Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery.

The degree and temporal context of variations in ribosome content during nutrient starvation of two copiotrophic marine bacteria, Vibrio alginolyticus and Vibrio furnissii, have been examined. The organisms were starved either by nutritional shift-down or by consumption of limiting nutrients resulting from growth into stationary phase. Measurements of the amount of hybridization to 16S rRNA-specific probes revealed that the cells retained between 10 and 26% of their original rRNA content after 15 days of starvation. In V. alginolyticus, losses in stationary-phase cells occurred rapidly (1 to 2 days), whereas cells shifted into starvation remained larger and retained considerably more rRNA. The ability of V. alginolyticus to recover from starvation was assessed after cells were maintained for 2, 8, and 15 days in nutrient-depleted medium. The pattern of recovery at the level of rRNA accumulation depended upon the duration of nutrient deprivation and the manner in which it was imposed. Stationary-phase cells starved for 2 days had only slight relative increases in rRNA levels after excess nutrients were added. As the duration of starvation lengthened to 8 and 15 days, increasingly greater amounts of rRNA (30 and 70 times preenrichment values, respectively) were transcribed after nutrient enrichment. Shift-down cells recovered from 2 and 8 days of starvation without extensive rRNA production. After 15 days, nutrient enrichment caused 16S rRNA levels to increase 30-fold. The results indicate that the mechanisms controlling starvation-survival in these marine bacterial species are linked to the physiological state at the onset of starvation and that the subsequent pattern of recovery will depend upon how starvation was initiated.     

Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods.

An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.     

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

AIM: Vibrio alginolyticus is an economically important micro-organism. The main aim of the present study was to develop a real-time polymerase chain reaction (PCR) assay for rapid, sensitive and effective quantification of V. alginolyticus in seawater and seafood. METHODS AND RESULTS: Purified DNA of V. alginolyticus, artificially inoculated seawater and seafood tissue homogenates were subjected to the gyrB-targeted real-time PCR assay. Natural seawater and seafood samples were analysed by this real-time PCR protocol. Specificity tests showed that positive result was obtained only with V. alginolyticus strains. The detection sensitivity was determined to be 0.4 pg of genomic DNA equivalent to 72 cells per PCR in pure culture and 100 cells in 1 ml of seawater or seafood tissue homogenates. Single cell detection is achieved after 3 h of sample enrichment. CONCLUSIONS: A sensitive and specific SYBR Green I-based real-time PCR assay targeting gyrB gene was successfully developed to quantify V. alginolyticus within 6 h in seawater and seafood samples. SIGNIFICANCE AND IMPACT OF THE STUDY: No report on the molecular-based method was available for quantitative detection of V. alginolyticus. This work will provide a novel method for evaluation of the risk of V. alginolyticus to marine environmental health and seafood safety.     

Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae.

The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation.     

Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods

An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.     

Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae

The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation.     

A polyphasic approach for the differentiation of environmental Vibrio isolates from temperate waters

Climate change and marine traffic lead to changing species communities in the oceans. Due to increasing seawater temperatures, pathogenic Vibrio species could become significant even in temperate waters. We classified mesophilic Vibrio isolates from the German Bight (North Sea) using a polyphasic approach with special emphasis on Vibrio parahaemolyticus. Matrix-assisted laser desorption/ionization time-of-flight MS was used as a primary screen to classify isolates, 16S rRNA gene and rpoB gene sequencing to identify species. Potential V. parahaemolyticus isolates were screened for regulatory or virulence-related genes (toxR, tlh, tdh, trh). To investigate genomic diversity, we applied repetitive-sequence-based PCRs. Results were evaluated and methods compared using multivariate statistical analysis. Most isolates were classified as V. parahaemolyticus or Vibrio alginolyticus. Reliable differentiation between both species was achieved by rpoB sequencing and toxR detection. Among the fingerprinting methods, ERIC-PCR showed the highest discriminatory power, displaying three separated clusters. These clusters represent the species V. parahaemolyticus, V. alginolyticus and one group in between. The frequent detection of V. parahaemolyticus in the German Bight reveals the urgency for further monitoring. In this context, a polyphasic approach, such as defined in this study, is needed to differentiate populations of V. parahaemolyticus and V. alginolyticus.     

Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities

Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100 degrees C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.     

Starvation-Survival Physiological Studies of a Marine Pseudomonas sp.

