The triggering effect of surfaces and surfactants on heat output,oxygen consumption and size reduction of a starving marine Vibrio

The marine Vibrio DW1 exhibited a positive response in heat output to a dialysis membrane surface in the presence of substrate (100 mM sodium glutamate) and, more particularly, in the absence of exogenous substrate (starvation conditions). The latter result paralleled the previously reported decrease in cell volume and increase in oxygen consumption by starving bacteria at a similar surface. Modified Morita's salts (MMS) did not extract nutrients from the dialysis membrane, but an artificial seawater containing tris buffer (ASW-tris) did extract surface active and nutrient materials from the membrane. The ASW-tris membrane extract and a commercial surfactant, Tween 85, were found to mimic the effects of the dialysis membrane surface by inducing a decrease in cell volume, and an increasing oxygen consumption and heat output of Vibrio DW1 even in the bulk liquid. The significance of the adsorption of naturally occurring surfactants at surfaces in relation to the behaviour of bacteria at the surfaces is discussed.Abbreviations ASW artificial seawater - MMS modified Morita's salts - NMMS nutrient modified Morita's salts - MMSG MMS plus 100 mM sodium glutamate     

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-¹⁴C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.Abbreviations ACP acyl carrier protein - FAME fatty acid methyl ester - FAS fatty acid synthetase - FID flame ionization detection - GLC gas-liquid chromatography - TLC thin-layer chromatography - In designations of fatty acids, such as 16:0, 16:1, etc the colon separates the number that denotes the number of carbon atoms and the number that denotes the number of double bonds, respectively, in the molecule - 16:0-CoA CoA ester of 16:0     

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.     

Vibrio navarrensis biotype pommerensis: a new biotype of V. navarrensis isolated in the German Baltic Sea

The determination of phenotypic and genotypic traits of a group of closely related Vibrio strains from the Baltic Sea did not allow species designation. DNA-DNA hybridization and fatty acid analysis revealed them as Vibrio navarrensis. Therefore we suggest these Vibrios to represent a new biotype, named V. navarrensis biotype pommerensis.     

Evolutionary relationships in Vibrio and Photobacterium as determined by immunological studies of superoxide dismutase

The amino acid sequence divergence of superoxide dismutases (SODs) from 22 species and five groups of Vibrio, Photobacterium, and a number of related organisms was determined by means of the microcomplement fixation technique and the Ouchterlony double diffusion procedure. Five reference antisera were used which had been prepared against the purified SODs from V. alginolyticus, V. splendidus II, V. fischeri, V. cholerae, and P. leiognathi. With a few exceptions the results were in agreement with past studies of other informational molecules and provided a comprehensive overview of evolutionary relationships in Vibrio and Photobacterium. The genus Vibrio was found to consist of a major group of primarily marine species which included V. fischeri, V. logei, V. splendidus, V. pelagius, V. nereis, V. campbellii, V. harveyi, V. natriegens, V. alginolyticus, V. parahaemolyticus, V. proteolyticus, V. fluvialis, V. vulnificus, V. nigripulchritudo, and V. anguillarum. On the outskirts of this large and relatively heterogeneous group were the fresh water and estuarine species V. cholerae and V. metschnikovii as well as the marine species V. gazogenes. A considerable distance from Vibrio were the related species of Photobacterium: P. phosphoreum, P. leiognathi, and P. angustum. Both genera were distant from species of Aeromonas as well as from Plesiomonas shigelloides, Escherichia coli, and Alteromonas hanedai, a luminous strict aerobe. The agreement between these and previous studies of evolution of informational molecules in Vibrio and Photobacterium is best explained by vertical evolution (involving no genetic exchange between species) rather than by its opposite — horizontal evolution.Non-Standard Abbreviations Anti-Plei, anti-Valg, anti-Vcho, anti-Vfis, anti-Vspl antisera to the Fe-containing superoxide dismutases from Photobacterium leiognathi strain 480, Vibrio alginolyticus strain 90, V. cholerae strain M 13, V. fischeri strain 61, and v. splendidus biotype II strain 2, respectively - AP alkaline phosphatase - ATCC American Type Culture Collection - GS glutamine synthetase - ImD immunological distance - NCMB National Colleciion of Marine Bacteria - NCTC National Collection of Type Cultures - NRC National Research Council of Canada Culture Collection - SDS sodium dodecyl sulfate - SOD superoxide dismutase     

Enzymatic,outer membrane proteins and plasmid alterations of starved <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> 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 μm. 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.     

