Alginolyticus Cells Research Articles

Management biofouling of uranium extraction from seawater by ultraviolet technique: Effect of UV wavelength

Marine biofouling is one of the primary limiting factors in uptake of uranium (U(VI)) from seawater. The effects of ultraviolet (UV) wavelength on its elimination capacity for marine biofouling were investigated in detail. V. alginolyticus was used as the representative of marine microorganisms in this work. UV254 irradiation can well reduce the adhesion of V. alginolyticus on poly(amidoxime) (PAO) surface and enhance U(VI) adsorption. The adsorption capacity of PAO for U(VI) increases from 40.7 mg/g to 67.1 mg/g at pH 8.2 and 298 K under UV254 irradiation condition. UV254 irradiation can well damage the DNA structure of V. alginolyticus by photochemistry reaction, and further reduce its extracellular polymeric substances (EPS). The increase of NO3- concentrations promote the yield of ˙OH by UV254 irradiation, and increase the inactivation of V. alginolyticus cells. Biological community results indicate that UV254 irradiation can intercept the reproduction and break the bottleneck of marine microorganisms, and would enhance the extraction of U(VI) from seawater.

Efficacy of vaccine from whole killed Vibrio alginolyticus cells on the immune response of white shrimp (Litopenaeus vannamei)

Efficacy of vaccine from whole killed Vibrio alginolyticus cells on the immune response of white shrimp (Litopenaeus vannamei)

CCCP Facilitates Aminoglycoside to Kill Late Stationary-Phase Escherichia coli by Elevating Hydroxyl Radical.

Improving the efficacy of existing antibiotics is significant for combatting antibiotic resistance that poses a major threat to human health. Carbonyl cyanide m-chlorophenylhydrazine (CCCP), a well-known protonophore for dissipating proton motive force (PMF), has been widely used to block the PMF-dependent uptake of aminoglycoside antibiotics and thus suppress aminoglycoside lethality. Here, we report that CCCP and its functional analog FCCP, but not other types of protonophores, unprecedently potentiate aminoglycosides (e.g., tobramycin and gentamicin) by 3-4 orders of magnitude killing of Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Vibrio alginolyticus cells in stationary phase but not these cells in exponential phase nor other 12 bacterial species we examined. Overall, the effect of CCCP on aminoglycoside lethality undergoes a gradual transition from suppression against E. coli exponential-phase cells to potentiation against late stationary-phase cells, with the cell growth status and culture medium being crucial. Consistently, disturbance of the PMF by changing transmembrane proton gradient (ΔpH) or electric potential (ΔΨ) also potentiates tobramycin. Nevertheless, CCCP neither increases the intracellular concentration of tobramycin nor decreases the MIC of the antibiotic, thus excluding that CCCP acts as an efflux pump inhibitor to potentiate aminoglycosides. Rather, we show that the combined treatment dramatically enhances the cellular level of hydroxyl radical under both aerobic and anaerobic culturing conditions, under which the antioxidant N-acetyl cysteine fully suppresses both hydroxyl radical accumulation and cell death. Together, these findings open a new avenue to develop certain protonophores as aminoglycoside adjuvants against pathogens in stationary phase and also illustrate an essential role of hydroxyl radical in aminoglycoside lethality regardless of aerobic respiration.

The antibacterial activity of clove Syzygium aromaticum extract and its effects on the survival rate of hybrid grouper Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂ infected with Vibrio alginolyticus

