Onset of ribosome degradation during cessation of growth in BHK-21/C13 cells.

When BHK-21/C13 cells growing exponentially in 10% serum are transferred to a medium containing only 0.25% serum, cell growth is decreased. After initial changes in RNA synthesis and degradation, protein content of the cultures reaches a plateau and eventually DNA synthesis is arrested. rRNA is relatively stable in exponentially growing cells. Immediately after 'step-down' rRNA degradation commences, but poly(A)-containing RNA does not appear to be degraded any faster than in control cells. Reutilization of RNA precursors has been independently measured and amounts to less than 1%/h for rRNA, insufficient to influence the conclusion that rRNA degradation begins almost immediately after 'step-down'. The degree of reutilization of uridine is much greater for poly(A)-containing RNA than for poly(A)-free RNA.     

Starved Tetrahymena thermophila cells that are unable to mount an effective heat shock response selectively degrade their rRNA.

Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock. An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins. Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells. Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment. We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment. However, selective rRNA degradation occurred in the nonsurviving cells during the heat shock and this presumably accounted for their inviability. A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.     

四膜虫S1营养生长和有性过程中cAMP的含量变化

用放射免疫法测定了四膜虫在群体生长各阶段和在有性过程中cAMP的含量变化。在群体生长的延迟期(Lag phase)之末,即将进入对数期时,cAMP含量出现一个峰值。随着对数期的进展,cAMP含量陆续下降,在对数期之末,cAMP含量降至最低值。群体进入静止期后,细胞内cAMP含量略有回升。 在饥饿诱导接台的过程中,细胞内cAMP含量在开始饥饿的很短时间内即迅速降至一个最低点,并且在整个接合过程中一直保持较低的水平。接合抑制剂伴刀豆球蛋白A(CoaA)和精胺(spermine)等在抑制接合的同时,也阻止饥饿细胞cAMP含量的迅速下降,使之不能达到在正制情况下接合出现时所达到的低水平。 在饥俄细胞的培养液中检出了较高量的cAMP。这可能有助于说明何以饥饿细胞的cAMP含量能够在很短时间内迅速下降。至于排出的cAMP对四膜虫的接合有无促进或其他作用,有特继续研究。     

Degradation of ribosomal RNA during starvation: comparison to quality control during steady-state growth and a role for RNase PH

Ribosomal RNAs are generally stable in growing Escherichia coli cells. However, their degradation increases dramatically under conditions that lead to slow cell growth. In addition, incomplete RNA molecules and molecules with defects in processing, folding, or assembly are also eliminated in growing cells in a process termed quality control. Here, we show that there are significant differences between the pathways of ribosomal RNA degradation during glucose starvation and quality control during steady-state growth. In both processes, endonucleolytic cleavage of rRNA in ribosome subunits is an early step, resulting in accumulation of large rRNA fragments when the processive exoribonucleases, RNase II, RNase R, and PNPase are absent. For 23S rRNA, cleavage is in the region of helix 71, but the exact position can differ in the two degradative processes. For 16S rRNA, degradation during starvation begins with shortening of its 3' end in a reaction catalyzed by RNase PH. In the absence of this RNase, there is no 3' end trimming of 16S rRNA and no accumulation of rRNA fragments, and total RNA degradation is greatly reduced. In contrast, the degradation pattern in quality control remains unchanged when RNase PH is absent. During starvation, the exoribonucleases RNase II and RNase R are important for fragment removal, whereas for quality control, RNase R and PNPase are more important. These data highlight the similarities and differences between rRNA degradation during starvation and quality control during steady-state growth and describe a role for RNase PH in the starvation degradative pathway.     

