Putative Spanner Function of the Vibrio PomB Plug Region in the Stator Rotation Model for Flagellar Motor.

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA (similar to MotA in Escherichia coli) and PomB (similar to MotB in E. coli), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after cross-linking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. IMPORTANCE The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by cross-linking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

Characterization of a CD59 in orange-spotted grouper (Epinephelus coioides).

CD59, a multifunctional glycoprotein, not only plays a regulatory role in complement cascades, but also participates in modulation of teleostean immunity. In this study, full length sequence of EcCD59 was obtained, comprising a 5'UTR of 163 bp, an ORF of 354 bp and a 3'UTR of 559 bp. EcCD59 gene encoded a polypeptide of 117 amino acids. Tissue-specific analysis revealed that the highest expression of EcCD59 mRNA was observed in muscle. Vibrio alginolyticus challenge can significantly increase EcCD59 mRNA expression in liver, kidney and spleen. EcCD59 distribution was detected by a combined approach using GFP-overexpression, immunofluorescence and ELISA assay, indicating that EcCD59 may be predominantly aggregated in cellular membrane. Both EcCD59 and EcCD59delGPI can directly bind to V. alginolyticus and decrease the in vitro growth of V. alginolyticus. Additionally, vibrio injection experiment indicated that the binding of EcCD59 or EcCD59delGPI to V. alginolyticus can restrict its growth rate in vivo. In this study, we found that EcCD59 may be involved in immune defense against vibrio infection in a complement-independent manner.

Localization and domain characterization of the SflA regulator of flagellar formation in Vibrio alginolyticus.

Many swimming bacteria use flagella as locomotive organelles. The spatial and numerical regulation of flagellar biosynthesis differs by bacterial species. The marine bacteria Vibrio alginolyticus use a single polar flagellum whose number is regulated positively by FlhF and negatively by FlhG. Cells lacking FlhF and FlhG have no flagellum. The motility defect in an flhFG deletion was suppressed by a mutation in the sflA gene that resulted in the production of multiple, peritrichous flagella. SflA is a Vibrio-specific protein. SlfA either facilitates flagellum growth at the cell pole or prevents flagellar formation on the cell body by an unknown mechanism. Fluorescent protein fusions to SflA localized to the cell pole in the presence of FlhF and FlhG, but exhibited both polar and lateral cell localization in ΔflhFG cells. Polar localization of SflA required the polar landmark protein HubP. Over-expression of the C-terminal region of SflA (SflAC ) in ΔflhFG ΔsflA cells suppressed the lateral flagellar formation. Our results suggest that SflA localizes with the flagella and that SflAC represses the flagellar initiation in ΔflhFG strains. A model is presented where SflA inhibits lateral flagellar formation to facilitate single polar flagellum assembly in V. alginolyticus cells.

Efficacy of cell free supernatant from Bacillus licheniformis in protecting Artemia salina against Vibrio alginolyticus and Pseudomonas gessardii.

Bacterial diseases are widespread in aquaculture farms and causative agents often adapt to biofilm mode of growth. These biofilms are detrimental to aquaculture species as they resist antibiotics and other agents that are used to control them. Two bacterial pathogens isolated from infected prawn samples were identified as Vibrio alginolyticus and Pseudomonas gessardii on the basis of morphological features, biochemical characteristics, 16S r RNA gene sequencing and phylogenetic analysis. Their pathogenic nature was confirmed by performing in vivo challenge experiments using Artemia salina as a model system. Seven days post infection, the mortality observed with V. alginolyticus and P. gessardii was 97 ±â€¯4.08% and 77.5 ±â€¯5.24%, respectively. The isolates formed extensive biofilms on polystyrene and glass surfaces. These infections could be controlled in an effective manner by using the cell free supernatant (CFS) of a tropical marine epizoic strain of Bacillus licheniformis D1 that is earlier reported to contain an antimicrobial protein (BLDZ1). The CFS inhibited biofilms in an efficient manner (82.35 ±â€¯1.69 and 82.52 ±â€¯1.11% for V. alginolyticus and P. gessardii, respectively) on co-incubation. In addition, pre-formed biofilms of V. alginolyticus and P. gessardii were also removed (84.53 ±â€¯1.26 and 67.08 ±â€¯1.43%, respectively). Fluorescence and scanning electron microscopic studies confirmed the antibiofilm potential of this protein on glass surfaces. The antibiofilm nature was due to the anti-adhesion and antimicrobial properties exhibited by the CFS. Treatment of A. salina with CFS (6 h prior to infections) was effective in protecting larvae against infections by field isolates. This study highlights the significance of marine natural products in providing alternative biofilm controlling agents to tackle infections and decreasing the usage of antibiotics in aquaculture settings.