Vibrio alginolyticus which becomes the main cause of vibriosis disease in grouper culture and causes great economic loss in Asian aquaculture industries. This study investigated the antibacterial activity of clove extract and the effect of adding clove powder to the diet on survival cantang grouper infected with V. alginolyticus. The clove extraction used a maceration method. Two dose levels of clove powder were used, namely 10 and 15 g kg−1. The control treatments without clove powder supplementation contained positive control (CP) and negative control treatment (CN). The results found that the clove extract contained five major compounds. The highest compound was phenol, 2-methoxy-4-(2-propenyl)-Eugenol (64.07%). Phytochemical analysis of clove extract contained phenolics, flavonoids, and tannins at (28.53 ± 0.00) mg/g, (0.38 ± 0.00) mg/g, and (0.15 ± 0.00) %, respectively. The diameter of the clove extract inhibition zone was significantly different (P < 0.05) in all treatments. The Scanning Electron Microscopy (SEM) result presents that the clove extract can alter the V. alginolyticus cell morphology. The dietary supplementation of clove powder improves the survival rate significantly higher (P<0.05) post-challenge test. The conclusion of this research is that clove extract has antibacterial activity that can inhibit growth and cause cell morphological damage to V. alginolyticus. The application of clove powder at a dose of 15g kg-1 was able to improve the survival value which was a higher post-challenge test.
 
 Keyword: antibacterial activity, clove, grouper, phytochemical, Vibrio alginolyticus

Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of NpHM4, a Derived Peptide of Nautilus pompilius Hemocyanin.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 μM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

Membrane-disruptive engineered peptide amphiphiles restrain the proliferation of penicillins and cephalosporins resistant Vibrio alginolyticus and Vibrio parahaemolyticus in instant jellyfish

Over 1 billion people rely on seafood as their primary source of protein and Vibrio spp. is the most important pathogen of foodborne disease outbreak related to consumption of seafood. Moreover, the rise of drug-resistant Vibrio strains further increases the difficulty of clinical treatment. However, there is no food preservative available to combat drug-resistant Vibrio spp. in seafood. In this study, we designed and synthesized three novel antibacterial peptides (ABPs) derived from zp3 (GIIAGIIIKIKK-NH 2 ). One of them, 3 K 5 K 7 G (GIKAKIGIKIKK-NH 2 ), had bioactivity against all tested penicillins and cephalosporins resistant Vibrio with minimal inhibitory concentration (MIC) values at 2–16 μM, performing better than its template zp3 . 3 K 5 K 7 G demonstrated favourable antibacterial capability in instant jellyfish, killing more than 99% Vibrio alginolyticus cells after 64 μM treatment for 4 h. Peptide 3 K 5 K 7 G could disrupt the cell membrane, resulting in rapid membrane potential dissipation. Next, this molecule could penetrate inside cytoplasm and further bind with DNA. Importantly, 3 K 5 K 7 G showed low cytotoxicity to mammalian cells (95% Vero cells survived in the presence of 160 μM 3 K 5 K 7 G ). Collectively, 3 K 5 K 7 G has the potential to be developed into a promising food-grade antibacterial additive, especially against those resistant Vibrio spp. in seafood. • 3 K 5 K 7 G performed better antibacterial activity than its template zp3 with α-helix structure. • 3 K 5 K 7 G had MIC values at 2–16 μM to penicillins and cephalosporins resistant Vibrio. • 3 K 5 K 7 G killed more than 99% V. alginolyticus in instant jellyfish after 4 h treatment. • 3 K 5 K 7 G disrupted the cell membrane, resulting in rapid membrane potential dissipation. • 3 K 5 K 7 G had low cytotoxicity to mammalian cells.

Effect of booster vaccination on immunoprotection in European seabass vaccinated against vibriosis