Starved cultures of a marine Pseudomonas sp. showed a 99.9% decrease in viable cell count during the first 25 days of starvation, yet the culture maintained 10⁵ viable cells per ml for over 1 year. The physiological responses of populations of a marine Pseudomonas sp. to nutrient starvation were observed for periods of up to 40 days. At various intervals during starvation, the numbers of total, viable, and respiring cells were determined within the cultures. The ATP content, endogenous respiration rate, uptake rates, and percent respiration for exogenous glucose and glutamate were determined throughout the starvation period to characterize the physiological changes in the cells. It was observed that, after initial adjustment periods, all parameters tested reached stabilized states after 18 to 25 days of starvation. The results indicate that the actively respiring subpopulation, rather than the viable or total cell numbers, is the most appropriate denominator for interpretation of observed activities on an individual cell basis.     

Differential effects of temperature and starvation on induction of the viable-but-nonculturable state in the coral pathogens Vibrio shiloi and Vibrio tasmaniensis

We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20 degrees C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17 degrees C), the VBNC state was not induced by incubation at 4 degrees C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30 degrees C), was induced into the VBNC state by incubation at 4 degrees C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20 degrees C, and V. shiloi became VBNC after incubation for 126 days at 20 degrees C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.     

Physiological responses to starvation in the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256

Sphingomonas sp. strain RB2256 is representative of the ultramicrobacteria that proliferate in oligotrophic marine waters. While this class of bacteria is well adapted for growth with low concentrations of nutrients, their ability to respond to complete nutrient deprivation has not previously been investigated. In this study, we examined two-dimensional protein profiles for logarithmic and stationary-phase cells and found that protein spot intensity was regulated by up to 70-fold. A total of 72 and 177 spots showed increased or decreased intensity, respectively, by at least twofold during starvation. The large number of protein spots (1,500) relative to the small genome size (ca. 1.5 Mb) indicates that gene expression may involve co- and posttranslational modifications of proteins. Rates of protein and RNA synthesis were examined throughout the growth phase and up to 7 days of starvation and revealed that synthesis was highly regulated. Rates of protein synthesis and cellular protein content were compared to ribosome content, demonstrating that ribosome synthesis was not directly linked to protein synthesis and that the function of ribosomes may not be limited to translation. By comparing the genetic capacity and physiological responses to starvation of RB2256 to those of the copiotrophic marine bacterium Vibrio angustum S14 (J. Ostling, L. Holmquist, and S. Kjelleberg, J. Bacteriol. 178:4901-4908, 1996), the characteristics of a distinct starvation response were defined for Sphingomonas strain RB2256. The capacity of this ultramicrobacterium to respond to starvation is discussed in terms of the ecological relevance of complete nutrient deprivation in an oligotrophic marine environment. These studies provide the first evidence that marine oligotrophic ultramicrobacteria may be expected to include a starvation response and the capacity for a high degree of gene regulation.     

F0F1-ATPase from Vibrio alginolyticus. Subunit composition and proton pumping activity.

An F0F1-ATPase was isolated from the membranes of the marine bacterium Vibrio alginolyticus. Homology between the subunits of the F0-complexes from E. coli and V. alginolyticus was found using antibodies against subunits a, b and c of the E. coli F0F1-ATPase. The F0F1-complex from V. alginolyticus was reconstituted into proteoliposomes, which were competent in ATP-dependent proton uptake. This process was inhibited by triphenyltin, DCCD, and venturicidin. Na+ did not affect proton translocation.     

An investigation of aquatic bacteria capable of utilizing chitin as the sole source of nutrients

R. OSAWA AND T. KOGA. 1995. A total of 48 bacterial strains capable of utilizing chitin (a polymer of N -acetyl-D-glucosamine [NAG]) as a sole source of nutrients were isolated from river and marine waters in Tokushima. These bacteria were identified as Vibrio fluvialis, V. parahaemolyticus, V. alginolyticus, V. mimicus, Listonella anguillarum and Aeromonas hydrophila . All strains were found positive for chitinase and chitobiase activities, and capable of utilizing NAG as a sole source of carbon and nitrogen.     

Starvation-induced modulations in binding protein-dependent glucose transport by the marine Vibrio sp. S14

The uptake kinetics of D-glucose were examined in the marine Vibrio sp. S14 during a period of 168 h of complete energy and nutrient starvation. Two glucose transport systems were distinguished in Vibrio sp. S14: a low affinity system (Km = 4.6 +/- 0.9 microM) at the onset of starvation, and a high affinity system (Km = 0.55 +/- 0.15 microM) after 168 h of starvation. Both systems had a narrow substrate specificity, and both were osmotic shock-sensitive.     

Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956.

