title: The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid-encoded system of Vibrio anguillarum?

doi: 10.1002/mbo3.65

Figure Lengend Snippet: Figure 3. Characterization of the angR gene in Vibrio harveyi HY01. (A) The angR gene is essential for the anguibactin production in V. harveyi HY01. Vibrio anguillarum CC9-16ΔfvtAΔfetA was used as an anguibactin indicator strain. EDDA (40 lmol/L) and Cm (10 lg/mL) were added into AB media with the indicator strain. Five microliters of an overnight culture of V. harveyi HY01 (pMMB208), V. harveyi HY01ΔangR (pMMB208), V. harveyi HY01ΔangR (pMMB208-angR), Vibrio anguillarum 775 (pJM1) (pMMB208), and V. anguillarum 96F (pMMB208) grown in AB broth with Cm and 1 lL of 1 mg/mL ferric ammonium citrate (FAC) were spotted on the plates and were incubated at 25°C. Presence of growth halos around spots was checked after 24-h incubation. V. anguillarum 775 (pJM1) (pMMB208) and FAC are positive controls. Vibrio anguillarum 96F (pMMB208) is a vanchrobactin producer and was used as a negative control. (B) Anguibactin production promotes the growth of V. harveyi in iron-limiting conditions. Fifty microliters of overnight culture (adjusted OD600 to 1) grown in 5 mL AB broth with Cm (10 lg/mL) was inoculated into AB broth containing Cm (10 lg/mL) and IPTG (1 mmol/L) (AB) or with addition of 10 lg/mL ferric ammonium citrate (FAC10), 30 lmol/L dipyridyl (DIP30), 60 lmol/L dipyridyl (DIP60), or 90 lmol/L dipyridyl (DIP90). OD600 was measured after 24-h incubation at 25°C. Experiments were repeated five times, and the error bars show standard deviation.

Article Snippet: Strains and plasmids Characteristics Reference or source Vibrio anguillarum strains 775 (pJM1) Wild type, Washington (serotype O1, pJM1) Crosa (1980) 775 (pJM1)-pMMB 775 (pJM1) harboring pMMB208 Naka et al. (2008) 96F-pMMB Vanchrobactin producer (serotype O1, plasmidless) harboring pMMB208 Naka et al. (2008) HNVA-8 CC9-16ΔfvtAΔfetA (anguibactin indicator strain) Naka and Crosa (2012) Vibrio harveyi strains ATCC BAA-1116 Marine (Ocean) isolate Lin et al. (2010) HY01 Dead, luminescing shrimp isolate Lin et al. (2010) HNVH-1 HY01ΔangR This study HNVH-2 HY01ΔangRΔfatA This study CAIM 148 Diseased shrimp (Penaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 513T Dead, luminescing amphipod (Talorchestia sp.) isolate V. harveyi type strain ATCC 14126 Lin et al. (2010) CAIM 1075 Oyster (Crassostrea gigas) isolate Lin et al. (2010) CAIM 1766 Sea horse (Hippocampus ingens) liver isolate Lin et al. (2010) CAIM 1792 Diseased shrimp (Litopenaeus vannamei) lesion isolate Lin et al. (2010) Vibrio campbellii strains 42A Healthy coral (Mussismilia hispida) isolate Lin et al. (2010) CAIM 115 Shrimp (Litopenaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 198 Shrimp (Litopenaeus sp.) hepatopancreas isolate Lin et al. (2010) CAIM 519T Seawater isolate V. campbellii type strain ATCC 25920 Lin et al. (2010) CAIM 1500 Snapper (Lutjanus guttatus) liver isolate Lin et al. (2010) Escherichia coli strains DH5a F , /80lacZDM15, endA1, recA1, hsdR17, (rK mK +), supE44, thi-1, gyrA96, relA1, D(lacZYA-argF)U169, k Laboratory stock S17-1kpir k-pir lysogen; thi pro hsdR hsdM+recA RP4 2-Tc::Mu-Km::Tn7 (Tpr Smr) Simon et al. (1983) Plasmids pGEM-T Easy A vector for the cloning of PCR products with blue/white screening, Apr Promega pBluescript II Cloning vector, Ampr Stratagene pBluescript-Km Cloning vector, Kmr This study pDM4 Suicide plasmid sacB gene, R6K origin, Cmr Milton et al. (1996) pHN11 pDM4 harboring ΔangR of V. harveyi HY01 This study pHN12 pDM4 harboring ΔfatA of V. harveyi HY01 This study pMMB208 A broad-host-range expression vector; Cmr IncQ lacIq Ptac; polylinker from M 13mp19 Morales et al. (1991) pHN13 pMMB208 harboring V. harveyi angR This study pHN14 pMMB208 harboring V. harveyi fatA This study ATCC and CAIM strains were obtained from American Type Culture Collection (http://www.atcc.org) and Collection of Aquatic Important Microorganisms (http://www.ciad.mx/caim), respectively.

