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.     

Starvation-Survival Processes of a Marine Vibrio

Levels of DNA, RNA, protein, ATP, glutathione, and radioactivity associated with [³⁵S]methionine-labeled cellular protein were estimated at various times during the starvation-survival process of a marine psychrophilic heterotrophic Vibrio sp., Ant-300. Values for the macromolecules were analyzed in terms of total, viable, and respiring cells. Electron micrographs (thin sections) were made on log-phase and 5.5-week-starved cells. On a per-cell basis, the levels of protein and DNA rapidly decreased until a constant level was attained. A second method in which radioactive sulfur was used for monitoring protein demonstrated that the cellular protein level decreased for approximately 2.5 weeks and then remained constant. An initial decrease in the RNA level with starvation was noted, but with time the RNA (orcinol-positive material) level increased to 2.5 times the minimum level. After 6 weeks of starvation, 45 to 60% of the cells remained capable of respiration, as determined by iodonitrotetrazolium violet-formazan granule production. Potential respiration and endogenous respiration levels fell, with an intervening 1-week peak, until at 2 weeks no endogenous respiration could be measured; respiratory potential remained high. The cell glutathione level fell during starvation, but when the cells were starved in the presence of the appropriate amino acids, glutathione was resynthesized to its original level, beginning after 1 week of starvation. The cells used much of their stored products and became ultramicrocells during the 6-week starvation-survival process. Ant-300 underwent many physiological changes in the first week of starvation that relate to the utilization or production of ATP. After that period, a stable pattern for long-term starvation was demonstrated.     

Survival of a Psychrophilic Marine Vibrio Under Long-Term Nutrient Starvation

Ant-300, a psychrophilic marine vibrio isolated from the surface water of the Antarctic convergence, was starved for periods of more than 1 year. During the first week of starvation, cell numbers increased from 100 to 800% of the initial number of cells. Fifty percent of the starved cells remained viable for 6 to 7 weeks while a portion of the population remained viable for more than 1 year. During the first 2 days of starvation, the endogenous respiration of the cells decreased over 80%. After 7 days, respiration had been reduced to 0.0071% total carbon respired per hour and remained constant thereafter. After 6 weeks of starvation, 46% of the cellular deoxyribonucleic acid had been degraded. Observation of the cellular deoxyribonucleic acid with Feulgen staining before starvation showed the average number of nuclear bodies per cell varied from 1.44 to 4.02 depending on the age of the culture. A linear relationship was found between the number of nuclear bodies per cell and the increase in cell numbers upon starvation. Our data suggest that Ant-300 is capable of surviving long periods of time with little or no nutrients and is therefore well adapted for the sparse nutrient conditions of the colder portions of the open ocean.     

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.     

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.     

Tetrazolium reduction in Saccharomyces cerevisiae

Summary The tetrazolium salt, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) was used to determine viable respiring cells in batch cultures of Saccharomyces cerevisiae. Respiring cells reduce INT to water insoluble iodonitrotetrazolium formazan (INT-formazan) which is deposited within the respiring cell. The INT-formazan granules can be observed by brightfield microscopy. This allows a rapid quantitative determination of the percentage of respiring cells and total cells within the same microscopic field.In actively growing batch cultures of S. cerevisiae, the respiring cell count was equal to the total cell count for the first 72 h of the growth cycle. After 144 h of incubation only 22.7% of the total cell numbers were actively respiring.     

Long-Term Starvation Survival of Rod and Spherical Cells of Arthrobacter crystallopoietes

Spherical and rod-shaped cells of Arthrobacter crystallopoietes, harvested during exponential growth, were subjected to total starvation for periods of time as long as 80 days. Viability measurements were made by plate count and slide culture procedures. Both cell forms remained 100% viable for 30 days. Thereafter, viability of rods and spheres decreased equally at a slow rate. After 60 days of starvation, more than 65% of both cell forms were viable. No significant cell lysis occurred as evidenced by microscopic examination, the small amount of 260-nm absorbing material found in the starvation buffer, and stability of radioactively labeled deoxyribonucleic acid in the cells. Endogenous respiration decreased 80-fold during the first 2 days of starvation, accompanied by a 30% decrease in dry weight of the cells. Thereafter, cellular carbon was oxidized to CO(2) at the constant level of 0.03%/hr over the remaining 78-day starvation period.     

Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry

Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied on seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and in pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured on agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil.     

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.     