Chromatin Immunoprecipitation Sequencing Technology Reveals Global Regulatory Roles of Low-Cell-Density Quorum-Sensing Regulator AphA in the Pathogen Vibrio alginolyticus.

Quorum sensing (QS) is an important regulatory system in virulence expression and environmental adaptation in bacteria. The master QS regulators (MQSR) LuxR and AphA reciprocally control QS gene expression in vibrios. However, the molecular basis for the regulatory functions of AphA remains undefined. In this study, we characterized its regulatory roles in Vibrio alginolyticus, an important zoonotic pathogen causing diseases in marine animals as well as in humans. AphA is involved in the motility ability, biofilm formation, and in vivo survival of V. alginolyticus Specifically, AphA is expressed at low-cell-density growth phases. In addition, AphA negatively regulates the expression of the main virulence factor, alkaline serine protease (Asp), through LuxR. Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) detected 49 enriched loci harboring AphA-binding peaks across the V. alginolyticus genome. An AphA-specific binding motif was identified and further confirmed by electrophoretic mobility shift assay (EMSA) and mutagenesis analysis. A quantitative real-time PCR (qRT-PCR) assay further validated the regulation of AphA on these genes. AphA binds directly to the aphA promoter and negatively regulates its own expression. Moreover, AphA directly regulates genes encoding adenylate cyclase, anti-σD, FabR, and the small RNA CsrB, revealing versatile regulatory roles of AphA in its physiology and virulence. Furthermore, our data indicated that AphA modulates motility through the coordinated function of LuxR and CsrB. Collectively, the findings of this work contribute to better understanding of the regulatory roles of AphA in QS and non-QS genes. IMPORTANCE: In this work, we determined that AphA, the master regulator of QS at low cell density, plays essential roles in expression of genes associated with physiology and virulence in V. alginolyticus, a Gram-negative pathogen for humans and marine animals. We further uncovered that 49 genes could be directly regulated by AphA and a 19-bp consensus binding sequence was identified. Among the 49 genes, the QS and other non-QS-associated genes were identified to be regulated by AphA. Besides, the small RNA CsrB was negatively regulated by AphA, and AphA regulate motility abilities through both CsrB and LuxR. Taken together, the findings of this study improve our understanding of the complex regulation network of AphA and QS.

PtSerpin from the swimming crab Portunus trituberculatus, a putative regulator of prophenoloxidase activation with antibacterial activity.

Serpin or serine protease inhibitor is the largest family of protease inhibitors involved in many innate immune pathways, particularly the prophenoloxidase (proPO) activating system in arthropod. Here, we report the molecular and functional characterization of PtSerpin identified from the swimming crab Portunus trituberculatus. The genomic sequence encoding mature peptide of PtSerpin gene contained two exons of 84 and 1098 bp separated by one intron of 111 bp. The recombinant PtSerpin (rPtSerpin) with a predicted size of 44 kDa was expressed in Escherichia coli system, purified and assayed for its activities. The rPtSerpin exhibited inhibitory activity against trypsin in a dose-dependent manner, but did not affect chymotrypsin, which could define a role for PtSerpin as a trypsin inhibitor. The rPtSerpin could inhibit the growth of Gram-negative bacterium Vibrio alginolyticus, but not the tested Gram-positive bacterium and fungus. Further phenoloxidase (PO) assay showed PO activity was dramatically increased in hemocyte lysate supernatant of P. trituberculatus upon bacterial challenge. The rPtSerpin could depress the crab proPO system activation in vitro, and it could lead to 100% inhibition of PO activity under the concentration of 8.62 µM. Moreover, the rPtSerpin was able to inhibit the PO activity induced by rPtcSP and rPtSPH1. These results together indicate that PtSerpin is a potential trypsin inhibitor and may participate in crab innate immunity by the inhibition of bacterial growth and the regulation of proPO system.

VscO, a putative T3SS chaperone escort of Vibrio alginolyticus, contributes to virulence in fish and is a target for vaccine development.

