Recovery from nutrient starvation by a marine Vibrio sp.

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.     

Synthesis of membrane and periplasmic proteins during starvation of a marine Vibrio sp

Changes in membrane and periplasmic protein profiles induced by starvation conditions in the marine Vibrio sp. S14 were examined by one-dimensional gel electrophoresis. Analysis by densitometry resolved at least six periplasmic proteins, nine outer membrane proteins, and four cytoplasmic membrane proteins induced at various times during 120 h of nutrient and energy starvation. Eight of these were also synthesized by heat- and/or ethanol-shocked cells. Pulse-labelling indicated that the starvation-induced proteins were not products of degradation, and that their synthesis was differently modulated during starvation. The most pronounced changes occurred during the initial hours of nutrient and energy deprivation. The correlation between the initial changes in protein composition and utilization of the intracellular energy reserve poly-beta-hydroxybutyrate is discussed. The rate of proteolysis during the initial hours of starvation was approximately 16 times greater than that during exponential growth.     

Heat-labile bacterial alkaline phosphatase from a marine Vibrio sp

Psychrophilic organisms have successfully adapted to various low-temperature environments such as cold ocean waters. Catalysts with increased catalytic efficiencies are produced, generally at the expense of thermal stability due to fewer non-covalent stabilizing interactions. A marine bacterial strain producing a particularly heat-labile alkaline phosphatase was selected from a total of 232 strains isolated from North-Atlantic coastal waters. From partial 16S rRNA sequences the strain was characterized as a Vibrio sp. An alkaline phosphatase was purified 151-fold with 54% yield from the culture medium using a single step affinity chromatography procedure on agarose-linked L-histidyldiazobenzylphosphonic acid. The active enzyme was a 55 +/- 6 kDa monomer. The enzyme had optimal activity at pH 10 and was strikingly heat-labile with a half-life of 6 min at 40 degrees C and 30 min at 32 degrees C. This enzyme from Vibrio sp. had a higher turnover number (k(cat)) and higher apparent Michaelis-Menten factor (K(m)) than the enzyme from Escherichia coli, a clear-indication of cold-adaptation. Inorganic phosphate was a competitive inhibitor with a relatively high K(i) value of 1.7 mM. Low affinity for phosphate may contribute to higher turnover rates due to more facile release of product.     

Responses to multiple-nutrient starvation in marine Vibrio sp. strain CCUG 15956.

The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.     

Isolation and characterization of chitinase from a marine bacterium Vibrio sp.

A chitinase was separated from the culture broth of Vibrio sp. 11211 isolated from sediment from the South China Sea. The chitinase was purified 18.3-fold with 33% recovery by ammonium sulphate precipitation and chromatography. The subunit molecular weight of the enzyme was estimated by SDS-PAGE to be about 30kDa. The enzyme showed optimum pH at 6.5 and optimum temperature at 50^°C, and was stable in the pH range of 4 to 9 and at the temperature below 40^°C.     

Isolation of a carbon starvation regulatory mutant in a marine Vibrio strain.

A carbon starvation-responding lac fusion of the marine Vibrio sp. strain S14 was used as a reporter strain in order to identify genes critical in the regulation of the carbon starvation response. Interestingly, sequence data together with an altered phenotype with respect to the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) imply that one of the genes (csrS) identified by this approach is an Escherichia coli spoT equivalent. Complementary data suggest that the function encoded by the csrS gene is essential for the successful development of starvation and stress resistance.     

Morphological characterization of small cells resulting from nutrient starvation of a psychrophilic marine vibrio.

Upon starvation, Ant-300, a psychrophilic marine vibrio, was observed to decrease in size and change in shape from a rod to a coccus. After 3 weeks of starvation 50% of the starved population was able to pass through a filter with a pore size of 0.4 mum. Electron microscopy of thin sections of the small cells revealed normal cell structure except for an enlarged periplasmic space. When inoculated into a fresh medium, starved cells growth without a significant lag and regained "normal" size and shape within 48 h.     

Extracellular nuclease produced by a marine bacterium. II. Purification and properties of extracellular nuclease from a marine Vibrio sp.

Extracellular nuclease produced by a marine Vibrio sp., strain No. 2, was purified by salting out with ammonium sulfate and by chromatography on a DEAE-cellulose column and twice on a Sephadex G-200 column. The nuclease was eluted as a single peak in which the deoxyribonuclease (DNase) activity and ribonuclease (RNase) activity appeared together. Polyacrylamide disc gel electrophoresis showed a single band of stained protein which had both DNase and RNase activity. The molecular weight of the enzyme was estimated to be 100 000 daltons. When using partially purified enzyme from the DEAE-cellulose column, the optimum pH for activity was 8.0, and the enzyme was activated strongly by 0.05 M Mg2+ ions and stabilized by 0.01 M Ca2+ ion. These concentrations of Mg2+ and Ca2+ ions are similar to those of the two cations in seawater. Indeed, the enzyme revealed high activity and strong stability when kept in seawater. The presence of particulate matter, such as cellulose powder, chitin powder. Hyflosupercel, Kaolin, and marine mud increased the stability of the enzyme. When the hydrostatic pressure was increased from 1 to 1000 atmospheres, the decrements of the enzyme activity were more pronounced at 30 and 40 degrees C than at 25 or 50 degrees C. The enzyme activity was restored after decompression to 1 atm at 30 degrees C.     

