Marine Vibrio Sp Research Articles

Aerobactin production by a planktonic marine Vibrio sp

Production of siderophores by marine bacteria could potentially contribute to metal complexation by organic compounds in seawater. Direct isolation of planktonic marine bacteria on siderophore indicator plates showed that siderophore producers are present. The siderophore‐producing organisms that could be detected on this medium were more numerous in the mixed layer than in deeper water at this sampling site. One strain (DS40M5) was characterized and found to be a marine Vibrio sp. A siderophore produced by Vibrio sp. DS40M5 is aerobactin, a compound not previously known from planktonic marine bacteria.

The carbon starvation stimulon in the marine Vibrio sp. S14 (CCUG 15956) includes three periplasmic space protein responders

Summary: Growth arrest and long-term starvation in the marine Vibrio sp. S14 induce alterations of the cell envelope as well as a sequential expression of starvation-specific polypeptides. The induction and accumulation of three dominant carbon-starvation-induced periplasmic space protein responders of molecular mass 120 (Cspl), 37 (Csp5) and 30 kDa (Csp6), were examined under different starvation and stress conditions. All three proteins were increasingly synthesized in response to carbon and multiple-nutrient starvation, but not during nitrogen and phosphorus starvation, nor in response to the imposition of stress conditions. Csp1 exhibited a more than 100-fold increased relative induction by carbon and multiple-nutrient starvation and was continuously synthesized throughout the experiment. Induction of Csp1 was completely repressed by all conditions other than carbon starvation. A less than fivefold increase in induction was monitored for the transient responders Csp5 and Csp6 within the first few hours of carbon starvation. Interestingly, Csp6 exhibited an increased transient induction at the onset as well as after 24 h of starvation. Accumulation of the three proteins was monitored by Western blot analysis using specific antisera. Of the conditions tested, long-term accumulation of these proteins was only detected when Vibrio sp. S14 cells were exposed to carbon and multiple-nutrient starvation. These results are discussed in relation to the recent finding that formation of starvation- and stress-resistant marine Vibrio is induced by carbon but not nitrogen or phosphorus starvation, and that Csp1, Csp5 and Csp6 will allow detailed studies of the carbon starvation stimulon.

Bacteria starved for prolonged periods develop increased protection against lethal temperatures

A marine Vibrio sp. DW1 and two Escherichia coli strains, K165 (htpR−) and Sc122 (htpR+) were submitted to heat stress after different times of starvation. All three bacterial strains developed starvation-mediated cross protection against heat. While two hours (Vibrio sp. DW1) and 24 hours (E. coli) of starvation gave near maximal protection, prolonged periods of non-growth offered increased protection. Chloramphenicol was added, at different times of starvation, to investigate the dependence on de novo protein synthesis for survival after heat stress during prolonged starvation. An obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found. Starvation-induced cross protection against heat may be dependent on protein synthesis in the initial phase of starvation while after prolonged starvation the continuous protection offered is suggested not to be mediated by de novo protein synthesis at these times.

Bacteria starved for prolonged periods develop increased protection against lethal temperatures

A marine Vibrio sp. DW1 and two Escherichia coli strains, K165 (htpR−) and Sc122 (htpR+) were submitted to heat stress after different times of starvation. All three bacterial strains developed starvation-mediated cross protection against heat. While two hours (Vibrio sp. DW1) and 24 hours (E. coli) of starvation gave near maximal protection, prolonged periods of non-growth offered increased protection. Chloramphenicol was added, at different times of starvation, to investigate the dependence on de novo protein synthesis for survival after heat stress during prolonged starvation. An obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found. Starvation-induced cross protection against heat may be dependent on protein synthesis in the initial phase of starvation while after prolonged starvation the continuous protection offered is suggested not to be mediated by de novo protein synthesis at these times.

Bacteria starved for prolonged periods develop increased protection against lethal temperatures

A marine Vibrio sp. DW1 and two Escherichia coli strains, K165 ( htpR −) and Sc122 ( htpR +) were submitted to heat stress after different times of starvation. All three bacterial strains developed starvation-mediated cross protection against heat. While two hours ( Vibrio sp. DW1) and 24 hours ( E. coli) of starvation gave near maximal protection, prolonged periods of non-growth offered increased protection. Chloramphenicol was added, at different times of starvation, to investigate the dependence on de novo protein synthesis for survival after heat stress during prolonged starvation. An obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found. Starvation-induced cross protection against heat may be dependent on protein synthesis in the initial phase of starvation while after prolonged starvation the continuous protection offered is suggested not to be mediated by de novo protein synthesis at these times.