Physiological and chemical gradients in a Pseudomonas putida 54G biofilm degrading toluene in a flat plate vapor phase bioreactor

A Pseudomonas putida 54G biofilm was grown on toluene vapor supplied as the sole external carbon and energy source in a flat plate biofilm reactor. Enumerations of cells in the biofilm were made using culture techniques (selective and nonselective for toluene) and microscopic techniques (total and respiring cells), and an analysis of the progression of the state of the culture was made by examination of various fractions of the populations. Long-term exposure to higher levels of toluene produced the following trends: (i) lower fraction of total cells that respired; (ii) lower fraction of culturable cells that also grew on toluene; (iii) higher fraction of respiring cells that could not grow on toluene plates; and (iv) a relatively constant fraction of total cells that could not be cultured on toluene. Respiration rate was determined using oxygen microsensors, and the fraction of the total respiration that was not associated with toluene uptake increased with higher toluene exposure. A combination of cryosectioning and respiration rate data was used to demonstrate that more respiring cells and a higher respiration rate both occurred at the base of the film, suggesting a deterioration in physiological state with continued exposure to toluene. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 361-371, 1997.     

Bacterial Abundance, Activity, and Viability in the Eutrophic River Warnow, Northeast Germany

The River Warnow is the drinking water source for the city of Rostock. Its eutrophic status is accompanied by high amounts of bacteria, which may reach up to 24 x 10(6) cells mL(-1) as recorded during a seasonal study in 2002. Because the river is eutrophic and also heavily loaded with organic matter, this burden is a problem for drinking water purification, as it must be removed completely to not trigger new bacterial growth in the pipeline network. Therefore, restoration measures in the river have to be planned, and bacteria have to be favored as decomposers. That includes the investigation of the physiological state of bacteria in situ. Viable and active cells in the lower reaches of River Warnow were estimated using a broad set of methods. Intact bacteria were investigated by the LIVE/DEAD BacLight bacterial viability kit, containing a mixture of permeant and impermeant nucleic acid stains. Cells with ribosomes were visualized by fluorescence in situ hybridization with the EUB338 oligonucleotide probe. Intact cells and ribosome-containing bacteria represented 24% of total numbers stained by 4'6,-diamidino-2-phenylindole (DAPI) or 66 and 62%, respectively, in relation to all bacteria visualized by the LIVE/DEAD kit. Both fractions were considered as viable, although the fraction of RIB + bacteria is most likely underestimated by the protocol applied. 5-Cyano-2,3-ditolyltetrazolium chloride (CTC) was applied to mark respiring bacteria. The esterase substrate CellTracker Green 5-chloromethylfluorescein diacetate showed cells with intracellular hydrolytic activity. Whereas 1.5% of DAPI-stained bacteria were observed as respiring, 3.8% exhibited intracellular hydrolytic activity on average. If these active fractions were calculated as the percentages of intact cells, much higher fractions of 5.4% were respiring and 16% hydrolytic. Temperature was a main factor influencing total and viable cell numbers simultaneously. The results confirm that there are different states of viable and active cells in natural bacterioplankton communities. However, it remains unclear why fractions of viable and active cells were rather low in this eutrophic river in comparison to similar waters. We recommend to carefully address cells as viable in contrast to nonviable, i.e., dead. As viable cells may be active or inactive with respect to many different activities, e.g., substrate uptake, respiration, hydrolysis, and cell deviation, it is necessary to choose the method to visualize active cells according to the question to be answered.     

Effect of Interfaces on Small, Starved Marine Bacteria

The copiotrophic marine Vibrio sp. strain DW1, shown previously in batch culture to increase in numbers at the onset of starvation and then to form viable small cells with low endogenous respiration, appears to have a survival advantage at interfaces. Vibrio sp. strain DW1 behaved differently at interfaces compared with the aqueous phase under starvation conditions: (i) small cells were observed at an air-water interface without nutrients, (ii) nutrients added to the air-water interface quickly produced larger cells at the surface, (iii) motility persisted many hours longer at the solid-water interface of a dialysis membrane in a microchamber at the onset of starvation, and (iv) regrowth and division at the solid-liquid interface occurred quickly and at nutrient concentrations too low to permit growth in the aqueous phase. It was concluded that, if small starved cells from copiotrophic bacteria can reach an interface, additional survival mechanisms become available to them: (i) interfaces constitute areas of favorable nutrient conditions, and (ii) interfaces lacking a sufficient amount of nutrient, nevertheless, trigger cells to become smaller, thus increasing their surface/volume ratio and the packing density.     

