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.     

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.     

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pH_c) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pH_c and the central nutrient signalling cAMP‐Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pH_c correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pH_c showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.     

Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae.

Metabolic changes have been investigated during continuous growth of yeast cells inoculated in glucose-containing medium until the cells entered the stationary phase in response to glucose exhaustion. Well in advance of glucose exhaustion, a transition phase was observed, characterized by a decrease in the growth rate and a progressive reduction of protein and RNA accumulation. Two-dimensional gel analysis of the proteins synthesized during this stage showed that the pattern of proteins remained similar to that of log-phase cells. When the cells entered the stationary phase, protein accumulation was 10% of that in log-phase cells, and incorporation of labeled RNA precursor was undetectable. Analysis of protein synthesis gave evidence that the synthesis of 95% of the proteins present in log-phase cells was arrested in stationary-phase cells. Among the 20 proteins whose synthesis continues throughout the stationary phase were identified actin, aldehyde dehydrogenase, enolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and five heat shock proteins. In addition, the synthesis of six new proteins was observed. The occurrence of these new proteins in stationary-phase cells is presumed to result from the release of carbon catabolite repression due to glucose exhaustion.     

Regulation of proliferation by the budding yeast Saccharomyces cerevisiae

Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state. In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication. This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell. Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle. In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase. Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells. Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.     

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.     

Respiration and cyanogenesis inPseudomonas aeruginosa

The respiratory ability of batch cultures ofPseudomonas aeruginosa strain 9-D2 peaks during midlog phase at 3.8 nmol O₂/min/10⁸ cells. This ability declines in late log phase, just prior to the time the culture begins to produce cyanide. The respiration of this organism is particularly sensitive to cyanide inhibition during midlog-phase growth, but is extremely resistant to this compound in stationary phase. These inhibition patterns are biphasic for each of these situations and indicate several respiratory responses to HCN. Addition of cyanide to midlog-phase cells resulted in the production of a stationary-phase type of cyanide respiration pattern in 2 h. A non-cyanideproducing mutant of this organism produced significantly less of the cyanide-resistant respiration components.     

Regulation of cell-cycle initiation in yeast by nutrients and protein synthesis.

Arrested Saccharomyces cerevisiae cells initiate the cell cycle in an asynchronous mode. The asynchronous manner of cycle initiation generates variability in cell-cycle times of individual cells. Limiting concentrations of adenine, methionine or histidine regulate the rate of cycle initiation in auxotrophs. A sigmoidal curve of rate vs. concentration is obtained for each of the three substances. Moreover, the three curves have similar Hill coefficients of 2.4, suggesting that a common intermediate requiring adenine, methionine and histidine regulates cell-cycle initiation in yeast. Low concentrations of cycloheximide reduce the rate of cycle initiation of arrested cells that are released from the block in a similar way as limiting nutrients. It thus appears that the common intermediate that requires the limiting nutrients depends upon protein synthesis. The rate of cycle initiation is more sensitive to cycloheximide or nutrient limitation than is protein synthesis. It is also affected by limiting nutrients to a much greater extent than is the overall rate of protein accumulation (i.e., net protein synthesis). Hence the mechanism that controls cycle initiation does not depend on the overall synthesis or accumulation of proteins in the cell. It may depend on synthesis of particular proteins whose production or function requires the limiting nutrients. The high sensitivity of cycle initiation to a decrease in the rate of protein synthesis could explain the ability of yeast cells to complete the cycle and arrest at stationary phase upon depletion of medium components. The cells cannot initiate the cycle although their protein synthesis capacity remains sufficiently high to allow traversal of the rest of the cell cycle.     

Role of Escherichia coli heat shock proteins DnaK and HtpG (C62.5) in response to nutritional deprivation.

Because of the highly conserved pattern of expression of the eucaryotic heat shock genes hsp70 and hsp84 or their cognates during sporulation in Saccharomyces cerevisiae and development in higher organisms, the role of the Escherichia coli homologs dnaK and htpG was examined during the response to starvation. The htpG deletion mutant was found to be similar to its wild-type parent in its ability to survive starvation for essential nutrients and to induce proteins specific to starvation conditions. The dnaK103 mutant, however, was highly susceptible to killing by starvation for carbon and, to a lesser extent, for nitrogen and phosphate. Analysis of proteins induced under starvation conditions on two-dimensional gels showed that the dnaK103 mutant was defective for the synthesis of some proteins induced in wild-type cells by carbon starvation and of some proteins induced under all starvation conditions, including the stationary phase in wild-type cells. In addition, unique proteins were synthesized in the dnaK103 mutant in response to starvation. Although the synthesis of some proteins under glucose starvation control was drastically affected by the dnaK103 mutation, the synthesis of proteins specifically induced by nitrogen starvation was essentially unaffected. Similarly, the dnaK103 mutant was able to grow, utilizing glutamine or arginine as a source of nitrogen, at a rate approximate to that of the wild-type parent, but it inefficiently utilized glycerol or maltose as carbon sources. Several differences between the protein synthetic pattern of the dnaK103 mutant and the wild type were observed after phosphate starvation, but these did not result in a decreased ability to survive phosphate starvation, compared with nitrogen starvation.     