Physiological and molecular adaptation to starvation and recovery from starvation by the marine Vibrio sp. S14

Physiological and molecular adaptation to starvation and recovery from starvation by the marine Vibrio sp. S14

Physiological and molecular adaptation to starvation and recovery from starvation by the marine Vibrio sp. S14

Physiological and molecular adaptation to starvation and recovery from starvation by the marine Vibrio sp. S14

Functional mRNA half-lives in the marine Vibrio sp. S14 during starvation and recovery

The decay rate of the potential to synthesize proteins after inhibition of transcription with rifampicin (Rif) was analysed at different times of energy and nutrient starvation for the marine Vibrio sp. S14. The decline of protein synthesis following Rif treatment is due to the instability of mRNA and permits an estimate of the functional mRNA half-life. The half-life of the mRNA pool was found to increase 6-fold (from 1·7 to 10·3 min) during a period of 24 h of total energy and nutrient starvation. To resolve whether the increase in the mean mRNA half-life was a result of the stabilization of the entire pool or if proteins expressed during starvation were translated from very stable mRNAs, the half-lives of specific mRNAs were measured. It was found that the half-lives of mRNAs common to both growing and starving cells were increased between 2- and 3-fold during a period of 24 h starvation, and that some starvation-specific proteins were encoded by extremely long-lived mRNAs (up to 70 min). The possible role of stabilization of mRNA as a mechanism to economize protein synthesis during starvation conditions is discussed

Macromolecular synthesis during recovery of the marine Vibrio sp. S14 from starvation

The recovery of Vibrio sp. S14 cells from energy- and nutrient-starvation was monitored after the addition of glucose minimal medium. Upshift experiments were done throughout a starvation period of 200 h to determine whether cells were more responsive to nutrient addition at the onset of starvation, or if the previously described programme of starvation-induced cellular reorganization had to be completed before cells could become committed to recovery following nutritional upshifts. The kinetics of macromolecular synthesis (RNA, protein and DNA), the rate of respiration and changes in median cell volume in response to nutritional upshifts at different times of starvation were examined. The relative rates of RNA and protein synthesis increased immediately upon addition of glucose minimal medium; the increase in protein synthesis was not dependent on a parallel increase in RNA synthesis, indicating that the starved cells may have an excess of protein synthesizing machinery, including stable RNA and functional ribosomes. The subsequent increase in the rate of DNA replication was initiated approximately 60 min before the first apparent cell division at approximately one doubling of the theoretical minimal cell volume (Vu). Two-dimensional gel electrophoresis was used to demonstrate the fate of starvation-specific proteins during the upshift, and also the synthesis of recovery-induced proteins that were not found in starving cells. Most starvation-inducible proteins were repressed immediately at the onset of the nutritional upshift, while 11 of the 21 recovery-induced proteins identified were expressed exclusively during the maturation phase and were subsequently repressed at the onset of regrowth. The possible role of such maturation-specific proteins in the rapid formation of a reproductive cell committed to growth and division is discussed.

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.

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

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

Specific inhibition of the Na(+)-driven flagellar motors of alkalophilic Bacillus strains by the amiloride analog phenamil

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus strains, was found to cause growth inhibition; therefore, the use of amiloride for the isolation of motility mutants was difficult. On the other hand, phenamil, an amiloride analog, inhibited motor rotation without affecting cell growth. A concentration of 50 microM phenamil completely inhibited the motility of strain RA-1 but showed no effect on the membrane potential, the intracellular pH, or Na(+)-coupled amino acid transport, which was consistent with the fact that there was no effect on cell growth. Kinetic analysis of the inhibition of motility by phenamil indicated that the inhibition was noncompetitive with Na+ in the medium. A motility mutant was isolated as a swarmer on a swarm agar plate containing 50 microM phenamil. The motility of the mutant showed an increased resistance to phenamil but normal sensitivity to amiloride. These results suggest that phenamil and amiloride interact at different sites on the motor. By examining various bacterial species, phenamil was found to be a specific and potent inhibitor for the Na(+)-driven flaggellar motors not only in various strains of alkalophilic Bacillus spp. but also in a marine Vibrio sp.

Role of Protein Synthesis in the Cell Division and Starvation Induced Resistance to Autolysis of a Marine Vibrio during the Initial Phase of Starvation

Starvation of a marine Vibrio sp. S14 for carbon, nitrogen and phosphorus resulted in a fourfold increase in cell number during the first 6 h in the starvation regime. This initial cell division of non-growing cells was dependent on both DNA and peptidoglycan synthesis as deduced from inhibition experiments using nalidixic acid and ampicillin. Inhibition of protein synthesis by the addition of chloramphenicol led to the cessation of both cell division and DNA synthesis after 40–60 min in the starvation regime. Starvation also induced resistance against autolytic cell wall degradation. Resistance to ampicillin-induced murein degradation was most extensive in the portion of the cell wall that was synthesized after the onset of starvation and was dependent on de novo protein synthesis. The amount of d-alanine per unit dry weight increased twofold during 24 h of starvation and an increased resistance to lysis induced by sonication was observed during this period. It is suggested that the fourfold increase in cell number during the first six hours of starvation requires proteins synthesized de novo and that new rounds of DNA replication may be initiated during non-growth subsequent to 40–60 min of starvation. While the rate of DNA synthesis during the initial 40–60 min was unaffected by the blockage of protein synthesis, the mechanism conferring autolysis resistance was effectively inhibited.