Changes in Cellular States of the Marine Bacterium Deleya aquamarina under Starvation Conditions

In this study, we have used different fluorescent dyes and techniques to characterize the heterogeneity and changes of the physiological states encountered by the marine bacterium Deleya aquamarina during a 92-day starvation survival experiment at 20 and 5(deg)C. Changes of physiological states were investigated on a single-cell basis by flow cytometry and epifluorescence microscopy in conjunction with fluorescent dyes specific for various cellular functions and constituents. Heterogeneities within populations with regard to functions (respiration, substrate responsiveness, enzymatic activity, and cytoplasmic membrane permeability), constituent (DNA), and cell volume (light scatter) were compared to the evolution of viable plate counts (CFU). At 20(deg)C, CFU changes were divided into three stages corresponding to stability up to day 13 followed by a rapid drop between days 13 and 42 and then by stabilization at a level of 10 to 20% during the remaining survival period. Most of the cellular fractions showing a metabolic activity were close to the evolution of the culturable cells, suggesting the absence of viable but nonculturable cells. On the other hand, cells with selective cytoplasmic membrane permeability but without any metabolic activity were observed, and this stage was followed by DNA alteration occurring at different rates after the loss of membrane cytoplasmic permeability. We observed a greater maintenance of culturability, physiological functions, DNA, and cellular volume at the lower temperature. These results have different ecological implications from both methodological and conceptual viewpoints.     

Metabolic activity of Flavobacterium strain P25 during starvation and after introduction into bulk soil and the rhizosphere of wheat

Abstract: The physiological state of introduced Flavobacterium strain P25 cells was determined in starvation cultures, in bulk soil, and in the rhizosphere of wheat using direct viable counts (DVC; based on cell elongation after use of nalidixic acid and substrate addition, resulting in a potential activity measurement) and the redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC; based on respiration without substrate additions, resulting in an in situ activity measurement). Both methods clearly demonstrated that the metabolic activity of Flavobacterium P25 cells decreased during starvation, followed by increased activity after amendment with substrate. This confirmed the applicability of DVC and CTC methods to Flavobacterium P25. Both DVC and CTC methods showed that the percentage of active cells in an introduced Flavobacterium P25 population in rhizosphere soil was lower than that in bulk soil in the first 1–2 weeks after planting wheat seedlings. After two weeks, the percentage of metabolically active cells in the P25 population in rhizosphere soil was higher than in bulk soil. Since different aspects of cellular physiology are measured when applying DVC and CTC, the impact of variations in environmental factors on the metabolic state of introduced strains may be monitored closely by these methods.     

The Survival of Marine Bacteria under Starvation Conditions

The survival under starvation conditions of two selected strains of marine bacteria, a yellow Pseudomonas sp. (strain 95A) and an unidentified oxidative peritrichate Gram negative rod (strain 41), was investigated. The 50% survival times of suspensions in phosphate buffer depended on cell density and were often more than 20 d. A capacity to scavenge atmospheric nitrogenous compounds led to a marked increase in the viability of cell suspensions of 10⁴ cells/ml. Intracellular poly-β-hydroxybutyrate (PHB) prolonged the survival of strain 95A. Strain 41 contained more intracellular protein and this was degraded during starvation in ammonia-free air. Prolonged survival was not explicable in terms of low adenylate charge states. The 'maintenance energy'requirements of strains 95A and 41 in chemostat cultures were 0.042 and 0.04 g glucose/g dry wt/h respectively, compared with dilution-rate-dependent values of 0.051 to 0.856 for Escherichia coli. The low maintenance energy requirements would not alone explain the long viability. Thus no peculiar physiological property such as nitrogen-scavenging, ability to survive at the expense of intracellular PHB or protein, abnormally low cellular protein content, low maintenance energy requirements or a low adenylate charge state fully account for the starvation resistance of these marine bacteria.     

Formation of Stable Bdelloplasts as a Starvation-Survival Strategy of Marine Bdellovibrios

Several wild-type isolates of marine bdellovibrios formed stable bdelloplasts when they infected gram-negative bacterial prey under certain culture conditions. Synchronous predator-prey cultures and low nutrient concentrations increased the yield of stable bdelloplasts. The bdellovibrio cells retained in the stable bdelloplasts showed a high survival capacity in nutrient-depleted saline solution (10% viable Bdellovibrio cells after 3 months at 25°C), whereas Bdellovibrio attack-phase cells kept under the same starvation conditions lost viability more quickly (1% viable cells after 48 h). The addition of yeast extract to a stable bdelloplast suspension induced lysis of the bdelloplasts and release of motile infecting attack-phase Bdellovibrio cells. Other substances, such as free amino acids, protein hydrolysates, NH₄⁺, carbohydrates, and organic amines, did not induce such a release. Stable bdelloplasts were highly hydrophobic and had a lower endogenous respiration rate than attack-phase cells. In general, stable bdelloplasts were almost as sensitive to temperature changes, desiccation, sonication, tannic acid, and Triton X-100 treatment as attack-phase cells. Electron microscopy of stable bdelloplasts did not reveal any extra cell wall layer, either in the bdelloplast envelope or in the retained Bdellovibrio cells, unlike the bdellocysts of the soil bacterium Bdellovibrio sp. strain W. We propose that formation of stable bdelloplasts is a survival strategy of marine bdellovibrios which occurs in response to nutrient- and prey-poor seawater habitats.     