Starved Tetrahymena Synthesize and Maintain an Excess of 5s rRNA in Their Cytoplasm1

We have measured the rate of accumulation of newly synthesized 5s ribosomal RNA (5s rRNA) in Tetrahymena thermophila cells in early log phase growth and in cells that had been starved in a dilute salt solution. From these measurements we have determined the rates of synthesis and levels of accumulation of 5s rRNA relative to 5.8s rRNA in these two different cell populations. In growing cells 5s rRNA is transcribed and accumulated in a 1:1 molar ratio when compared with 5.8s rRNA. In contrast, in starved cells, 5s rRNA is produced at a rate which is about 15% higher than that seen for 5.8s rRNA. This excess 5s rRNA accumulates in the cytoplasm in a non-ribosomal form and is maintained in the cell as long as the cell remains in a starved condition. The role this excess 5s rRNA may play in the control of 5s rRNA gene expression is discussed.     

Magnesium Starvation of Aerobacter aerogenes II. Rates of Nucleic Acid Synthesis and Methods for Their Measurement

The rates of synthesis of Aerobacter aerogenes nucleic acids were estimated during incubation of the bacteria in a Mg(++)-free medium. Deoxyribonucleic acid (DNA) synthesized during Mg(++) starvation, or in the preceding exponential growth, remained acid-precipitable for 2.5 hr before breaking down to acid-soluble products during a period of many hours. Rates of DNA synthesis were calculated by correcting the net amounts of DNA per milliliter to values that would have appeared had there been no decay. After the first few hours, this rate was constant, the amount of DNA present at the start of Mg(++) starvation being synthesized every 130 min. Rates of synthesis of total ribonucleic acid (RNA) were established in two ways: (i) by measurements of the incorporation of exogeneous uracil and glucose carbon into RNA, and (ii) by the accumulation of transfer RNA (tRNA), since this component is stable during Mg(++) starvation. After the first few hours, this rate was constant, the amount of RNA present at the start of Mg(++) starvation being synthesized about every 120 min. Fractionation by gradient centrifugation revealed that at all times of starvation the ratio of newly synthesized tRNA-rRNA was the same as it was during exponential growth. Furthermore, newly synthesized ribosomal RNA (rRNA) became a part of polysomal structures. Thus, in the absence of Mg(++), DNA, tRNA, and rRNA were synthesized in the same relative proportions as during exponential growth, at rates close to one-half the instantaneous rates of synthesis in the bacteria growing exponentially at the start of starvation.     

Clostridium cellulolyticum Viability and Sporulation under Cellobiose Starvation Conditions

Depending on the moment of cellobiose starvation, Clostridium cellulolyticum cells behave in different ways. Cells starved during the exponential phase of growth sporulate at 30%, whereas exhaustion of the carbon substrate at the beginning of growth does not provoke cell sporulation. Growth in the presence of excess cellobiose generates 3% spores. The response of C. cellulolyticum to carbon starvation involves changes in proteolytic activities; higher activities (20% protein degradation) corresponded to a higher level of sporulation; lower proteolysis (5%) was observed in cells starved during the beginning of exponential growth, when sporulation was not observed; with an excess of cellobiose, an intermediate value (10%), accompanied by a low level of sporulation, was observed in cells taken at the end of the exponential growth phase. The basal percentage of the protein breakdown in nonstarved culture was 4%. Cells lacking proteolytic activities failed to induce sporulation. High concentrations of cellobiose repressed proteolytic activities and sporulation. The onset of carbon starvation during the growth phase affected the survival response of C. cellulolyticum via the sporulation process and also via cell-cellulose interaction. Cells from the exponential growth phase were more adhesive to filter paper than cells from the stationary growth phase but less than cells from the late stationary growth phase.     

High survival efficiency and ribosomal RNA decaying pattern of Desulfobacter latus, a highly specific acetate-utilizing organism, during starvation