RpoS is necessary for both the positive and negative regulation of starvation survival genes during phosphate, carbon, and nitrogen starvation in Salmonella typhimurium.

The starvation stress response of Salmonella typhimurium encompasses the genetic and physiologic changes that occur when this bacterium is starved for an essential nutrient such as phosphate (P), carbon (C), or nitrogen (N). The responses to the limitation of each of these nutrients involve both unique and overlapping sets of proteins important for starvation survival and virulence. The role of the alternative sigma factor RpoS in the regulation of the starvation survival loci, stiA, stiB, and stiC, has been characterized. RpoS (sigma S) was found to be required for the P, C, and N starvation induction of stiA and stiC. In contrast, RpoS was found to be required for the negative regulation of stiB during P and C starvation-induced stationary phase but not during logarithmic phase. This role was independent of the relA gene (previously found to be needed for stiB induction). The role of RpoS alone and in combination with one or more sti mutations in the starvation survival of the organism was also investigated. The results clearly demonstrate that RpoS is an integral component of the complex interconnected regulatory systems involved in S. typhimurium's response to nutrient deprivation. However, differential responses of various sti genes indicate that additional signals and regulatory proteins are also involved.     

The alternative sigma factor sigmaE controls antioxidant defences required for Salmonella virulence and stationary-phase survival

Bacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors sigmaS (sigma38, RpoS) and sigmaH (sigma32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor sigmaE (sigma24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the sigmaE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking sigmaE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both sigmaE and sigmaS is non-viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary-phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of sigmaE-deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the sigmaE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.     

Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae.

Starvation of cells of the yeast Saccharomyces cerevisiae causes cessation of proliferation and acquisition of characteristic physiological properties. The stationary-phase state that results represents a unique developmental state, as shown by a novel conditional phenotype (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987): mutant cells cannot proliferate at the restrictive temperature when stimulated to reenter the mitotic cell cycle from stationary phase but are unaffected and continue proliferation indefinitely if transferred to the restrictive temperature during exponential growth. We have exploited this reentry mutant phenotype to demonstrate that the same stationary-phase state is generated by nitrogen, sulfur, or carbon starvation and by the cdc25-1 mutation, which conditionally impairs the cyclic AMP-mediated signal transduction pathway. We also show that heat shock, a treatment that elicits physiological perturbations associated with stationary phase, does not cause cells to enter stationary phase. The physiological properties associated with stationary phase therefore do not result from residence in stationary phase but from the stress conditions that bring about stationary phase.     

Surface hydrophobic and hydrophilic protein alterations in Candida albicans

Abstract Cell surface hydrophobicity influences pathogenesis of Candida albicans . Previous studies suggested that stationary-phase hydrophilic and hydrophobic cells, obtained by growth at 37 and 23°C, respectively, may have similar hydrophobic proteins. However, whether hydrophilic and hydrophobic surface proteins differ during the growth cycle at 37°C is unknown. Freeze-fracture analysis revealed surface fibrillar layer differences between hydrophobic late-lag and hydrophilic stationary-phase yeast cells grown at 37°C. Hydrophilic protein differences were also observed between these populations. However, similar hydrophobic proteins were detected among the late-lag and stationary phase cells grown at 37°C and hydrophobic stationary-phase cells grown at 23°C. These results suggest that hydrophobic proteins remain constant but hydrophilic proteins vary during growth. Thus, conversion from surface hydrophilicity to hydrophobicity by C. albicans may only require alterations in the hydrophilic fibrillar protein components.     

The impact of culture medium on the development and physiology of biofilms of Pseudomonas fluorescens formed on polyurethane paint

Microbial biofilms cause the deterioration of polymeric coatings such as polyurethanes (PUs). In many cases, microbes have been shown to use the PU as a nutrient source. The interaction between biofilms and nutritive substrata is complex, since both the medium and the substratum can provide nutrients that affect biofilm formation and biodeterioration. Historically, studies of PU biodeterioration have monitored the planktonic cells in the medium surrounding the material, not the biofilm. This study monitored planktonic and biofilm cell counts, and biofilm morphology, in long-term growth experiments conducted with Pseudomonas fluorescens under different nutrient conditions. Nutrients affected planktonic and biofilm cell numbers differently, and neither was representative of the system as a whole. Microscopic examination of the biofilm revealed the presence of intracellular storage granules in biofilms grown in M9 but not yeast extract salts medium. These granules are indicative of nutrient limitation and/or entry into stationary phase, which may impact the biodegradative capability of the biofilm.     

TOR controls translation initiation and early G1 progression in yeast.

Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early G1 phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (G0). A G1 cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced G1 arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the G1 arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, G1 progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early G1 progression and growth in the absence of nutrients.