Changes in viability, respiratory activity and morphology of the marine Vibrio sp. strain S14 during starvation of individual nutrients and subsequent recovery

Abstract Starvation for different individual nutrients revealed various morphotypes of Vibrio sp. strain S14. Carbon or multiple-nutrient starved cells formed ultramicrocells with low respiratory activity and high culturability. In contrast, cells starved for nitrogen or phosphorus formed filaments or swollen rods with large inclusion bodies of PHB. These cells exhibited a 3–4 log decrease in culturability, nevertheless many were actively respiring and direct viable counts equalled at least 80% of the original number of cells. After 2–3 days of prolonged incubation, microcolonies appeared at approximately the same number of cells as at the onset of starvation. A nutrient-induced increase in respiratory activity, after 120 h of starvation, was instantaneous for cells starved for carbon or multiple-nutrients, but cells depleted of nitrogen or phosphorus exhibited a lag period of at least 3 h.     

Non-toluene-associated respiration in a Pseudomonas putida 54G biofilm grown on toluene in a flat-plate vapor-phase bioreactor

Non-toluene-associated respiration (NTAR) within a Pseudomonas putida 54G biofilm growing on toluene as sole external carbon source was evaluated using oxygen microelectrodes in a flat-plate vapor-phase biological reactor. Two fluorescent probes, 2,4-diamidino-2-phenylindole and 5-cyano-2,3-ditolyltetrazolium chloride, were used to evaluate the number of total and respiring cells respectively within the biofilm. Biofilm samples were also analyzed for viable and toluene-culturable cells by spread-plating on non-selective and selective media respectively. Fractions of viable stressed, respiring and non-respiring cells within the biofilm were evaluated. The NTAR rate was positively correlated with the fraction of viable stressed and non-respiring cells within the biofilm, which suggested the capability of some cells to grow at the expense of leakage and lysis products coming from injured and dead cells. This effect was more pronounced at higher toluene concentration. Results suggest that NTAR should be incorporated into mathematical models of biofilm reactors degrading volatile organic carbon compounds. Received: 4 January 1997 / Received revision: 20 March 1997 / Accepted: 27 March 1997     

The Relationship Between Electron Transport Activity as Measured by CTC Reduction and CO2 Production in Mixed Microbial Communities

Abstract CTC (5-cyano-2,3-ditolyl tetrazolium chloride) is a redox indicator that facilitates the detection of microbial electron transport activity due to the fluorescence and water insolubility of the reduced CTC-formazan (CTF). The goal of this work was to establish the relationship between the CTC response (both the numbers of CTF-containing cells and the fluorescence intensity of CTF per cell) and respiration in mixed microbial communities. To obtain CTF-containing cell numbers over a range of respiration rates, aerobic bioreactors with on-line CO₂ monitoring were batch fed ground wheat at slow, intermediate, and fast retention times. Samples were taken before and after feeding, and throughout starvation cycles. Each sample was treated with 25 mm CTC, and either supplemented with 10% R2A, or left unsupplemented. CTF-containing cell numbers showed a weak and inconsistent response to transient pulses in respiration, and decreased during long-term starvation at all three retention times. The degree of starvation within the microbial community could be estimated using the ratio of supplemented to unsupplemented CTF-containing cell population. Total fluorescence intensity per cell was consistently higher at peaks of CO₂ production, but did not decrease as dramatically as total cell numbers did in response to starvation. The results indicate the importance of concurrent examination of both the numbers and total fluorescence intensity of CTF-containing cells. Received: 3 September 1996; Accepted: 18 December 1996     

Starvation-Induced Thermal Tolerance as a Survival Mechanism in a Psychrophilic Marine Bacterium

Carbon-starved cultures of strain Ant-300, a psychrophilic marine vibrio isolated from the Antarctic Convergence, were compared with their nonstarved counterparts for resistance to heat. Specifically, starved and unstarved cells were exposed to 17°C, which is 4°C above the maximum growth temperature, and compared with cells maintained at the optimum temperature (5 to 7°C). Total cell counts, direct viable-cell counts, and plate counts were monitored. At a temperature of 17°C, viability (as indicated by plate counts) was lost within 40 h, with direct viable-cell counts indicating less than 5% viability at this time. However, when cells were carbon starved for 1 week prior to heat challenge, significant plateability was maintained for more than 6 days; direct viable-cell counts of starved cells maintained at 17°C indicated the presence of viable cells for at least 12 days. Because starvation is the normal physiological state of copiotrophic, heterotrophic bacteria in oligotrophic marine waters, these data suggest that starvation conditions may be a significant factor in providing heat tolerance to psychrophiles.