In farmed saltwater fish, vaccination is considered the first barrier against pathogenic agents. In Egypt, the vaccine application can diminish the dilemma of summer mass kills of farmed marine fish. The efficacy of booster vaccination with a bivalent vaccine against vibriosis was studied in European seabass. Fish were vaccinated by direct immersion (DI) in water containing bivalent Vibrio alginolyticus and Vibrio parahemolyticus whole cells killed vaccine. Immersion vaccination is more applicable under semi-intensive fish culture, but less potent than injection. Accordingly, to increase antigen uptake and conferred protection, vaccinated fish randomly received booster vaccination via direct immersion (DI imm.) or intraperitoneal injection (i.p.) at 21 days post-prime vaccination (21 dpv). Vaccinated and unvaccinated fish were monitored for 3 months and challenged with V. alginolyticus and/or V. parahemolyticus at 42 dpv. Specific and non-specific immune responses were evaluated in blood samples. At 52 dpv (10 days post-challenge), the relative immunoglobulin T (IgT) expression was assessed in the gills, hindgut, head kidney and spleen. Notably, imm. + imm. and imm. + i.p. vaccination yielded significantly higher specific antibody titre at 70 dpv, whereas the former was associated with significantly higher IgT expression in the gills and hindgut (p < .05) than the latter. High IgT levels in mucosal organs (gills and hindgut) might be the first indicator of the involvement of IgT in mucosal immune responses. Relative per cent survival among booster-vaccinated fish ranged from 70% to 90%. The results support the use of this vaccination protocol as a highly effective tool to protect seabass from vibriosis and to improve fish health and environment. Booster doses are strongly recommended under farming environments.

Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects.

Vibrio alginolyticus (V. alginolyticus) is a common pathogen for humans and marine aquatic animals. Vibriosis of marine aquatic animals, caused by V. alginolyticus, has become more prevalent globally in recent years. Hence, a safe and effective vaccine is urgently needed for the control of this disease. Here, the strain 16‐3 isolated from the large yellow croaker (Larimichthys crocea) suffered from canker was identified as V. alginolyticus based on morphological, biochemical, and 16S rDNA sequencing analysis. Then, recombinant temperature‐controlled lysis plasmid pBV220‐lysisE was electroporated into the strain 16‐3 to generate V. alginolyticus bacterial ghosts (VaBGs) by inducing lysis gene E expression, and the safety and immune effects of VaBGs were further investigated in mice and large yellow croaker. The results showed that VaBGs were as safe as formalin‐killed V. alginolyticus cells (FKC) to mice and fish. Compared with FKC and PBS groups, significant elevations of the serum agglutinating antibody titer, serum bactericidal activity, lymphocyte proliferative responses, and levels of four different cytokines (Th1 type: IL‐2, TNF‐α; Th2 type: IL‐4 and IL‐6) in serum were detected in the VaBGs group, indicating that a Th1/Th2‐mediated mixed immune response was elicited by the VaBGs. More importantly, after challenged with the parent strain 16‐3, VaBGs‐vaccinated mice and fish showed higher protection than FKC‐vaccinated mice, the relative percent of survival (RPS) being 60%, 66.7% and 40%, respectively. Taken together, this is the first demonstration that the newly constructed V. alginolyticus ghosts may be developed as a safe and effective vaccine against V. alginolyticus infection in aquaculture.

INCREASE OF IMMUNITY CANTIK HYBRID GROUPER JUVENILES BY LIPOPOLYSACCHARIDE (LPS)

Disease is one of obstacle in fish culture. Immunity of fish against disease can be increased by using immunostimulant. The purpose of this experiment to increasing immunity of cantik hybrid grouper (cross breeding of tiger grouper, Epinephelus fuscoguttatus female with marbled grouper, E. polyphekadion male) to the diseases by using immunostimulant. Treatments were LPS cell walls of V. alginolyticus and commercial LPS was also used in this study, as a-control without LPS was used saline water (0.85% NaCl). The LPS was delivered by injection intra-muscularly with concentration of 0.1 mL/fish. This experiment was design by completely randomized with 3 replicates. Results showed that LPS were effective to increase titer antibody values and survival rates of cantik hybrid grouper. The titer antibody values of fish after 90 days rearing period were 128 (LPS from V. alginolyticus), 64 (commercial LPS), and only 4 in the control fish. The survival rates at the end of experiment were 88.67 ± 3.62% (LPS from V. alginolyticus), 85.22±5.93% (commercial LPS), and only 50.13 ± 6.11% in the control fish. Relative percentage survival of fish following challenged with live V. alginolyticus were 77.46% in the received LPS from V. alginolyticus cell wall and 71.12% in the fish received commercial LPS. It is suggested that LPS effective to increase immunity of cantik hybrid grouper against bacterial infection. Keywords: cantik hybrid grouper, immunity, lipopolysaccharide, Vibrio alginolyticus