黄色粘球菌发育阶段外膜蛋白表达的变化

【目的】黄色粘球菌是研究原核发育的一种模式生物,对其膜蛋白的研究仍然十分缺乏。【方法】利用6种预测软件,在黄色粘球菌的基因组中筛选编码外膜蛋白(OMP)的基因。根据报告基因lacZ,检测这些基因在营养性生长和发育阶段的表达。【结果】基于生物信息学分析,筛选出11个编码外膜蛋白的基因。其中2个基因(MXAN3106和MXAN3883)在发育阶段表达量上升,它们分别编码Secretin家族和Fimbrial usher protein (FUP)家族转运蛋白。其余9个基因在发育起始阶段表达量降低或保持较低水平,它们均编码TonB依赖型受体或外排蛋白。【结论】这些数据提示,黄色粘球菌由生长到发育的转换过程,伴随着膜蛋白表达的显著变化。     

Development of duplex PCR for the detection of Salmonella and Vibrio in drinking water

We report here a rapid protocol for the detection of Vibrio and Salmonella in drinking water using a duplex PCR reaction. The developed protocol can detect as few as 500 cells in a single reaction, which has been achieved by optimizing the temperature steps and magnesium chloride concentration for the reactions. The described PCR protocol could detect Vibrio and Salmonella spiked in drinking water.     

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m²) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.     

Vibrio mimicus are the reservoirs of the heat-stable enterotoxin gene (nag-st) among species of the genus Vibrio

Using a 0.27 kb DNA probe specific for the heat-stable enterotoxin gene (nag-st) of Vibrio cholerae non-O1, 1109 strains representing 17 species of the genus Vibrio, isolated from clinical and environmental sources were examined. The nag-st gene was preponderantly associated with strains classified as V. mimicus; 16.8% of these strains hybridized. It was more frequent in the clinical isolates (22.6%) than in the environmental isolates (13.7%). The incidence of nag-st gene-positive strains of V. mimicus isolated from different countries was uniformly high and ranged between 8.7% (Bangladesh) and 57.1% (environmental strains from USA). The incidence of the nag-st gene was much lower among strains of V. cholerae non-O1 (3.6%). Probe-positive and-negative strains of V. mimicus and V. cholerae non-O1 were used to evaluate the performance of the conventional suckling mouse assay for detection of the NAG-ST enterotoxin. Of the 31 probe-positive strains, only five (16.1%) yielded a positive fluid accumulation ratio (FA ratio) when neat heated culture supernatant was used to perform the suckling mouse assay. All the 31 probe-positive strains gave a positive FA ratio when 20-fold concentrated and heated culture supernatants of the strains were used to perform the suckling mouse assay. The need to concentrate (by at least 20-fold) the culture supernatant of strains of V. mimicus and V. Cholerae non-O1 was identified as an important step to obtain consistent results when using the suckling mouse assay for detection of NAG-ST.P. Yuan, A. Ogawa and T. Takeda are with the Department of Infectious Disease Research, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154, Japan; P. Yuan is also with the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. T. Ramamurthy and G.B. Nair are with the National Institute of Cholera and Enteric Diseases, Calcutta, India. T. Shimada is with the National Institute of Health, Tokyo 141, Japan. S. Shinoda is with the Faculty of Pharmaceutical Sciences, Okayama University, Japan.     

Growth characteristics of an obligately psychrophilic Vibrio sp.

The growth characteristics of an obligately psychrophilic Vibrio sp. have been studied in a chemostat with glucose or lactose as the limiting substrate over a temperature range 0–23°C. Vibrio AF-1 has an optimum growth temperature of 15°C and maximum growth temperature which is dependent upon the carbon source. On glucose growth ceases at 20°C whereas on lactose growth continues to 23°C. Growth rate is also a function of the carbon source provided. When grown on glucose, fructose, sucrose, maltose and galactose _max values of 0.046 h^-1 at 15°C were recorded whereas on lactose, mannose, ribose and xylose _max values of 0.020 h^-1 were obtained. Substrate affinities (K _s ) for the 9 sugars also fall into 2 divisions as for _max and are temperature dependent. Those sugars which support a high growth rate have highest K _s values at 0°C whereas these which give a low growth rate show maximum affinities at 15°C. Vibrio AF-1 produces the maximum cell yield (0.6 g/g sugar consumed) at temperature <8°C irrespective of the carbon source utilised and correlated with maximum rates of sugar uptake and minimum O₂ consumption. Maintenance energy determination on glucose grown cells show that at 2° C 2% of the carbon input is used for maintenance whereas at 20°C the requirement increases to 10% of the carbon input.     