Techniques: Incubation, Negative Control, Standard Deviation

Journal: MicrobiologyOpen

Article Title: The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid-encoded system of Vibrio anguillarum?

doi: 10.1002/mbo3.65

Figure Lengend Snippet: Figure 4. The fatA gene in Vibrio harveyi HY01 encodes the ferric-anguibactin outer membrane receptor protein. (A) The fatA gene is involved in the ferric-anguibactin transport in V. harveyi HY01. V. harveyi HY01ΔangR (pMMB208), V. harveyi HY01ΔangRΔfatA (pMMB208), and V. harveyi HY01ΔangRΔfatA (pMMB208-fatA) were used as indicator strains. DIP (100 lmol/L), IPTG (1 mmol/L), and Cm (10 lg/mL) were supplemented into AB medium with indicator strains. Five microliters of overnight culture of V. harveyi HY01 (pMMB208) and Vibrio anguillarum 775 (pJM1) (pMMB208) and 1 lL of 1 mg/mL ferric ammonium citrate (FAC) were spotted on the plates. Presence of growth halos around spots was checked after 24-h incubation at 25°C. (B) The FatA protein locates on the outer membrane of V. harveyi. Vibrio harveyi HY01ΔangR (pMMB208), V. harveyi HY01ΔangRΔfatA (pMMB208), and V. harveyi HY01ΔangRΔfatA (pMMB208-fatA) were grown in AB medium until exponential phase (OD600 ~0.3). Outer membrane proteins were then extracted using sarkosyl as described in Materials and Methods. Western blots were performed using anti-V. anguillarum FatA polyclonal antibody.

Article Snippet: Strains and plasmids Characteristics Reference or source Vibrio anguillarum strains 775 (pJM1) Wild type, Washington (serotype O1, pJM1) Crosa (1980) 775 (pJM1)-pMMB 775 (pJM1) harboring pMMB208 Naka et al. (2008) 96F-pMMB Vanchrobactin producer (serotype O1, plasmidless) harboring pMMB208 Naka et al. (2008) HNVA-8 CC9-16ΔfvtAΔfetA (anguibactin indicator strain) Naka and Crosa (2012) Vibrio harveyi strains ATCC BAA-1116 Marine (Ocean) isolate Lin et al. (2010) HY01 Dead, luminescing shrimp isolate Lin et al. (2010) HNVH-1 HY01ΔangR This study HNVH-2 HY01ΔangRΔfatA This study CAIM 148 Diseased shrimp (Penaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 513T Dead, luminescing amphipod (Talorchestia sp.) isolate V. harveyi type strain ATCC 14126 Lin et al. (2010) CAIM 1075 Oyster (Crassostrea gigas) isolate Lin et al. (2010) CAIM 1766 Sea horse (Hippocampus ingens) liver isolate Lin et al. (2010) CAIM 1792 Diseased shrimp (Litopenaeus vannamei) lesion isolate Lin et al. (2010) Vibrio campbellii strains 42A Healthy coral (Mussismilia hispida) isolate Lin et al. (2010) CAIM 115 Shrimp (Litopenaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 198 Shrimp (Litopenaeus sp.) hepatopancreas isolate Lin et al. (2010) CAIM 519T Seawater isolate V. campbellii type strain ATCC 25920 Lin et al. (2010) CAIM 1500 Snapper (Lutjanus guttatus) liver isolate Lin et al. (2010) Escherichia coli strains DH5a F , /80lacZDM15, endA1, recA1, hsdR17, (rK mK +), supE44, thi-1, gyrA96, relA1, D(lacZYA-argF)U169, k Laboratory stock S17-1kpir k-pir lysogen; thi pro hsdR hsdM+recA RP4 2-Tc::Mu-Km::Tn7 (Tpr Smr) Simon et al. (1983) Plasmids pGEM-T Easy A vector for the cloning of PCR products with blue/white screening, Apr Promega pBluescript II Cloning vector, Ampr Stratagene pBluescript-Km Cloning vector, Kmr This study pDM4 Suicide plasmid sacB gene, R6K origin, Cmr Milton et al. (1996) pHN11 pDM4 harboring ΔangR of V. harveyi HY01 This study pHN12 pDM4 harboring ΔfatA of V. harveyi HY01 This study pMMB208 A broad-host-range expression vector; Cmr IncQ lacIq Ptac; polylinker from M 13mp19 Morales et al. (1991) pHN13 pMMB208 harboring V. harveyi angR This study pHN14 pMMB208 harboring V. harveyi fatA This study ATCC and CAIM strains were obtained from American Type Culture Collection (http://www.atcc.org) and Collection of Aquatic Important Microorganisms (http://www.ciad.mx/caim), respectively.