Molecular architecture of the sheathed polar flagellum in Vibrio alginolyticus

Vibrio species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. Vibrio swimming motility is driven by a polar flagellum covered with a membranous sheath, but this sheathed flagellum is not well understood at the molecular level because of limited structural information. Here, we use Vibrio alginolyticus as a model system to study the sheathed flagellum in intact cells by combining cryoelectron tomography (cryo-ET) and subtomogram analysis with a genetic approach. We reveal striking differences between sheathed and unsheathed flagella in V. alginolyticus cells, including a novel ring-like structure at the bottom of the hook that is associated with major remodeling of the outer membrane and sheath formation. Using mutants defective in flagellar motor components, we defined a Vibrio-specific feature (also known as the T ring) as a distinctive periplasmic structure with 13-fold symmetry. The unique architecture of the T ring provides a static platform to recruit the PomA/B complexes, which are required to generate higher torques for rotation of the sheathed flagellum and fast motility of Vibrio cells. Furthermore, the Vibrio flagellar motor exhibits an intrinsic length variation between the inner and the outer membrane bound complexes, suggesting the outer membrane bound complex can shift slightly along the axial rod during flagellar rotation. Together, our detailed analyses of the polar flagella in intact cells provide insights into unique aspects of the sheathed flagellum and the distinct motility of Vibrio species.

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.

The RNA Chaperone Hfq Is Involved in Colony Morphology, Nutrient Utilization and Oxidative and Envelope Stress Response in Vibrio alginolyticus.

Hfq is a global regulator that is involved in environmental adaptation of bacteria and in pathogenicity. To gain insight into the role of Hfq in Vibrio alginolyticus, an hfq deletion mutant was constructed in V. alginolyticus ZJ-T strain and phenotypically characterized. Deletion of hfq led to an alteration of colony morphology and reduced extracellular polysaccharide production, a general impairment of growth in both rich medium and minimal media with different carbon sources or amino acids, enhanced sensitivity to oxidative stress and to several antibiotics. Furthermore, a differential transcriptomic analysis showed significant changes of transcript abundance for 306 protein coding genes, with 179 genes being up regulated and 127 down-regulated. Several of these changes could be related to the observed phenotypes of the mutant. Transcriptomic data also provided evidence for the induction of the extracytoplasmic stress response in absence of Hfq. Altogether, these findings point to broad regulatory functions for Hfq in V. alginolyticus cells, likely to underlie an important role in pathogenicity.

Speed-dependent chemotactic precision in marine bacteria

Chemotaxis underpins important ecological processes in marine bacteria, from the association with primary producers to the colonization of particles and hosts. Marine bacteria often swim with a single flagellum at high speeds, alternating "runs" with either 180° reversals or ∼90° "flicks," the latter resulting from a buckling instability of the flagellum. These adaptations diverge from Escherichia coli's classic run-and-tumble motility, yet how they relate to the strong and rapid chemotaxis characteristic of marine bacteria has remained unknown. We investigated the relationship between swimming speed, run-reverse-flick motility, and high-performance chemotaxis by tracking thousands of Vibrio alginolyticus cells in microfluidic gradients. At odds with current chemotaxis models, we found that chemotactic precision-the strength of accumulation of cells at the peak of a gradient-is swimming-speed dependent in V. alginolyticus Faster cells accumulate twofold more tightly by chemotaxis compared with slower cells, attaining an advantage in the exploitation of a resource additional to that of faster gradient climbing. Trajectory analysis and an agent-based mathematical model revealed that this unexpected advantage originates from a speed dependence of reorientation frequency and flicking, which were higher for faster cells, and was compounded by chemokinesis, an increase in speed with resource concentration. The absence of any one of these adaptations led to a 65-70% reduction in the population-level resource exposure. These findings indicate that, contrary to what occurs in E. coli, swimming speed can be a fundamental determinant of the gradient-seeking capabilities of marine bacteria, and suggest a new model of bacterial chemotaxis.