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio. Wang and Li contributed equally to this work.     

Isolation and characterization of actinomycetes antagonistic to pathogenic Vibrio spp. from nearshore marine sediments

Summary A total of 94 actinomycete strains were isolated from the marine sediments of a shrimp farm, 87.2% belonged to the genus Streptomyces, others were Micromonospora spp. Fifty-one percent of the actinomycete strains showed activity against the pathogenic Vibrio spp. strains. Thirty-eight percent of marine Streptomyces strains produced siderophores on chrome azurol S (CAS) agar plates. Seven strains of Streptomyces were found to produce siderophores and to inhibit the growth of Vibrio spp. in vitro. Two of them belonged to the Cinerogriseus group, the most frequently isolated group of Streptomyces. The results showed that streptomycetes could be a promising source for biocontrol agents in aquaculture.     

Changes in phenolic metabolism of tobacco plants during short-term boron deficiency

The effects of boron (B) deficiency on several phenolics and enzyme activities involved in the biosynthesis of these compounds were investigated in tobacco plants (Nicotiana tabacum L. cv. Gatersleben). The levels of phenylpropanoids (mainly the caffeic acid esters, chlorogenic acid and its isomers) as well as phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and polyphenoloxidase (PPO, EC 1.14.18.1) activities were determined in plants subjected to B starvation for 1–7 d. The results presented here show that a short-term B deficiency causes both quantitative and qualitative changes in the phenolic metabolism of tobacco plants, which are especially evident after 3 d of B starvation. Although the concentration of B decreased from the onset of B starvation, root B level was less affected than leaf B by a short-term B deficiency. The concentration of phenylpropanoids as well as PAL and PPO activities increased mainly in the leaves of tobacco plants during B starvation. Moreover, leaves starved of B for 7 d showed the accumulation of new compounds, one of which was identified as caffeoylputrescine. In addition, a positive correlation between PAL activity and phenylpropanoid concentration was observed in tobacco leaves, especially after 5–7 d of B starvation, suggesting that an increase in PAL activity during B starvation could be responsible for the enhancement in the levels of phenylpropanoids.     

Changes in membrane glycoproteins during fruit-body formation inPhysarum polycephalum

Sporangia formation ofPhysarum polycephalum was induced by starvation and illumination, and the morphogenic process during the differentiation was studied by scanning electron microscopy. Plasma membranes were prepared from these differentiating plasmodia and the membrane proteins were analyzed by polyacrylamide gel electrophoresis. Many glycoproteins appeared during the fruit-body formation. Of these a protein of apparent molecular mass of 66 kD was prominent in sporangia forming stage which showed a high affinity to RCA lectin. Inhibition of the glycosylation and processing of these glycoproteins resulted in the prevention of fruit-body formation suggesting that the synthesis of these membrane components is a prerequisite process for the sporangia formation in the slime mold.     

Detection of Vibrio cholerae O1 in the aquatic environment in Brazil employing direct immunofluorescence microscopy

Culturing and immunofluorescence (FA) methods for detection of Vibrio cholerae O1 in samples collected from the aquatic environment at selected sites in Brazil were compared. Of the samples examined, 90% were positive for V. cholerae O1 by FA but none was positive by culture, although strains of V. cholerae other than O1 strains were readily isolated. Evidence for V. cholerae O1 being autochthonous to the aquatic environment of Brazil is presented. Furthermore, FA methods are recommended for cholera surveillance programmes directed at the natural environment.M.T. Martins is with the Department of Microbiology, I.C.B. II, University of Sao Paulo, Sao Paulo, S.P. CEP 05508, Brazil. P.S. Sanchez and M.I.Z. Sato are with the State Agency for Environmental Control-CETESB, Sao Paulo, S.P. CEP 05459, Brazil. P.R. Brayton and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD 20742, USA; R.R. Colwell is also with the Center of Marine Biotechnology, Maryland Biotechnology Institute, 600 East Lombard St, Baltimore, MD 21202, USA.     

