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.     

Responses of Marine Bacteria Under Starvation Conditions at a Solid-Water Interface

Size changes during starvation of 17 marine bacterial isolates at a solid-water interface and in the liquid phase were examined. Twelve rod-shaped, hydrophilic bacteria decreased in size more rapidly at the solid surface than in the liquid phase, a result parallel to that observed previously for one of the strains at an air-water interface. On the other hand, three rod-shaped, hydrophobic bacteria diminished in size more rapidly in the liquid phase than at the solid-water interface. The rapid size decrease (defined here as the dwarfing phase) in either situation appeared to be an active process which occurred more rapidly when the cells were in an early stage of logarithmic growth at the onset of starvation. Dwarfing was reversibly inhibited by low temperature and low pH but was not inhibited by chloramphenicol. Three coccoidal bacteria showed little tendency to become smaller upon starvation in the liquid phase or at a surface.     

Aerobic and Anaerobic Starvation Metabolism in Methanotrophic Bacteria

The capacity for anaerobic metabolism of endogenous and selected exogenous substrates in carbon- and energy-starved methanotrophic bacteria was examined. The methanotrophic isolate strain WP 12 survived extended starvation under anoxic conditions while metabolizing 10-fold less endogenous substrate than did parallel cultures starved under oxic conditions. During aerobic starvation, the cell biomass decreased by 25% and protein and lipids were the preferred endogenous substrates. Aerobic protein degradation (24% of total protein) took place almost exclusively during the initial 24 h of starvation. Metabolized carbon was recovered mainly as CO(inf2) during aerobic starvation. In contrast, cell biomass decreased by only 2.4% during anaerobic starvation, and metabolized carbon was recovered mainly as organic solutes in the starvation medium. During anaerobic starvation, only the concentration of intracellular low-molecular-weight compounds decreased, whereas no significant changes were measured for cellular protein, lipids, polysaccharides, and nucleic acids. Strain WP 12 was also capable of a limited anaerobic glucose metabolism in the absence of added electron acceptors. Small amounts of CO(inf2) and organic acids, including acetate, were produced from exogenous glucose under anoxic conditions. Addition of potential anaerobic electron acceptors (fumarate, nitrate, nitrite, or sulfate) to starved cultures of the methanotrophs Methylobacter albus BG8, Methylosinus trichosporium OB3b, and strain WP 12 did not stimulate anaerobic survival. However, anaerobic starvation of these bacteria generally resulted in better survival than did aerobic starvation. The results suggest that methanotrophic bacteria can enter a state of anaerobic dormancy accompanied by a severe attenuation of endogenous metabolism. In this state, maintenance requirements are presumably provided for by fermentation of certain endogenous substrates. In addition, low-level catabolism of exogenous substrates may support long-term anaerobic survival of some methanotrophic bacteria.     

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.     

Bacteria in Oligotrophic Environments: Starvation Survival Lifestyle

World Journal of Microbiology and Biotechnology -     

Survival of Bacteria on Metal Surfaces

Survivor curves were determined for Serratia marcescens, Sarcina lutea, Pasteurella tularensis, and P. pestis deposited from the airborne state onto metallic surfaces and subsequently stored at various humidities and temperatures. Cells of all species tested remained alive longest in a dry atmosphere, except that cells of S. marcescens survived best in a saturated atmosphere. Survival decreased most rapidly at the intermediate humidity level for three of the test organisms, yet P. tularensis died most rapidly in a saturated atmosphere. An increase in temperature decreased survival of P. pestis and P. tularensis.     

Catalase Activity of Psychrophilic Bacteria Grown at 2 and 30 C

Catalase activity was measured in resting-cell suspensions of psychrophilic bacteria grown at 2 and at 30 C. Enzyme activity decreased in both cell-suspension types as harvest age increased. At comparable physiological age, cells grown at 2 C had more catalase than cells grown at 30 C.     