Purification and characterization of alginate lyase from marine Vibrio sp. YWA

Extracellular alginate lyase secreted by marine Vibrio sp.YWA,isolated from decayedLaminaria japonica,was purified by a combination of ammonium sulfate precipitation and diethylaminoethyl-Sephacel column chromatography.The results show that the molecular mass of alginate lyase wasapproximately 62.5 kDa,with an optimal pH and temperature at pH 7.0 and 25℃,respectively.K_m wasapproximately 72.73 g/L.The activity of the enzyme was enhanced by EDTA and Zn~(2 ),but inhibited by Ba~(2 ).The substrates specificity analysis shows that it was specific for hydrolyzing poly-β-D-1,4-mannuronate inalginate.     

Enhanced yeast immobilization by nutrient starvation

Saccharomyces uvarum NRRL Y1347 cells were immobilized in a porous support. Cell loadings of up to 600 mg dry cell/g support or 70 mg dry cell/cm³ support were obtained. Starvation in a marine environment increased the adhesion strength of immobilized cells.     

ATP level of a starving surface-bound and free-living marine Vibrio sp.

Abstract ATP levels of three marine isolates (a Vibrio sp., Serratia marcescens , and an unidentified rodshaped organism) were measured during nutrient-limiting conditions for free-living bacteria or cells adhered to a glass/liquid interface. In all strains there was a higher content of ATP per biovolume (numbers × cell volume) for cells starved at the glass/liquid interface compared with those in the bulk phase. Leakage from viable cells and the formation of a conditioning film are suggested only partly to explain the elevated levels of ATP per biovolume at the surface.     

Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp. QY101

Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides.     

The induction of stress proteins in three marine Vibrio during carbon starvation

Abstract The induction of DnaK and GroEL homologous proteins by heat-shock and long-term carbon starvation was studied in Vibrio vulnificus, Vibrio sp. strain S14, and Vibrio sp. strain DW1. In each Vibrio strain one protein (60 kDa) reacted with antibodies against Escherichia coli -GroEL and two proteins, DnaK (69 kDa) and Sis1 (62-60 kDa), reacted with antibodies against E. coli -Dnak. The carbon starvation elicited induction of the stress proteins was strain-specific, suggesting that the induction of stress proteins like DnaK and GroEL in marine Vibrios might not be a uniform starvation response. It appears as of these proteins, only DnaK in Vibrio sp. strain S14 remains induced after long-term carbon starvation in the three marine bacterial strains that were tested.     

Partial characterization of an exopolysaccharide secreted by a marine bacterium,Vibrio neocaledonicus sp. nov., from New Caledonia

Aims

Exopolysaccharides (EPS) are industrially valuable molecules with numerous useful properties. This study describes the techniques used for the identification of a novel Vibrio bacterium and preliminary characterization of its EPS.

Methods and Results

Bioprospection in marine intertidal areas of New Caledonia followed by screening for EPS producing brought to selection of the isolate NC470. Phylogenetic analysis (biochemical tests, gene sequencing and DNA–DNA relatedness) permitted to identify NC470 as a new member of the Vibrio genus. The EPS was produced in batch fermentation, purified using the ultrafiltration process and analysed by colorimetry, Fourier Transform Infrared spectroscopy, gas chromatography, Nuclear Magnetic Resonance and HPLC‐size exclusion chromatography. This EPS exhibits a high N‐acetyl‐hexosamines and uronic acid content with a low amount of neutral sugar. The molecular mass was 672 × 10³ Da. These data are relevant for possible technological exploitation.

Conclusions

We propose the name Vibrio neocaledonicus sp. nov for this isolate NC470, producing an EPS with an unusual sugar composition. Comparison with other known polymers permitted to select applications for this polymer.

Significance and Impact of the Study

This study contributes to evaluate the marine biodiversity of New Caledonia. It also highlights the biotechnological potential of New Caledonia marine bacteria.     

Purification and characterization of beta-1,3-xylanase from a marine bacterium, Vibrio sp. XY-214

beta-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp. XY-214 by ammonium sulfate precipitation and successive chromatographies. The enzyme had a pl of 3.6 and a molecular mass of 52 kDa. The enzyme had the highest level of activity at pH 7.0 and 37 degrees C. The enzyme activity was completely inhibited by Cu2+, Hg2+, and N-bromosuccinimide. The enzyme hydrolyzed beta-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-beta-D-xyloside, beta-1,4-xylan, beta-1,3-glucan, or carboxymethyl cellulose.     

Glucose upshift of carbon-starved marine Vibrio sp. strain S14 causes amino acid starvation and induction of the stringent response.

The physiological status of carbon-starved cells of the marine Vibrio sp. strain S14 has been investigated by the analysis of their immediate response to carbon and energy sources. During the first minute after glucose addition to 48-h-starved cells, the pools of ATP and GTP increased rapidly, and the [ATP]/[ADP] ratio reached the level typical for growing cells within 4 min. The total rates of RNA and protein synthesis increased initially but were inhibited 4 to 5 min after glucose addition by the induction of the stringent response. A mutation in the relA gene abolished stringent control during the recovery and significantly prolonged the lag phase, before the starved cells regrew, after the addition of a single source of carbon. However, both the wild-type and the relA cells regrew without a significant lag phase when given glucose supplemented with amino acids. On the basis of these results, it is suggested that carbon-starved cells are deficient in amino acid biosynthesis and that ppGpp and the stringent response are involved in overcoming this deficiency, presumably by depressing the synthesis of amino acid biosynthetic enzymes. Furthermore, the data suggest that the starved cells primarily are starved for energy, and evidence is presented that the step-up in the rate of protein synthesis after refeeding is partially dependent on de novo RNA synthesis.