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 10 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.     

Exoprotease Activity of Two Marine Bacteria during Starvation

Exoprotease activity during 120 h of total energy and nutrient starvation was examined in two marine bacteria, Vibrio sp. strain S14 and Pseudomonas sp. strain S9. The activity was determined by spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder (hide powder azure). Starved cells of both strains (5 h for S14, and 4 or 24 h for S9) showed greater extracellular proteolytic activity than at the onset of starvation. The exoprotease activity of cells starved for longer periods of time then decreased, but was found to be present at significant levels throughout the starvation period studied (120 h). The accumulation of exoprotease activity in the bulk phase during starvation indicated that both strains constitutively excreted extracellular proteases. As deduced from experiments with chloramphenicol, de novo protein synthesis during starvation was required for the production and/or release of the exoproteases into the surrounding environment. The degradation of hide powder azure allowed an immediate increase in respiration rate, also by long-term-starved cells. This suggests that metabolic systems are primed to respond to the availability of substrates, allowing the cells to recover rapidly. The regulation of exoprotease activity was also studied and found to be different in the two strains. Casamino Acids repressed exoprotease activity in Pseudomonas sp. strain S9, whereas a mechanism similar to catabolite repression was found for Vibrio sp. strain S14 in that glucose repressed activity and cyclic AMP reversed this effect. The exoproteases appeared to be metalloproteinases because the addition of EDTA to cell-free starvation supernatants from both strains significantly inhibited the activity of the proteases.     

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.     

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.     

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.     

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.     

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.     

Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization following Formazan Reduction

Respiring Pseudomonas spp. in milk were quantified within 6 h by fluorescence in situ hybridization (FISH) with vital staining. FISH with an oligonucleotide probe based on 16S rRNA sequences was used for the specific detection of Pseudomonas spp. at the single cell level. 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to estimate bacterial respiratory activity. The numbers of respiring Pseudomonas cells as determined by FISH with CTC staining (CTC-FISH) were almost the same or higher than the numbers of CFU as determined by the conventional culture method.     

Staphylococcus sp. KW-07 contains nahH gene encoding catechol 2,3-dioxygenase for phenanthrene degradation and a test in soil microcosm

We isolated three species of phenanthrene-degrading bacteria from oil-contaminated soils and marine sediment, and assessed the potential use of these bacteria for bioremediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs). Based on 16S rDNA sequences, these bacteria were Staphylococcus sp. KW-07 and Pseudomonas sp. CH-11 from soil, and Ochrobactrum sp. CH-19 from the marine sediment. By PCR amplification, catechol 2,3-dioxygenase genes (nahH genes) mediating PAH degradation in the chromosome of Staphylococcus sp. KW-07 and Ochrobactrum sp. CH-19, and in plasmid DNA of Pseudomonas sp. CH-11 were detected. All isolates had a similar optimal growth temperature (25 °C) and optimal growth pH (7.0) in a minimal salt medium (MSM) with 0.1% (w/v) phenanthrene as the sole source of carbon and energy. Pseudomonas sp. CH-11 and Staphylococcus sp. KW-07 degraded 90% of added phenanthrene in 3 days and Ochrobactrum sp. CH-19 degraded 90% of the phenanthrene in 7 days under laboratory batch culture conditions. However, Staphylococcus sp. KW-07 was the most effective among the three strains in degradation of phenanthrene in soil. After inoculation of 1 × 10¹¹ cells of Staphylococcus sp. KW-07, over 90% degradation of 0.1% phenanthrene (0.1 g/100 g soil) was achieved after 1 month at 25 °C. The results collectively suggest that the Staphylococcus sp. KW-07 strain isolated may be useful in bioremediation of PAH-contaminated soils.     

Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli

Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by real-time polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5–1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods.     

The effects of cortisol on the primary response of mouse spleen cell cultures to heterologous erythrocytes

Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro.Cortisol treatment in vivo reduced spleen cell numbers by 88% after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells.In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10⁶ cells occurred 24 hr later than in controls and averaged, respectively, 27% and 141% of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen.The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture.     

Responses of Ruminococcus flavefaciens, a Ruminal Cellulolytic Species, to Nutrient Starvation

Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39°C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST₅₀) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST₅₀ = 6.6 h at a dilution rate of 0.33 h⁻¹) than for suspensions that had been limited by nitrogen (ST₅₀ = 9.5 h at a dilution rate of 0.33 h⁻¹). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST₅₀ = 7.1 h) than for nitrogen-limited cells (mean ST₅₀ = 12 h). Although it has relatively low ST₅₀ values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.     

Long-term starvation-induced loss of antibiotic resistance in bacteria

Escherichia coli, Pseudomonas fluorescens, and aPseudomonas sp. strain 133B containing the pSa plasmid were starved in well water for up to 523 days. There were two patterns of apparent antibiotic resistance loss observed. InPseudomonas sp. strain 133B, there was no apparent loss of antibiotic resistance even after starvation for 340 days. InE. coli, by day 49 there was a ten-fold difference between the number of cells that would grow on antibiotic- and nonantibiotic-containing plates. However, over 76% of the cells that apparently lost their antibiotic resistance were able to express antibiotic resistance after first being resuscitated on non-selective media. By day 523, only 12% of these cells were able to express their antibiotic resistance after being resuscitated. After starvation for 49 days, cells that could not grow on antibiotic medium even after resuscitation, showed a permanent loss of chloramphenicol (Cm) resistance but retained resistance to kanamycin (Km) and streptomycin (Sm). Restriction enzyme digests show that a 2.5 to 3.0 Kb region from map location 12.5 to 15.5 Kb was deleted. This coincides with the 2.5 Kb reduction in plasmid size observed in 3 isolates that had lost antibiotic resistance after starvation for 49 days.Published as Technical Paper #9224, Oregon Agricultural Experiment Station.     