Abstract: Exponentially grown Desulfobacter latus cells were transferred to anaerobically prepared minimum medium without a carbon or energy substrate and incubated under anaerobic conditions. Changes in 16S ribosomal RNA (rRNA) of individual cells and the viable fraction in a population were monitored. The cell preparation was stained with a phylogenetic DNA probe labelled with fluorescent dye and the fluorescence of each cell was determined with confocal scanning laser microscope. Viable cells were defined as those capable of reducing a tetrazolium salt (the INT method [1]). The viability of a Desulfobacter starvation culture decreased to 85% in 48 h, but further decrease was not observed during prolonged starvation. The mean amount of 16S rRNA in individual cells decreased exponentially for 48 h to 30% the mean value obtained for exponentially growing cells, but did not decrease by prolonged starvation. About 30% of the mean content of 16S rRNA in growing cells was found in the starved cell population, suggesting that most individual cells in the starved population were not metabolically active. The difference between gross pixel intensity of cells having <8% of 16S rRNA in growing cells and those with a negative control probe was not significant. Thus, non-viable cells may not show positive signals by phylogenetic staining.     

Survival of parental and genetically modified derivatives of a soil isolated Pseudomonas fluorescens under nutrient-limiting conditions

Wild type, a rifampicin-resistant mutant and three genetically modified derivatives of the soil isolate Pseudomonas fluorescens R2f were starved in pure cultures for periods of up to 70 d. Cells were starved after harvesting at a point early in the stationary phase of the growth curve and all five strains demonstrated the ability to survive nutrient deprivation and resuscitate rapidly when growth nutrients became available. No difference in total counts and metabolic activity was detected between the strains. Plate counts were similar for all strains up to day 35. Wild type and the rifampicin-resistant mutant strain showed greater recovery than the genetically modified strains on day 70. During the starvation period there was a significant decrease in cell lengths of all five strains, however, there was no significant difference between the strains. The shape of the starved cells varied with the growth phase at which they had been harvested. Cells taken from early stationary phase and starved produced predominantly rod-shaped cells whereas those taken from early log phase and starved produced small round cells. In experiments when the rifampicin-resistant mutant and the genetically modified strain Art-3 were starved at early log phase the cells were significantly smaller than respective cultures not exposed to the nutrient limiting conditions, and there was no significant difference in the response of the two strains. None of the cultures produced ultramicrobacteria, and none of the cultures entered a non-culturable state. Starvation at different cell densities did not affect the recoverability of the cells. The results of this study demonstrate that responses to starvation conditions by the genetically modified and parental strains are similar.     

Studies on the endogenous metabolism and senescence of starved Sarcina lutea

1. When washed suspensions of Sarcina lutea are starved aerobically in phosphate buffer at the growth temperature of 37 degrees , the rate of endogenous oxygen consumption decreases to very low values after 10hr., although many of the cells survive for 40hr. If starvation is prolonged further, the bacteria die at a rate of approximately 1.5% of the initial viable population per hour. 2. Oxidation of intracellular free amino acids accounts for most of the observed endogenous oxygen uptake but RNA is also utilized and a portion of the component bases and pentose is degraded and presumably oxidized. Ammonia appears in the supernatant and some pentose and ultraviolet-absorbing nucleotide are released from the cells. DNA, protein and polysaccharide are not measurably degraded. 3. Survival can be correlated with the ability of aerobically starved bacteria to oxidize exogenous l-glutamate and glucose. When starved under nitrogen for 40hr. cells continue to oxidize their endogenous reserves at undiminished rates when transferred to aerobic conditions; on prolonging anaerobic starvation the rate of oxidation declines during the period of most rapid loss of viability. 4. In the presence of Mg(2+), RNA degradation during aerobic starvation is almost completely suppressed without affecting the period for which the bacteria survive. 5. Cells grown in peptone supplemented with glucose accumulate reserves of polysaccharide which are metabolized in aerobic starvation, together with free amino acids. Ammonia is evolved and RNA is degraded to a greater extent than in peptone-grown suspensions. Bacteria rich in polysaccharide survive less well than those which are deficient in the polymer; the reason for this phenomenon has yet to be established. 6. In peptone medium, endogenous oxygen uptake and the concentration of intracellular free amino acids decline as growth progresses and they continue to decrease when the organism is held in stationary phase. Under the conditions used, the endogenous Q(o2) and free amino acid pool of cells grown in peptone with 2% (w/v) glucose did not decline so markedly and the bacteria contained large amounts of polysaccharide at all stages of growth.     

Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria.

The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined. Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes. Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days. Biofilm populations of N. europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium. Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation. Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent. Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h. AHL production by N. europaea was detected by using a luxR-luxAB AHL reporter system. The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions.     

Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown.

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing "stepdown" in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active beta-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in "stepped-down" rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of beta-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.     

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.     

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.     

Nucleo-cytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain.

The metabolism of high-molecular-weight RNA in the nuclear and cytoplasmic fractions of newborn and adult rat brain was investigated after the intracranial administration of [32P]Pi. In young brain, a considerable proportion of the newly synthesized radioactive RNA is transferred to the cytoplasm, in contrast with the adult brain, where there appears to be a high intranuclear turnover. Electrophoretic analysis of the newly synthesized RNA showed that processing of the rRNA precursor to yield the 28S and 18S rRNA may be more rapid in the adult than in the young, although most of the adult rRNA in the nucleus is not transferred to the cytoplasm. In young brain, processing is probably tightly coupled to transport of rRNA into the cytoplasm, so that 28S and 18S rRNA are not subjected to possible degradation within the nucleus. Polyadenylated RNA turns over in concert with high-molecular-weight RNA in the nuclei of the adult rat brain. In the cytoplasm the polyadenylated RNA has a higher turnover rate relative to rRNA. In the young brain the polyadenylated RNA is transferred to the cytoplasm along with rRNA, although polyadenylated RNA is transported into the cytoplasm at a faster rate. The nuclear and cytoplasmic polyadenylated RNA species of young brain are larger than their corresponding adult counterparts. These results suggest that there are considerable changes in the regulation of the nucleo-cytoplasmic relationship of rRNA and polyadenylated RNA during the transition of the brain from a developing replicative phase to an adult differentiated and non-dividing state.     

Synthesis of native 60S and 40S ribosomal subunits in Yoshida rat ascites hepatoma AH-130 cells: correlation with the rate of cell growth

The 60Sn and 40Sn subunit ribosome synthesis declined significantly in Yoshida rat ascites hepatoma AH-130 cells from the log phase to the plateau phase of the in vivo growth. Two main classes of 40Sn particles with protein/RNA ratios of 1.82 (p2) and 1.20 (p3) and a minor "heavy" one with protein/RNA ratio of 0.96 (p1) could be distinguished reproducibly by their ultraviolet absorption after sucrose zone sedimentation. The p2 particles appeared the dominating class in log phase cells. In plateau phase cells a decrease of p2 and an increase of p3 particles was observed. Under these conditions the p1 particles and the peaks corresponding to 60Sn subunits and to 80S ribosomes were also increased. Newly synthetized 40Sn particles banded in the p3 region of the gradient and p2 particles originated from them. These particles entered into the ribosomal cycle and contained poly(A) RNA. Formation of radioactive 80S couples by subunits entering into the ribosomal cycle was markedly stimulated in log phase cells and almost completely blocked in cells at the plateau phase of growth.     

Stability of protein and ribonucleic acid in Bacillus stearothermophilus.

The turnover of protein in a prototrophic strain of Bacillus stearothermophilus during exponential growth in a salts medium with glucose or succinate as carbon source was about 4 %/h and in a richer nutrient broth medium about 23 %/h. Protein degradation under non-growing conditions conformed to a similar pattern. The turnover of RNA (non-messenger) was about 1 %/h in salts medium and about 9 %/h in nutrient broth. The turnover of protein and RNA in the thermophile is thus moderate rather than massive. This conclusion was confirmed by measurement of the decay of a specific enzyme, isocitrate lyase, in the prototroph and of the overall protein turnover in a non-prototrophic strain of B. stearothermophilus. The half-lives of a number of enzyme systems in intact cells of the prototrophic thermophile at its optimum growth temperature showed some variation but indicated a significant rate of inactivation. Such decay of protein in vivo apparently accounts for the moderate protein turnover observed during growth.