Carbon starvation induces the development of a starvation- and stress-resistant cell state in marine Vibrio sp. strain S14 (CCUG 15956). The starved cells remain highly responsive to nutrients during prolonged starvation and exhibit instantaneous severalfold increases in the rates of protein synthesis and RNA synthesis when substrate is added. In order to elucidate the physiological basis for the survival of cells that are starved for a long time, as well as the capacity of these cells for rapid and efficient recovery, we analyzed the ribosome content of carbon-starved Vibrio sp. strain S14 cells. By using direct chemical measurements of the amounts of ribosomal particles in carbon-starved cultures, we demonstrated that ribosomes were lost relatively slowly (half life, 79 h) and that they existed in large excess over the apparent demand for protein synthesis. After 24 h of starvation the total rate of protein synthesis was 2.3% of the rate during growth, and after 3 days this rate was 0.7% of the rate during growth; the relative amounts of ribosomal particles at these times were 81 and 52%, respectively. The ribosome population consisted of 90% 70S monoribosomes, and no polyribosomes were detected in the starved cells. The 70S monoribosomes were responsible for the bulk of the protein synthesis during carbon starvation; some activity was also detected in the polyribosome size region on sucrose density gradients. We suggest that nongrowing carbon-starved Vibrio sp. strain S14 cells possess an excess protein synthesis capacity, which may be essential for their ability to immediately initiate an upshift program when substrate is added.     

Polynucleotide sequence relationships among Japanese and American strains of Vibrio parahaemolyticus

Polynucleotide sequence relationships between two reference Vibrio parahaemolyticus strains isolated from Japanese and American gastroenteritis patients were investigated by use of (32)P-DNA/DNA reassociation in free solution. In addition, these strains were similarly compared with 22 other strains of estuarine and marine vibrios, including 11 strains previously identified as V. parahaemolyticus (2 Japanese, 1 of unknown location, and 8 American strains obtained from diverse geographical locations and sources in North America), 3 strains of V. alginolyticus, and 8 of Vibrio spp. Deoxyribonucleic acid (DNA) from the Japanese and American gastroenteritis isolates showed high relative levels of intraspecific duplex formation (92 to 93%) when reassociated, reciprocally, at 60 C. Heterologous DNA duplexes exhibited thermal elution midpoint [Tm(e)] values comparable to those obtained from homologous duplexes (88.0) when thermally eluted from hydroxyapatite, thus indicating high base-pair complementarity. Other V. parahaemolyticus strains showed DNA homologies of 85% or greater, with correspondingly high Tm(e) values (86.0 to 88.0) for the heteroduplexes formed. DNA of two of three V. alginolyticus strains (ATCC 17749 and 166-70) was 55 to 60% homologous to reference V. parahaemolyticus DNA preparations; Vibrio sp. strain 5144 (originally classified as V. parahaemolyticus biotype 2 and subsequently as V. alginolyticus strain 5144) showed only 24 to 26% DNA homology to the same reference DNA. These data provide evidence that Vibrio sp. strain 5144 is genetically distinct from the other V. alginolyticus strains used in this study. Three bioluminescent strains thought to be closely related to V. parahaemolyticus demonstrated only 24 to 31% DNA homology to the reference V. parahaemolyticus DNA. These data firmly establish the existence in some Atlantic and Gulf Coast estuaries of organisms genetically very similar to V. parahaemolyticus, the causative agent of "shirasu" food poisoning in Japan.     

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.     

Chemotactic responses of Vibrio alginolyticus to algal extracellular products.

A capillary assay was used to evaluate the chemotactic responses of Vibrio alginolyticus to three common algal extracellular products. Acrylate and glycolate attracted the motile marine bacterium. The peak response occurred with 10(-2) M of each chemical. Acrylic and glycolic acid also attracted V. alginolyticus, with the peak response occurring at 5 x 10(-4) M of each chemical. Higher concentrations of the organic acids resulted in a decreased response. The bacteria also displayed positive chemotaxis to dimethyl sulfide.     

Peritoneal inflammatory cells in plaice, Pleuronectes platessa L.: effects of stress and endotoxin

When plaice were injected intraperitoneally with either oyster glycogen or live Vibrio alginolyticus an acute cellular inflammatory response was observed. The duration of these responses, 7 and 15 days respectively, exceeded the time course of the mammalian cellular inflammatory reaction. Peak leucocyte numbers were found at 2–3 days and neutrophils, which were phagocytic, were more numerous than macrophages. Although the increase in macrophage numbers was less marked, these cells appeared more actively phagocytic than neutrophils. Cortisol injections and environmentally-induced stress caused a significant reduction in the extent of inflammatory cell infiltrates, while endotoxin significantly enhanced the response.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.