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.

Relative changes in incorporation rates of leucine and methionine during starvation survival of two bacteria isolated from marine waters

Two copiotrophic Gram-negative bacteria isolated from marine waters, S14 and Vibrio sp. DW1, were examined for changes in the rate of protein synthesis in the initial phase of energy and nutrient deprivation. The incorporation of [3H] leucine into the trichloroacetic acid (TCA)-insoluble material was examined as a method for estimating rates of protein synthesis. The incorporation of methionine was measured and compared with the results of leucine incorporation. Protein synthesis was demonstrated throughout a period of 120 h of starvation. The incorporation rate was related to the time of starvation and decreased subsequent to an initial increase during the first few hours of dormancy. Control experiments with proteinase K and chloramphenicol demonstrated that the labelled amino acids were preferentially incorporated into proteins. It was also demonstrated that the uptake of amino acids was not a rate-limiting step. During the first hours of starvation the ratio of the protein to the dry weight of the S14 cells increased parallel to the increase in the amino acid incorporation rate. The increased activity of the protein-synthesising system during the first hours of nutrient and energy depletion indicates the presence of an active cellular response to the downshift conditions. Furthermore, these findings are consistent with the increased respiratory activity during the first hours of starvation, which has previously been observed for the bacteria examined in this study.

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

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

Arginine Uptake by a Psychrophilic Marine Vibrio sp. During Starvation-induced Morphogenesis

The effects of starvation on the arginine transport system in ANT-300, a psychrophilic marine Vibrio sp., were investigated. A rapid rate of uptake was detected on the first day of starvation, followed by a much lower but constant rate of uptake until day 35. Transport was found to require maintenance of a proton gradient across the membrane, but not to involve ATP. Respiration of the transported arginine increased during the 35 d of starvation. Thus, ANT-300 is able to maintain a high-affinity, active transport system for arginine during extended periods of starvation even in the absence of an exogenously supplied energy source.

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.

Pathogenesis of Experimental Vibriosis in Larval American Oysters, Crassostrea virginica

Larval oysters were experimentally infected with isolates of pure cultures of marine Vibrio sp. These animals were studied live, histologically, with the immunofluorescent test and with electron microscopy. All inoculated groups, but no control groups, demonstrated decreased growth and/or high mortality.One bacterial isolate attached preferentially to larval shell and destroyed mantle tissue as it grew along the internal surface of the shell. Phagocytes consumed invading bacterial cells but were ultimately overwhelmed by the infection.The second bacterial isolate caused velar damage in young larvae that remained active. In these larvae, the velar cells became detached or internally disorganized and lost their rectractor muscle insertions. In more advanced larvae, detachment of absorptive cells of the digestive gland was the earliest change observed and seemed to result from attachment of bacterial antigens to the cell surface. In both younger and more advanced larvae, food cycling and nutrient utilization were disrupted early in the disease process. The older larvae showed a general atrophy.Clinical signs such as mantle cell detachment could be detected early in the disease. Other clinical observations and the immunofluorescent test were also useful in the early diagnosis of vibriosis in larval oysters.Key words: vibriosis, larval oysters, Crassostrea virginica, histology, electron microscopy, ultrastructure, immunofluorescence, oyster hatcheryDes larves d'huîtres ont été infectées expérimentalement par des isolats de cultures pures de Vibrio sp. marins. Ces animaux furent étudiés en vie, histologiquement, à l'aide du test d'immunofluorescence et enfin au microscope électronique. Tous les groupes inoculés, mais non les groupes témoins, accusent un déclin de croissance et/ou une mortalité élevée.

Extracellular nuclease produced by a marine bacterium. I. Extracellular deoxyribonuclease formation by a marine Vibrio sp.

Deoxyribonuclease (DNase) activity was found in the culture fluids of numerous marine bacteria isolated from seawater. Among these ogranisms, marine bacterium, Vibrio sp., strain No. 2, showed the highest deoxyribonucleic acid-hydrolyzing activity. This organism requires salts of seawater for both growth and extracellular DNase formation. The DNase activity could not be detected in the synthetic seawater culture liquid lacking magnesium ion, and DNase activity decreased in a calcium-deficient medium. The optimum temperature for the growth of this organism was between 15 and 25 degrees C. The formation of extracellular DNase was the greatest at 20 degrees C and less activity was found at 10 and 30 degrees C.