Type III secretion system (T3SS) in Vibrio alginolyticus is essential for its pathogenesis. VscO's homologous proteins FliJ, InvI and YscO have been suggested to be putative chaperone escorts although its function in V. alginolyticus is unclear. To investigate the physiological role of VscO, a mutant strain of V. alginolyticus with an in-frame deletion of the vscO gene was constructed in the present study. One finding was that the mRNA expression levels of SycD, VopB and VopD proteins decreased in the ΔvscO mutant. In addition, the ΔvscO mutant showed an attenuated swarming ability and a ten-fold decrease in the virulence to fish. However, the ΔvscO mutant showed no difference in the biofilm formation and ECPase activity. Complementation of the mutant strain with the vscO gene could restore the phenotypes of the wild-type strain. Finally, the recombinant VscO protein caused a high antibody titer and an effective protection against lethal challenge with the wild-type strain V. alginolyticus. These results indicated that VscO protein has a specific role in the pathogenesis of V. alginolyticus and it may be a candidate antigen for development of a subunit vaccine against vibriosis.

Characterization of the periplasmic region of PomB, a Na+-driven flagellar stator protein in Vibrio alginolyticus.

The stator proteins PomA and PomB form a complex that couples Na(+) influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB(C)). To investigate the function of PomB(C), a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB(C) fragment consisting of residues 135 to 315 (PomB(C5) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB(ΔL)) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na(+) influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB(ΔL) stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal α-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a "plug" segment is important for the control of Na(+) influx through the stator complex as well as for stator assembly.

Roles of Hfq in the stress adaptation and virulence in fish pathogen Vibrio alginolyticus and its potential application as a target for live attenuated vaccine.

Vibrio alginolyticus has brought about severe economic damage to the mariculture industry by causing vibriosis in various fish species in South China. The virulent determinants of this bacterium have not been well characterized except the exotoxin alkaline serine protease, Asp. In addition, the mechanism of virulence regulation in V. alginolyticus remains largely unknown apart from a Vibrio harveyi-like quorum sensing (QS) system which is established to manipulate the expression of various virulence-related genes. Hfq, an sRNA chaperone, is an important post-transcriptional regulator in a variety of bacteria. Here, the roles of Hfq were characterized in regulating the stress resistance and pathogenesis in V. alginolyticus. We demonstrated that the hfq deletion mutant became more sensitive to several environmental stresses, including osmotic stress, ethanol, temperature shift, and iron starvation. The deletion of hfq abrogated the motility and biofilm formation in this bacterium. Hfq negatively regulated the expression of main virulence factor, Asp, through QS system. The results also indicated that Hfq modulated the survival and multiplication of V. alginolyticus in fish. Hfq thus appears to be a new pleiotropic regulator of pathogenesis in V. alginolyticus. Moreover, high immunoprotective rate was achieved with a single dose of injection or immersion vaccination with live hfq mutant, suggesting the mutant's merits as a valuable vaccine candidate against V. alginolyticus.

Occurrence of Vibrio parahaemolyticus and Vibrio alginolyticus in the German Bight over a seasonal cycle.

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10(3) N l(-1) and MPNs up to 240 N g(-1) were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.

A selective and differential medium for Vibrio alginolyticus.

In this paper, we present a selective and differential medium, termed Vibrio alginolyticus (VAL) agar, developed for the isolation and identification of V. alginolyticus. The presence of bile salts, high salinity and high incubation temperature allows the selective growth of moderately halophilic Vibrio species. Differentiation of bacteria is achieved by identifying species capable of sucrose fermentation, made visible by the pH indicator bromocresol purple. In this study, all of the 26 strains of V. alginolyticus and only three of the 99 strains representing 30 species (including 19 Vibrio species) other than V. alginolyticus were able to grow in the VAL medium. The remaining three strains could be further differentiated from V. alginolyticus according to colour or the diameter of colonies produced on VAL agar plates. Colonies isolated from shellfish rearing water and infected shrimp through the use of VAL agar plates were all positively identified as V. alginolyticus by conventional tests and 16S rDNA sequencing. The testing of specificity and differentiation capability of VAL shows the potential of the agar as a medium for the primary isolation of V. alginolyticus from pathological and environmental samples.

Roles of rpoN in biofilm formation of Vibrio alginolyticus HN08155 at different cell densities.

RpoN (δ54) as a global regulator controls crucialvirulence-associated phenotype, which can regulate flagellum and exopolysaccharides (EPS) during pathogenic biofilm formation. However, the knowledge of the roles of rpoN in biofilm formation of V. alginolyticus is limited, especially at different cell densities. Herein, deletion mutant strain ΔrpoN, complementary strain ΔrpoN-C and negative control strain ΔrpoN-Z were constructed to investigate the effects of rpoN on biofilm formation of V. alginolyticus HN08155 based on flagellum and EPS at different cell density conditions. The results showed that all of strains can form biofilm, and biofilms of strains with rpoN were formed at low cell density (LCD) and detached at high cell density (HCD), while those of ΔrpoN and ΔrpoN-Z were absent at LCD and accumulated excessively with a spotty pellicle at HCD without detaching. The EPS contents of strains with rpoN was greater than that of ΔrpoN and ΔrpoN-Z at LCD, while the opposite trends were observed at HCD. The expression levels of rpoN were quantified, which were consistent with the trend of biofilm formation. It's worth noting that absence of rpoN resulted in the failure of biofilm detachment, lacking of flagellum and decreasing motility, indicating that rpoN was not necessary for biofilm formation, but it was essential for biofilm detachment.