Techniques: Membrane, Incubation, Western Blot

Journal: MicrobiologyOpen

Article Title: The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid-encoded system of Vibrio anguillarum?

doi: 10.1002/mbo3.65

Figure Lengend Snippet: Figure 5. Evaluation of anguibactin production from various strains. Vibrio anguillarum CC9-16ΔfvtAΔfetA was used as an anguibactin indicator strain. EDDA (40 lmol/L) was added into AB media with the indicator strain. Higher concentration of agarose (1%) was supplemented into AB media to reduce swarming of some strains. Overnight culture grown in AB broth was spotted on the plates, and the plates were incubated at 25°C. Presence of growth halos around spots was checked after 24-h incubation. Clear zone around the spot observed in Vibrio harveyi CAIM 513T exhibits swarming but not anguibactin production. Anguibactin production was never detected in anguibactin production negative strains shown in this figure even after incubation for several days. Experiments were repeated three times, and this figure shows a representative.

Article Snippet: Strains and plasmids Characteristics Reference or source Vibrio anguillarum strains 775 (pJM1) Wild type, Washington (serotype O1, pJM1) Crosa (1980) 775 (pJM1)-pMMB 775 (pJM1) harboring pMMB208 Naka et al. (2008) 96F-pMMB Vanchrobactin producer (serotype O1, plasmidless) harboring pMMB208 Naka et al. (2008) HNVA-8 CC9-16ΔfvtAΔfetA (anguibactin indicator strain) Naka and Crosa (2012) Vibrio harveyi strains ATCC BAA-1116 Marine (Ocean) isolate Lin et al. (2010) HY01 Dead, luminescing shrimp isolate Lin et al. (2010) HNVH-1 HY01ΔangR This study HNVH-2 HY01ΔangRΔfatA This study CAIM 148 Diseased shrimp (Penaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 513T Dead, luminescing amphipod (Talorchestia sp.) isolate V. harveyi type strain ATCC 14126 Lin et al. (2010) CAIM 1075 Oyster (Crassostrea gigas) isolate Lin et al. (2010) CAIM 1766 Sea horse (Hippocampus ingens) liver isolate Lin et al. (2010) CAIM 1792 Diseased shrimp (Litopenaeus vannamei) lesion isolate Lin et al. (2010) Vibrio campbellii strains 42A Healthy coral (Mussismilia hispida) isolate Lin et al. (2010) CAIM 115 Shrimp (Litopenaeus sp.) hemolymph isolate Lin et al. (2010) CAIM 198 Shrimp (Litopenaeus sp.) hepatopancreas isolate Lin et al. (2010) CAIM 519T Seawater isolate V. campbellii type strain ATCC 25920 Lin et al. (2010) CAIM 1500 Snapper (Lutjanus guttatus) liver isolate Lin et al. (2010) Escherichia coli strains DH5a F , /80lacZDM15, endA1, recA1, hsdR17, (rK mK +), supE44, thi-1, gyrA96, relA1, D(lacZYA-argF)U169, k Laboratory stock S17-1kpir k-pir lysogen; thi pro hsdR hsdM+recA RP4 2-Tc::Mu-Km::Tn7 (Tpr Smr) Simon et al. (1983) Plasmids pGEM-T Easy A vector for the cloning of PCR products with blue/white screening, Apr Promega pBluescript II Cloning vector, Ampr Stratagene pBluescript-Km Cloning vector, Kmr This study pDM4 Suicide plasmid sacB gene, R6K origin, Cmr Milton et al. (1996) pHN11 pDM4 harboring ΔangR of V. harveyi HY01 This study pHN12 pDM4 harboring ΔfatA of V. harveyi HY01 This study pMMB208 A broad-host-range expression vector; Cmr IncQ lacIq Ptac; polylinker from M 13mp19 Morales et al. (1991) pHN13 pMMB208 harboring V. harveyi angR This study pHN14 pMMB208 harboring V. harveyi fatA This study ATCC and CAIM strains were obtained from American Type Culture Collection (http://www.atcc.org) and Collection of Aquatic Important Microorganisms (http://www.ciad.mx/caim), respectively.