Light Enhances Survival of Dinoroseobacter shibae during Long-Term Starvation

Aerobic anoxygenic phototrophs (AAPs) as being photoheterotrophs require organic substrates for growth and use light as a supplementary energy source under oxic conditions. We hypothesized that AAPs benefit from light particularly under carbon and electron donor limitation. The effect of light was determined in long-term starvation experiments with Dinoroseobacter shibae DFL 12^T in both complex marine broth and defined minimal medium with succinate as the sole carbon source. The cells were starved over six months under three conditions: continuous darkness (DD), continuous light (LL), and light/dark cycle (LD, 12 h/12 h, 12 µmol photons m⁻² s⁻¹). LD starvation at low light intensity resulted in 10-fold higher total cell and viable counts, and higher bacteriochlorophyll a and polyhydroxyalkanoate contents. This coincided with better physiological fitness as determined by respiration rates, proton translocation and ATP concentrations. In contrast, LD starvation at high light intensity (>22 µmol photons m⁻² s⁻¹, LD conditions) resulted in decreasing cell survival rates but increasing carotenoid concentrations, indicating a photo-protective response. Cells grown in complex medium survived longer starvation (more than 20 weeks) than those grown in minimal medium. Our experiments show that D. shibae benefits from the light and dark cycle, particularly during starvation.     

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.     

Effect of Growth Rate and Starvation-Survival on the Viability and Stability of a Psychrophilic Marine Bacterium

Cell populations of the marine bacterium ANT-300, from either batch or continuous culture with dilution rates ranging from D = 0.015 h⁻¹ to D = 0.200 h⁻¹, were monitored for viability, direct counts, and optical density for 98 days under starvation conditions. Three stages of starvation survival were observed for each of the cell populations. Although direct counts remained at 2 × 10⁷ to 3 × 10⁷ cells ml⁻¹ throughout the starvation period, large fluctuations occurred in cell viability during stage 1 (0 to 14 days) of starvation survival. Stage 2 (14 to 70 days) involved an overall decrease in viability for each of the cell populations; the rate of viability loss was dependent upon the growth rate. Cell viability stabilized at approximately 0.3% of the direct count in stage 3 (70 to 98 days). Long-term starvation corresponded to the prolongation of stage 3 starvation survival. Cell volumes for each of the cell populations decreased with the length of the starvation period. However, the cell volume of starved cells was also dependent more on growth rate than on the length of the time starved. We hypothesize that the cell population with the slowest growth rate is most closely representative of cells found in the oligotrophic marine environment.     

Survival in Sterile Soil and Atrazine Degradation of Pseudomonas sp. Strain ADP Immobilized on Zeolite

In laboratory settings, the ability of bacteria and fungi to degrade many environmental contaminants is well proven. However, the potential of microbial inoculants in soil remediation has not often been realized because catabolically competent strains rarely survive and proliferate in soil, and even if they do, they usually fail to express their desired catabolic potential. One method to address the survival problem is formulating the microorganisms with physical and chemical support systems. This study investigates the survival of Pseudomonas sp. strain ADP in sterile soil and its retention of atrazine-degrading functionality. Assessment was conducted with free and zeolite-immobilized bacteria incorporated into the soil. Pseudomonas sp. strain ADP remained viable for at least 10 weeks when stored at 15°C in sterile soil. Cell numbers increased for both free and zeolite-immobilized bacteria during this period, except for free cells when grown in Miller's Luria-Bertani medium, which exhibited constant cell numbers over the 10 weeks. Only the zeolite-immobilized cell retained full functionality to degrade atrazine after 10 weeks in sterile soil regardless of the medium used to culture Pseudomonas sp. strain ADP. Functionality was diminished in free-cell inoculations except when using an improved culture medium. Survival of zeolite-immobilized Pseudomonas sp. strain ADP separated from the soil matrix after 10 weeks’ incubation was significantly (p < .05) greater than in soil inoculated with free cells or in the soil fraction inoculated by release from zeolite-immobilized Pseudomonas sp. strain ADP.     

Nitrogen Fixation Associated with Development and Localization of Mixed Populations of Cellulomonas sp. and Azospirillum brasilense Grown on Cellulose or Wheat Straw

Mixed cultures of Cellulomonas sp. and Azospirillum brasilense were grown with straw or cellulose as the carbon source under conditions favoring the fixation of atmospheric nitrogen. Rapid increases in cell numbers, up to 10⁹ cells per g of substrate, were evident after 4 and 5 days of incubation at 30°C for cellulose and straw, respectively. Nitrogen fixation (detected by acetylene reduction measured on parallel cultures) commenced after 2 and 4 days of incubation for straw and cellulose, respectively, and continued for the duration of the experiment. Pure cultures of Cellulomonas sp. showed an increase in cell numbers, but CO₂ production was low, and acetylene reduction was not detected on either cellulose or straw. Pure cultures of A. brasilense on cellulose showed an initial increase in cell numbers (10⁷ cells per g of substrate) over 4 days, followed by a decline presumably caused by the exhaustion of available carbon substrate. On straw, A. brasilense increased to 10⁹ cells per g of substrate over 5 days and then declined slowly; this growth was accompanied by acetylene reduction. Scanning electron micrographs of straw incubated with a mixed culture under the above conditions for 8 days showed cells of both species in close proximity to each other. Evidence was furnished that the close spatial relationship of cells from the two species facilitated the mutually beneficial association between them and thus increased the efficiency with which the products of straw breakdown were used for nitrogen fixation.