Conformational and dynamics simulation study of antimicrobial peptide hedistin-heterogeneity of its helix-turn-helix motif.

Hedistin is an antimicrobial peptide isolated from the coelomocytes of Nereis diversicolor, possessing activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The three-dimensional structure of hedistin in both aqueous solution and deuterated dodecylphosphocholine (DPC) micelles was examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. And, the early events of the antibacterial process of hedistin were simulated using palmitoyl-oleoyl-phophatidylcholine (POPC) lipid bilayers and molecular dynamics (MD) simulation methods. Hedistin lacks secondary structure in aqueous solution, however, in DPC micelles, it features with a heterogeneous helix-turn-helix moiety and exhibits obvious amphipathic nature. The turn region (residues Val9-Thr12) in the moiety is a four-residue hinge, lying in between the first N-terminal alpha-helix (residues Leu5-Lys8) and the second alpha-helix (residues Val13-Ala17) regions and causing an approximately 120 degrees angle between the axes of the two helices. The segmental and nonlinear nature of hedistin structure is referred to as the heterogeneity of its helix-turn-helix motif which was found to be corresponding to a kind of discrete dynamics behavior, herein coined as its dynamical heterogeneity, at the early stage (0-50 ns) of the MD simulations. That is, the first helix segment, prior to (at 310 K) or following (at 363 K) the second helix, binds to the lipid head-group region and subsequently permeates into the hydrophobic lipid tail region, and the hinge is the last portion entering the lipid environment. This result implies that hedistin may adopt a "carpet" model action when disrupting bacterial membrane.

Deletion of valR, a homolog of Vibrio harveyis luxR generates an intermediate colony phenotype between opaque/rugose and translucent/smooth in Vibrio alginolyticus.

A previous study has shown that Vibrio alginolyticus ZJ-51 undergoes colony phase variation between opaque/rugose (Op) and translucent/smooth (Tr). The AI-2 quorum-sensing master regulator ValR, a homolog to V. harveyi LuxR, was suggested to be involved in the transition. To investigate the role of ValR in the variation and in biofilm formation, an in-frame deletion of valR in both Op and Tr backgrounds was carried out. The mutants in both backgrounds showed an intermediate colony morphotype, where the colonies were less opaque/rugose but not fully translucent/smooth either. They also showed an intermediate level of motility. However, biofilm formation was severely decreased in both mutants and polar flagella were depleted also. Quantitative PCR showed that most of the genes related to flagellar and polysaccharide biosynthesis were upregulated in the mutant of Op background (Delta valR/Op) but downregulated in the mutant of Tr background (Delta valR/Tr) compared with their parental wild-type strains. This suggests that ValR may control biofilm formation by regulating flagellar biosynthesis and affect the expression of the genes involved in colony phase variation in V. alginolyticus.

Na+-NQR Confers Aminoglycoside Resistance via the Regulation of l-Alanine Metabolism.

Sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) functions as a unique redox-driven sodium pump, generating membrane potential, which is related to aminoglycoside antibiotic resistance. However, whether it modulates other metabolisms to confer antibiotic resistance is unknown. The present study showed that loss of nqrA or nqrF led to differential metabolomes with elevated resistance to aminoglycoside antibiotics. Decreased alanine, aspartate, and glutamate metabolism and depressed abundance of alanine were characterized as the most impacted pathway and crucial biomarker, respectively. Further data showed that higher viability was detected in ΔnqrA and ΔnqrF mutant strains than their parent strain ATCC 33787 in the presence of gentamicin but recovered by exogenous l-alanine. It proceeds by the following events. The loss of nqrA or nqrF led to the decrease of membrane potential, ATPase activity, and then ATP and cyclic AMP (cAMP), which reduced the cAMP/CRP (cAMP receptor protein) complex. The reduced cAMP/CRP complex promoted l-alanine catabolism and inhibited l-alanine anabolism, causing reduced levels of alanine. Reduced alanine affected the expression of antiporter families Atp and Mnh genes. Our results suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.IMPORTANCE The Na+-NQR complex functions as a unique redox-driven sodium pump, generating membrane potential directly. However, whether it mediates generation of membrane potential indirectly is unknown. The present study shows that the Na+-NQR complex impacts membrane potential through other antiporter families Atp and Mnh. It proceeds by ATP and then cAMP/CRP regulon, which inhibits l-alanine catabolism and promotes l-alanine anabolism. When the Na+-NQR complex is reduced as in antibiotic-resistant bacteria, l-alanine is depressed, which is related to the antibiotic resistance phenotypes. However, exogenous l-alanine reverts the phenotype and promotes antibiotic-mediated killing. These findings suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.

