A member of a novel family of yeast 'zn-finger' proteins mediates the transition from stationary phase to cell proliferation.

The cloning and molecular characterization of the GCS1 gene from the budding yeast Saccharomyces cerevisiae show that stationary phase is in fact a unique developmental state, with requirements to resume cell proliferation that can be distinct from those for maintenance of proliferation. Deletion of the GCS1 gene products a novel phenotype: stationary-phase mutant cells do not resume proliferation at a restrictive temperature of 15 degrees C, but mutant cells lacking Gcs1p that are proliferating at the permissive temperature of 29 degrees C continue to proliferate after transfer to 15 degrees C as long as nutrients are available. The GCS1 gene sequence predicts a 39 kDa polypeptide with a novel 'Zn-finger' motif. A point mutation within the finger motif produces a phenotype that mimics that of deletion of the GCS1 gene, showing that the finger motif is essential for full Gcs1p activity. Gcs1p and the products of two newly identified genes, SPS18 and GLO3, constitute a family of novel Zn-finger proteins.     

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.     

Isolation and Properties of a Temperature-Sensitive Sporulation Mutant of Bacillus subtilis

A thermosensitive sporulation mutant (t(s)-4) of Bacillus subtilis was isolated, and its morphological, physiological, and enzymatic properties were investigated. This mutant is able to grow equally well at 30 and 42 C, but is unable to sporulate at the higher temperature. Electron microscope studies have shown that the t(s)-4 mutant is blocked at stage zero of spore development. This was further confirmed by its inability to produce antibiotic when grown at the restrictive temperature and by the relatively low ribonucleic acid (RNA) and protein turnover during the stationary growth phase, characteristic for stage zero asporogenic mutants. At the permissive temperature, however, antibiotic production and RNA and protein turnover took place at the rate normally found in sporogenic strains of B. subtilis. The above properties were not altered in the parent strain when grown at either 30 or 42 C. By shifting cultures of the t(s)-4 mutant from 30 to 42 C and from 42 to 30 C at different stages of growth, we have been further able to show that the event affected at the high temperature takes place at a very early stage of spore development. As a consequence of this early block in the sporulation process, the t(s)-4 mutant grown at 42 C became defective in the late spore-specific enzymes involved in the biosynthesis of dipicolinic acid. This study suggests that the sporulation process is mediated by a regulatory protein which is altered in the thermosensitive mutant when grown at the restrictive temperature. As a result of this alteration, a pleiotropic phenotype is produced which has lost the ability to catalyze the late biochemical reactions required for spore formation.     

A Genetic Strategy to Demonstrate the Occurrence of Spontaneous Mutations in Nondividing Cells within Colonies of Escherichia Coli

A genetic strategy was designed to examine the occurrence of mutations in stationary-phase populations. In this strategy, a parental population of cells is able to survive under both permissive and restrictive conditions whereas mutants at a particular target locus exhibit a conditional-lethal phenotype. Thus, by growing the population to stationary phase under restrictive conditions and then shifting it to permissive conditions, mutations that had arisen in stationary phase can be studied without confounding effects caused by the occurrence of similar mutations during growth of the population. In two different applications of this strategy, we have studied the reversion to Lac(+) in stationary phase of several Lac(-) mutations in Escherichia coli. Our results indicate that a variety of spontaneous point mutations and deletions, particularly those that are sensitive to the mechanisms of replication slippage (for their generation) and methyl-directed mismatch repair (for their correction), can arise in nondividing populations of cells within a colony. The frequency of their occurrence was also elevated in mutS strains, which are defective in such mismatch repair. These data have relevance to the ongoing debate on adaptive or directed mutations in bacteria.     

Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12

Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(S) (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an rpoS strain had acquired a GASP mutation in lrp. This GASP allele, lrp-1141, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The lrp-1141 allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a lrp null allele generated by a transposon insertion has a fitness gain identical to that of the lrp-1141 allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the lrp-1141 GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the lrp GASP phenotype, and hence the lrp GASP phenotype is due to more global physiological changes.     