Isolation and some properties of cellulose-degrading Vibrio sp. LX-3 with agar-liquefying ability from soil

A new bacterium, Vibrio sp. LX-3 was isolated from soil, which was a facultatively anaerobic and polarly flagellated Gram-negative rod. Sodium ions stimulated its growth but were not an absolute requirement. The isolate could digest both crystalline cellulose and agar. Carboxymethylcellulase was produced extracellularly when various celluloses were used as carbon sources, but no reducing sugars were found in the culture on cellulose over the incubation period. Only low agarase activity could be detected in the broth of agar although the strain LX-3 strongly liquefied agar.     

Changes in lipid and carbohydrate metabolism during starvation in adult Manduca sexta

Summary Adult Manduca sexta feed very irregularly in the laboratory, and many adult males never feed. Feeding adults live longer and feeding females lay many more eggs; however, in both feeding (sugar water) and starving adults a decrease of metabolic reserves is observed. Carbohydrates disappear from hemolymph and from fat body. Fat body lipid also decreases, while hemolymph lipid concentration increases strongly in starving adults. The activity of fat body glycogen phosphorylase increases strongly in starving adult M. sexta. The activity of glycogen phosphorylase is correlated inversely with hemolymph sugar concentration. Injected trehalose inactivates glycogen phosphorylase within 2 h, and lowers the hemolymph lipid level within 6 h. In starving adult M. sexta, neither the activation of glycogen phosphorylase nor the increase of hemolymph lipid concentration depends on adipokinetic hormone, since cardiacectomy does not prevent the activation of glycogen phosphorylase nor the increase of hemolymph lipid level.Abbreviations AKH adipokinetic hormone - EDTA ethylenediamine tetraacetate Present address: Department of Biochemistry and Center for Insect Science, The University of Arizona, Tucson, AZ 85721, USA     

Membrane potential changes during transport of hexoses in Lemna gibba G1

The membrane potential (pd) of duck weed (Lemna gibba G1) proved to be energy dependent. At high internal ATP levels of 74 to 105 nmol ATP g^-1 FW, pd was between -175 and -265 mV. At low ATP levels of 23 to 46 nmol ATP g^-1 FW, pd was low, about -90 to -120 mV at pH 5.7, but -180 mV at pH 8. Upon addition of glucose in the dark or by light energy the low pd recovered to the high values. The active component of the pd was depolarized by the addition of hexoses in the dark and in the light. Hexose-dependent depolarization of the pd (= pd) followed a saturation curve similar to active hexose influx kinetics. Depolarization of the pd recovered in the dark even in the presence of the hexoses and with a 10fold enhancement in the light. Depolarization and recovery could be repeated several times with the same cell. Glucose uptake caused a maximum depolarization of 133 mV, fructose uptake half that amount, sucrose had the same effect as glucose. During 3-O-methylglucose and 2-deoxyglucose uptake the depolarizing effect was only slightly lower. The pd remained unchanged in the presence of mannitol. The glucose dependent pd and especially the rate of pd recovery proved to be pH-dependent between pH 4 and pH 8. It was independent of the presence of 1 mM KCl. Although no pH could be measured in the incubation medium, these results can be best explained by a H⁺-hexose cotransport mechanism powered by active H⁺ extrusion at the plasmalemma.Abbreviations LD longday - SD shortday - pd membrane electropotential difference - pd maximum membrane potential depolarization - L light - D dark - FW fresh weight - d days of culture of Lemna gibba - 1X perfusing solution without sugar, see methods     

Taxonomic identification,phenanthrene uptake activity,and membrane lipid alterations of the PAH degrading <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain Sphe3

This report describes phenanthrene uptake as well as the effect of phenanthrene on the membrane phospholipid and fatty acid composition in a newly isolated bacterial strain, Sphe3, that we taxonomically identified as Arthrobacter sp. Strain Sphe3 is able to utilize phenanthrene as a carbon source at high rates and appears to internalize phenanthrene with two mechanisms: a passive diffusion when cells are grown on glucose, and an inducible active transport system when cells are grown on phenanthrene as a sole carbon source. Active transport followed Michaelis-Menten kinetics, and it was amenable to inhibition by 2,4-dinitrophenol and sodium azide. Evidence provided here indicates that apart from inducing an active PAH uptake, the presence of phenanthrene elicits significant changes in membrane fluidity.