Bio-inspired antibacterial cellulose paper-poly(amidoxime) composite hydrogel for highly efficient uranium(vi) capture from seawater.

A bio-inspired cellulose paper-poly(amidoxime) composite hydrogel is explored via UV-polymerization. This hydrogel has a highly efficient uranium capture capacity of up to 6.21 mg g-1 for WU/Wdry gel and 12.9 mg g-1 for WU/Wpoly(amidoxime) in seawater for 6 weeks, due to its enhanced hydrophilicity, good hydraulic/ionic conductivity and broad-spectrum antibacterial performance.

Cloning, identification, and characterization of the rpoS-like sigma factor rpoX from Vibrio alginolyticus.

Vibrio alginolyticus ZJ-51 displays phase variation between opaque/rugose colonies (Op) and translucent/smooth colonies (Tr). These colony variants show great differences in biofilm formation and motility. In this study, a gene encoding for an rpoS-like sigma factor, rpoX, has been cloned and characterized. The absence of rpoX did not affect colony switching rate but did decrease biofilm formation in both the Op and the Tr variants. When challenged with hydrogen peroxide, the DeltarpoX in the Op background showed a slightly higher survival rate compared with the wild type, whereas survival was decreased in the Tr background. Deletion of rpoX in the Tr background resulted in a higher ability to resist ethanol challenges and to survive hyperosmolarity challenges, and in the Op background the opposite phenotype was observed. This indicates that the rpoX gene is involved in biofilm formation and stress response but the effects are controlled by colony phase variation in V. alginolyticus.

Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus.

Many pathogens undergo phase variation between rugose and smooth colony morphology or between opaque and translucent colony morphology, which is mainly due to the variation in the surface polysaccharides. In this study, Vibrio alginolyticus ZJ-51 displayed phase variation between opaque, rugose colonies (Op) and translucent, smooth colonies (Tr). Unlike the vibrios reported previously, Tr cells of ZJ-51 enhanced biofilm formation and motility, but they did not differ from Op cells in the quantity of surface polysaccharides produced. Real time PCR was used to analyze the expression of the genes involved in polysaccharide biosynthesis, flagellar synthesis, and the AI-2 quorum-sensing system. The results revealed that the K-antigen capsule gene cluster (which consists of homologs to the cpsA-K in Vibrio parahaemolyticus) and O-antigen polysaccharide gene cluster (which contains homologs to the wza-wzb-wzc) were significantly more transcribed in Tr cells. The AI-2 quorum-sensing genes showed enhanced expression in the Tr variant which also exhibited greater expression of genes associated with polar flagellar biosynthesis. These results suggest that colony phase variation might affect the virulence and survival ability in the stressful environment inhabited by V. alginolyticus.

Investigation of several virulence properties among Vibrio alginolyticus strains isolated from diseased cultured fish in Tunisia.

We analysed 34 Vibrio alginolyticus strains isolated from gilthead sea bream Sparus aurata L. and sea bass Dicentrarchus labrax L. cultured in fish farms on the Tunisian Mediterranean coast for the presence of several virulence properties such as extracellular products (ECP) production, growth in iron-limiting conditions and survival in fish serum. The results obtained with different substrates showed that ECP of V. alginolyticus were hydrolytic. The virulence was correlated with the ability of strains to grow in the presence of non-immune fish serum or under conditions of iron limitation. We further examined the presence of virulence genes homologous to those in V. cholerae (toxR, toxS, VPI and ace); toxR was found in 16 V. alginolyticus strains and toxS in 17 strains out of 34 analysed. A positive amplification for the virulence pathogenicity island (VPI) was produced by 12 V alginolyticus strains. Finally, the ace expected amplification fragment was found in 7 V. alginolyticus isolates. Thus, the pathogenicity of V. alginolyticus may be the result of a combination of all these factors.

Bioactive Compounds of Pseudoalteromonas sp. IBRL PD4.8 Inhibit Growth of Fouling Bacteria and Attenuate Biofilms of Vibrio alginolyticus FB3.

Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C16H24O2). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3.Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C16H24O2). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3.