Interaction of cAMP with the CDC25-mediated step in the cell cycle of budding yeast

Addition of exogenous cAMP to cultures of the start mutant cdc25-1 of Saccharomyces cerevisiae shifted to restrictive temperature causes a partial reversion of the mutated phenotype, with a marked increase of the percentage of budded cells. This effect is coupled to a progression in the cell cycle, as demonstrated by DNA histograms obtained by flow cytometry. Moreover cdc25 cells have a high intracellular cAMP content also at restrictive temperature, and no change in the cAMP content was seen during a transition from restrictive to permissive temperature. These data suggest that CDC25 gene product allows cell proliferation by interacting with a cAMP-mediated mechanism.     

A role for the umuDC gene products of Escherichia coli in increasing resistance to DNA damage in stationary phase by inhibiting the transition to exponential growth

The umuDC gene products, whose expression is induced by DNA-damaging treatments, have been extensively characterized for their role in SOS mutagenesis. We have recently presented evidence that supports a role for the umuDC gene products in the regulation of growth after DNA damage in exponentially growing cells, analogous to a prokaryotic DNA damage checkpoint. Our further characterization of the growth inhibition at 30 degrees C associated with constitutive expression of the umuDC gene products from a multicopy plasmid has shown that the umuDC gene products specifically inhibit the transition from stationary phase to exponential growth at the restrictive temperature of 30 degrees C and that this is correlated with a rapid inhibition of DNA synthesis. These observations led to the finding that physiologically relevant levels of the umuDC gene products, expressed from a single, SOS-regulated chromosomal copy of the operon, modulate the transition to rapid growth in E. coli cells that have experienced DNA damage while in stationary phase. This activity of the umuDC gene products is correlated with an increase in survival after UV irradiation. In a distinction from SOS mutagenesis, uncleaved UmuD together with UmuC is responsible for this activity. The umuDC-dependent increase in resistance in UV-irradiated stationary-phase cells appears to involve, at least in part, counteracting a Fis-dependent activity and thereby regulating the transition to rapid growth in cells that have experienced DNA damage. Thus, the umuDC gene products appear to increase DNA damage tolerance at least partially by regulating growth after DNA damage in both exponentially growing and stationary-phase cells.     

The effect of temperature onshibire ts cell clones in the compound eye ofDrosophila melanogaster

Summary We have analysed the effect of temperature on both developing and adult eye cell clones homozygous forshi ^ST139, a temperature-sensitive mutant ofDrosophila melanogaster. The mutant gene, autonomous in its cellular expression, causes structural modifications of ommatidial cells when adult clones of cells are exposed to the restrictive temperature (29°C) for several days. However, the mutant phenotype reverses to normal within 4 days at the permissive temperature (20°C). The results of pulse, shift-up and shift-down experiments show that the temperaturesensitive period for developing compound eye cells is from the late second instar up to the early pupa. Cytodifferentiation of compound eye cells is blocked by restrictive temperature treatment during this period, whereas cell proliferation does not seem to be directly affected. These results are discussed with regard to the other known aspects of the phenotype observed in mutant individuals.     

Introduction to the biology of the bacterial stationary phase: mechanism of general response to stress

This review is devoted to the biology of stationary-phase bacteria, occurring in a specific physiological state at which they arrive in the process of complex response to various kinds of stresses accompanying the retardation and cessation of growth and reproduction. A general account of the problem is presented. Special emphasis is placed on one of the metabolic mechanisms involved in the formation of the physiological state of stationary-phase bacteria and performing primarily protective functions (the so-called general response of cells to stresses). The relationship between this and other regulatory mechanisms involved in the transition of bacteria to the stationary phase and the maintenance of this phase is discussed.     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.     

An unusual suicidal interaction inEscherichia coli involving nucleoid protein H-NS

A conditional-lethal mutation (rpoB364) mapping to the gene that encodes the β-subunit of RNA polymerase was obtained inEscherichia coli. This mutation caused cell filamentation at the restrictive growth temperature and partial derepression of the osmotically regulatedproU operon at the permissive growth temperature. Even under the latter condition, transformants of therpoB364 mutant strain carrying the plasmid vector pACYC184, but not those carrying otherpolA-dependent multicopy plasmids such as pACYC177 or pBR322, were killed in early stationary phase; one class of suppressor mutants isolated as survivors within these transformant colonies were further derepressed forproU-lac expression, and the mutation in each of several independent clones of this class was mapped tohns, the gene that encodes the protein H-NS of theE. coli nucleoid. Thehns mutations did not suppress the conditional-lethal growth phenotype of therpoB364 mutant itself. On the other hand, intracellular overproduction of guanosine 3’, 5’-bispyrophosphate (ppGpp) in therpoB364 strain alleviated both the growth inhibition at the restrictive temperature and the pACYC184-mediated stationary-phase lethality. Upon subcloning into pUC19 or into pACYC177, a 105-bpXbal-HindIII fragment from pACYC184 was shown to be sufficient to confer therpoB364 hns ⁺-dependent lethal phenotype. We suggest that the level in stationary-phase cultures of a gene product(s) that interacts with the pACYC184 DNA fragment is altered in therpoB364 hns+derivative (compared to that inrpoB+ orrpoB364 hns strains) and that this results in cell suicide.     