An investigation on the immunoassays of an ammonia nitrogen-degrading bacterial strain in aquatic water

Probiotics have widely been used to maintain and improve water quality condition by removal of ammonia nitrogen in aquaculture. To determine the relationship between the efficiency and quantity of probiotics, the method for detection and quantification and safety of the shrimp Litopenaeus vannamei were explored using an ammonia nitrogen-degrading bacterial strain Vibrio alginolyticus. The polyclonal antibodies (Pabs) for immunosorbent assay (indirect ELISA) were used to quantify the probiotics. The results showed that: (1) the ammonia nitrogen degradation rate of the candidate strain was 67.49%; (2) no significant differences in the accumulative mortalities of the shrimp between the treatments and the control (P>0.05); and (3) for the use of indirect ELISA test, detection limit is 104CFUml−1 and an acceptable linear correlation (R2=0.95) was obtained with the range of quantification between l04 and 108CFUmL−1 of the specific target bacteria. The results suggest that the indirect ELISA is a very rapid and sensitive technique for detecting V. alginolyticus in the cultured water. Statement of relevanceA strain of V. alginolyticus with high ammonia–nitrogen degradation rate, which was safe for L. vannamei was acquired. Polyclonal antibodies (Pabs) with high titer were obtained. An indirect enzyme-linked immunosorbent assay (indirect ELISA) was set up to detect V. alginolyticus. The technique is an effective method to quantify the probiotics of V. alginolyticus cells in the cultured water.

Pressure-Speed Relationship of the Sodium-Driven Flagellar Motor of Vibrio Alginolyticus

The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Motor rotation rate and directions can be changed by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also an inhibitor of the rotation of flagellar motors [1, 2]. Our previous results indicated that the application of pressure inhibits the rate of ion tranlocation in the mechanochemical energy translation, but the detailed mechanism is still unknown. Here, we characterized the pressure dependence of the rotational speed of sodium-driven flagellar motor in swimming Vibrio alginolyticus cells. The motor in strain NMB136 exclusively rotates in counter-clockwise direction and propells the cell body forward. We monitored the pressure-induced effects on the behavior of the cells that swim freely in solution. The swimming speed exponentially decreased with the increment of pressure. The sodium concentration dependence of the swimming speed at each pressure was well described by a Michaelis-Menten kinetics. The applied pressures decreased the maximum velocity, but increased the Michalis constant. Our results showed that the motor has at least two pressure-sensitive reactions, one of which is the binding process of external sodium ions to the motor. Another is the post-sodium-binding process, suggesting sodium transit and/or its release to inside the cell.[1] Nishiyama M. and Y. Sowa. 2012. Microscopic Analysis of Bacterial Motility at High Pressure. Biophys. J.102:1872-1880.[2] Nishiyama M. et al. 2013. High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor. J. Bactetiol.195: 1809-1814.

Molecular cloning and expression of a heat-shock cognate 70 (hsc70) gene from swordtail fish (Xiphophorus helleri)