Growth at Low Ammonium Concentrations and Starvation Response as Potential Factors Involved in Niche Differentiation among Ammonia-Oxidizing Bacteria

In nature, ammonia-oxidizing bacteria have to compete with heterotrophic bacteria and plants for limiting amounts of ammonium. Previous laboratory experiments conducted with Nitrosomonas europaea suggested that ammonia-oxidizing bacteria are weak competitors for ammonium. To obtain a better insight into possible methods of niche differentiation among ammonia-oxidizing bacteria, we carried out a growth experiment at low ammonium concentrations with N. europaea and the ammonia oxidizer G5-7, a close relative of Nitrosomonas oligotropha belonging to Nitrosomonas cluster 6a, enriched from a freshwater sediment. Additionally, we compared the starvation behavior of the newly enriched ammonia oxidizer G5-7 to that of N. europaea. The growth experiment at low ammonium concentrations showed that strain G5-7 was able to outcompete N. europaea at growth-limiting substrate concentrations of about 10 μM ammonium, suggesting better growth abilities of the ammonia oxidizer G5-7 at low ammonium concentrations. However, N. europaea displayed a more favorable starvation response. After 1 to 10 weeks of ammonium deprivation, N. europaea became almost immediately active after the addition of fresh ammonium and converted the added ammonium within 48 to 96 h. In contrast, the regeneration time of the ammonia oxidizer G5-7 increased with increasing starvation time. Taken together, these results provide insight into possible mechanisms of niche differentiation for the ammonia-oxidizing bacteria studied. The Nitrosomonas cluster 6a member, G5-7, is able to grow at ammonium concentrations at which the growth of N. europaea, belonging to Nitrosomonas cluster 7, has already ceased, providing an advantage in habitats with continuously low ammonium concentrations. On the other hand, the ability of N. europaea to become active again after longer periods of starvation for ammonium may allow better exploitation of irregular pulses of ammonium in the environment.     

Aggregates of Resident Bacteria Facilitate Survival of Immigrant Bacteria on Leaf Surfaces

The fate of immigrant bacterial cells on leaves under stressful conditions was determined as a function of the anatomical features and the local spatial density of resident cells at their landing site. Pantoea agglomerans 299R was established on bean leaves and the survival of immigrant cells of Pseudomonas fluorescens A506 and Pseudomonas syringae B728a, as well as P. agglomerans itself, was determined by epifluorescence microscopy following subsequent exposure of plants to desiccation stress. Resident and immigrant bacterial strains constitutively expressed the cyan and the green fluorescent protein, respectively, and the viability of individual cells was assessed directly on leaf surfaces following propidium iodide staining. Although only a small fraction of the immigrant cells landed on established bacterial aggregates, their fate was usually strongly influenced by the presence of indigenous bacteria at the site at which they landed. Immigrants of P. agglomerans 299R or P. fluorescens A506 that arrived as solitary cells had about double the probability of survival when landing on aggregates formed by P. agglomerans 299R than when landing on uncolonized areas of the leaf surface. In contrast, the survival of P. syringae B728a was similar irrespective of whether it landed on colonized or uncolonized parts of a leaf. The nature of plant anatomical features at which immigrant bacteria landed also strongly influenced the fate of immigrant bacteria. The fraction of immigrant cells of each species tested that landed on veins, glandular trichomes, or epidermal cells altered by P. agglomerans that died was always less than when they landed on normal epidermal cells or at the base of hooked trichomes. Depending on the process by which immigrants arrive at a leaf, only a small fraction of cells may be deposited on existing bacterial aggregates. Although uncolonized sites differed greatly in their ability to influence the survival of immigrant cells, the fate of an immigrant bacterium will depend on the nature of the leaf structure on which it is deposited, and apparently indirectly on the amount of nutrients and water available at that site to support the development of bacterial aggregates.     

Control of Cell Growth in Bacteria: Experiments with Thymine Starvation

When cells of Escherichia coli THU were starved for thymine, they continued to grow without division for at least two successive volume doublings at their initial rate. Within experimental error this average rate of volume increase, 0.21 mum(3) per hr, was identical with that observed in control cultures during two generations of growth in the presence of thymine. This growth rate was also independent of the age of the cells at the time of starvation. These results are consistent with the hypothesis, proposed earlier, that growth rates are controlled by uptake sites for binding, transport, or accumulation of compounds into the cell, that the number of these sites is constant throughout most of the cell cycle, and that this number doubles near or at cell division.