Stationary phase in the yeast Saccharomyces cerevisiae.

Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis, and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. We propose that cell cycle arrest coordinated with the ability to remain viable in the absence of additional nutrients provides a good operational definition of starvation-induced stationary phase.     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.     

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.     

Light-induced lysis and carotenogenesis in Myxococcus xanthus

Burchard, Robert P. (University of Minnesota, Minneapolis), and Martin Dworkin. Light-induced lysis and carotenogenesis in Myxococcus xanthus. J. Bacteriol. 91:535-545. 1966.-Myxococcus xanthus, grown vegetatively in the light, developed an orange carotenoid after the cells entered stationary phase of growth; pigment content increased with age. Cells grown in the dark did not develop carotenoid and could be photolysed by relatively low-intensity light only during stationary phase; rate of photolysis increased with age. Photolysis adhered to the reciprocity law, was temperature-independent and oxygen-dependent, and required the presence of nonspecific, monovalent cations; it was inhibited by one of several divalent cations. Logarithmic-phase cells were photosensitized by 100,000 x g pellet preparations of sonic-treated stationary-phase cells grown in the light and dark. A porphyrin with a Soret band at 408 mmu was isolated from photosensitive cells; logarithmic-phase cells contained about 1/16 the amount of porphyrin of stationary-phase cells. The purified material had spectral and chemical properties of protoporphyrin IX and photosensitized logarithmic-phase cells. Its spectrum was similar to the action spectrum for photolysis. We concluded that protoporphyrin IX is the natural endogenous photosensitizer. Carotenogenesis was stimulated by light in the blue-violet region of the visible spectrum and was inhibited by diphenylamine, resulting in photosensitivity of the cells. Photoprotection by carotenoid was lost in the cold. A mutant which synthesized carotenoid in the light and dark was photosensitive only after growth in diphenylamine. The ecological significance of these phenomena is discussed.     

Involvement of the Global Regulator H-NS in the Survival of Escherichia coli in Stationary Phase

Long-term batch cultures of Escherichia coli grown in nutrient-rich medium accumulate mutations that provide a growth advantage in the stationary phase (GASP). We have examined the survivors of prolonged stationary phase to identify loci involved in conferring a growth advantage and show that a mutation in the hns gene causing reduced activity of the global regulator H-NS confers a GASP phenotype under specific conditions. The hns-66 allele bears a point mutation within the termination codon of the H-NS open reading frame, resulting in a longer protein that is partially functional. Although isolated from a long-term stationary-phase culture of the parent carrying the rpoS819 allele that results in reduced RpoS activity, the hns-66 survivor showed a growth disadvantage in the early stationary phase (24 to 48 h) when competed against the parent. The hns-66 mutant is also unstable and reverts at a high frequency in the early stationary phase by accumulating second-site suppressor mutations within the ssrA gene involved in targeting aberrant proteins for proteolysis. The mutant was more stable and showed a moderate growth advantage in combination with the rpoS819 allele when competed against a 21-day-old parent. These studies show that H-NS is a target for mutations conferring fitness gain that depends on the genetic background as well as on the stage of the stationary phase.     

Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14

The opportunistic pathogen Pseudomonas aeruginosa produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with P. aeruginosa infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by P. aeruginosa PA14 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NAD+ in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in P. aeruginosa PA14, indicating that pyocyanin may also influence the intracellular redox state by decreasing carbon flux through central metabolic pathways.     

Function of the sigma(E) regulon in dead-cell lysis in stationary-phase Escherichia coli

Elevation of active sigma(E) levels in Escherichia coli by either repressing the expression of rseA encoding an anti-sigma(E) factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase E. coli occurs in a sigma(E)-dependent fashion. We propose there is a novel physiological function of the sigma(E) regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.