Heat shock proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In the present study, a full-length cDNA, encoding the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), was isolated from swordtail fish (Xiphophorus helleri) and designated as XheHsc70. The Xhehsc70 cDNA was 2 104 bp long with an open reading frame of 1 941 bp, and it encoded a protein of 646 amino acids with a theoretical molecular weight of 70.77 kDa and an isoelectric point of 5.04. The deduced amino acid sequence shared 94.1%-98.6% identities with the Hsc70s from a number of other fish species. Tissue distribution results show that the Xhehsc70 mRNA was expressed in brain, heart, head kidney, kidney, spleen, liver, muscle, gill, and peripheral blood. After immunization with formalin-killed Vibrio alginolyticus cells there was a significant increase in the Xhehsc70 mRNA transcriptional level in the head kidney of the vaccinated fish compared with in the control at 6, 12, 24, and 48 h as shown by quantitative real time RT-PCR. Based on an analysis of the amino acid sequence of XheHsc70, its phylogeny, and Xhehsc70 mRNA expression, XheHsc70 was identified as a member of the cytoplasmic Hsc70 (constitutive) subfamily of the Hsp70 family of heat shock proteins, suggesting that it may play a role in the immune response. The Xhehsc70 cDNA sequence reported in this study was submitted to GenBank under the accession number JF739182.

Ballast waters treatment using UV/Ag–TiO 2 + O 3 advanced oxidation process with Escherichia coli and Vibrio alginolyticus as indicator microorganisms

Ballast water transfers aquatic species among ports and poses biological threat to the world's oceans. This work investigated the use of combined ultraviolet (UV)/Ag–TiO 2 + ozone (O 3) processes for ballast water treatment using Escherichia coli and Vibrio alginolyticus as indicator microorganisms. In all tests, the V. alginolyticus cells are more resistant to UV/O 3 inactivation than are the E. coli cells. Bacterial cells under stressing stimulated activities of antioxidants for their protection. The combined UV + O 3 and UV/Ag–TiO 2 + O 3 tests yield synergistic removal of both strains compared with individual tests. The combined UV/Ag–TiO 2 + O 3 process is potentially applicable for onboard treatments for ballast waters.

Conversion of mono-polar to peritrichous flagellation in Vibrio alginolyticus

Precise regulation of the number and positioning of flagella are critical in order for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. It has been shown that, in V. alginolyticus cells, the putative GTPase FlhF determines the polar location and production of flagella, while the putative ATPase FlhG interacts with FlhF, preventing it from localizing at the pole, and thus negatively regulating the flagellar number. In fact, no ΔflhF cells have flagella, while a very small fraction of ΔflhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of ΔflhFG cells were isolated. The mutation induced an increase in the flagellar number and, furthermore, most Vibrio cells appeared to have peritrichous flagella. The sequence of the flagella related genes was successfully determined, however, the location of the suppressor mutation could not been found. When the flhF gene was introduced into the suppressor mutant, multiple polar flagella were generated in addition to peritrichous flagella. On the other hand, introduction of the flhG gene resulted in the loss of most flagella. These results suggest that the role of FlhF is bypassed through a suppressor mutation which is not related to the flagellar genes.

Mutational Analysis of the GTP-binding Motif of FlhF which Regulates the Number and Placement of the Polar Flagellum in Vibrio alginolyticus

Precise regulation of the number and placement of flagella is critical for the mono-flagellated bacterium Vibrio alginolyticus to swim efficiently. We previously proposed a model in which the putative GTPase FlhF determines the polar location and generation of the flagellum, the putative ATPase FlhG interacts with FlhF to prevent FlhF from localizing to the pole, and thus FlhG negatively regulates the flagellar number in V. alginolyticus cells. To investigate the role of the GTP-binding motif of FlhF, we generated a series of alanine-replacement mutations at the positions that are highly conserved among homologous proteins. The results indicate that there is a correlation between the polar localization and the ability to produce flagella in the mutants. We investigated whether the mutations in the GTP-binding motif affected the ability to interact with FlhG. In contrast to our prediction, no significant difference was detected in the interaction with FlhG between the wild-type and mutant FlhFs. We showed that the GTP-binding motif of FlhF is important for polar localization of the flagellum but not for the interaction with FlhG.

Enzymatic, outer membrane proteins and plasmid alterations of starved Vibrio parahaemolyticus and Vibrio alginolyticus cells in seawater

The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 microm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.