Techniques: Concentration Assay, Incubation

Journal: International Aquatic Research

Article Title: Dietary short-chain organic acids enhanced resistance to bacterial infection and hepatopancreatic structural integrity of the giant freshwater prawn, Macrobrachium rosenbergii

doi: 10.1007/s40071-017-0177-y

Figure Lengend Snippet: Fig. 2 The mean cumulative mortality (%) of the giant freshwater prawn fed diets with different added organic acids and subsequently challenged with Vibrio harveyi over a 14-day period. Values are mean of triplicate groups of ten prawns. Different letters indicate significant difference (P \ 0.05)

Article Snippet: In preparation for the bacterial disease challenge, V. harveyi (strain no. ATCC 14126), which was stored in a Marine broth 2216 containing beads and 20% (v/v) glycerol (Qrec, New Zealand) (pH 7.4) at -80 C was grown on a TSA plate (Trypticase Soy Agar) at 28 C for 24-h. A single colony was selected and cultured on a TSA plate for another 24-h at 28 C before being transferred to a Tryptic Soy Broth for 24-h at 28 C. The broth cultures were then stored in an incubator shaker for 12 h. The broth culture was then centrifuged at 10,000 rpm for 15 min.

Techniques:

Journal: International Aquatic Research

Article Title: Dietary short-chain organic acids enhanced resistance to bacterial infection and hepatopancreatic structural integrity of the giant freshwater prawn, Macrobrachium rosenbergii

doi: 10.1007/s40071-017-0177-y

Figure Lengend Snippet: Fig. 3 Section of the hepatopancreas from the giant freshwater prawns fed the control diet (a, b) or LA diet (c, d) after being challenged with Vibrio harveyi for 2 weeks. The hepatopancreatic sections of the prawns fed the control diet show hemocyte infiltrations (HI) within the interstitial sinuses, a near collapse of the tubules (a) and separation of myoepithelial layer and epithelium (S) (b). The hepatopancreatic sections of the prawns fed the LA diet show a more normal tubule structure and lumen (L), but with some instances of abnormal lumen (ABL) (c) along with some hemocyte infiltrations (d). E cells (E), B cells (B), R cells (R). H&E stain. Magnification 920; bar 100 lm (a, c). Magnification 940; bar 50 lm (b, d)

Article Snippet: In preparation for the bacterial disease challenge, V. harveyi (strain no. ATCC 14126), which was stored in a Marine broth 2216 containing beads and 20% (v/v) glycerol (Qrec, New Zealand) (pH 7.4) at -80 C was grown on a TSA plate (Trypticase Soy Agar) at 28 C for 24-h. A single colony was selected and cultured on a TSA plate for another 24-h at 28 C before being transferred to a Tryptic Soy Broth for 24-h at 28 C. The broth cultures were then stored in an incubator shaker for 12 h. The broth culture was then centrifuged at 10,000 rpm for 15 min.

Techniques: Control, Staining