Magnesium Starvation of Aerobacter aerogenes III. Protein Metabolism

The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg(++)-deficient medium. Bacteria were exposed to leucine-H(3) during the exponential growth period preceding Mg(++) starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg(++) starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg(++) starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg(++) starvation was correspondingly less. During recovery from Mg(++) starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg(++) was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.     

Selective Autolysis of Nuclei as a Source of DNA Precursors in Paramecium aurelia Exconjugants*

The fate of labeled DNA in macronuclear fragments of starving Paramecium aurelia exconjugants was studied by quantitative autoradiography. Labeled material originally contained in DNA of macronuclear fragments is incorporated into macronuclear anlagen. During the starvation period the mean number of macronuclear fragments per cell decreased exponentially while there was an approximately exponential increase in the volume of macronuclear anlagen. Fragments appeared to be selectively and individually autolyzed. Labeled material originally contained in fragments was largely if not completely conserved through 108 hr of starvation during which more than 90% of the fragments were lost. Soluble labeled material was detectable after autolysis of fragments began, but finally almost all labeled material was incorporated into macronuclear anlagen.     

Ribosome biosynthesis in Tetrahymena thermophila. III. Regulation of ribosomal RNA degradation in growing and growth arrested cells

We have measured the turnover rate of ribosomal RNA in exponentially growing Tetrahymena thermophila cells, cells entering the plateau phase of growth, and nutrient-deprived (starved) cells. Ribosomal RNA is stable in cells in early log phase growth but it begins to turnover as the cells begin a deceleratory growth phase prior to entering a plateau state. Likewise, rRNA in cells transferred from early log phase growth to a starvation medium begins to be degraded immediately upon starvation. In both cases the degradation of rRNA exhibits biphasic kinetics. A rapid initial exponential degradation with a half time of nine and one-half hours lasting for six hours is followed by a slower exponential degradation with a half-life of 35 hours. When starved cells are transferred to fresh growth medium turnover of rRNA ceases. The evidence presented suggests that the alteration in degradation rate is a regulated process which is most likely independent of the cell cycle.     

The Proteomic Response of Bacillus pumilus Cells to Glucose Starvation

Since starvation for carbon sources is a common condition for bacteria in nature and it can also occur in industrial fermentation processes due to mixing zones, knowledge about the response of cells to carbon starvation is beneficial. The preferred carbon source for bacilli is glucose. The response of Bacillus pumilus cells to glucose starvation using metabolic labeling and quantitative proteomics was analyzed. Glucose starvation led to an extensive reprogramming of the protein expression pattern in B. pumilus. The amounts of proteins of the central carbon metabolic pathways (glycolysis and TCC) remained stable in starving cells. Proteins for gluconeogenesis were found in higher amounts during starvation. Furthermore, many proteins involved in acquisition and usage of alternative carbon sources were present in elevated amounts in starving cells. Enzymes for fatty acid degradation and proteases and peptidases were also found in higher abundance when cells entered stationary phase. Among the proteins found in lower amounts were many enzymes involved in amino acid and nucleotide synthesis and several NRPS and PKS proteins.     

Viability and Endogenous Substrates Used During Starvation Survival of Rhodospirillum rubrum

Cells of Rhodospirillum rubrum were grown photoorganotrophically and chemoorganotrophically and then starved for organic carbon and combined nitrogen under four conditions: anaerobically in the light and dark and aerobically in the light and dark. Illumination prolonged viability and suppressed the net degradation of cell material of phototrophically grown cells, but had no effect on chemotrophically grown cells that did not contain bacteriochlorophyll. The half-life survival times of carbohydrate-rich phototrophically grown cells during starvation anaerobically or aerobically in the light were 17 and 14.5 days, respectively. The values for starvation aerobically and anaerobically in the dark were 3 and 0.5 days, respectively. Chemotrophically grown cells had half-life survival times of 3 and 4 days during starvation aerobically in the light and dark, respectively, and 0.8 day during starvation anaerobically in the light or dark. Of all cell constituents examined, carbohydrate was most extensively degraded during starvation, although the rate of degradation was slowest for phototrophically grown cells starved anaerobically in the light. Phototrophically grown cells containing poly-beta-hydroxybutyrate as carbon reserve were less able to survive starvation anaerobically in the light than were carbohydrate-rich cells starved under comparable conditions. Light intensity had a significant effect on viability of phototrophically grown cells starving anaerobically. At light intensities of 320 to 650 lx, the half-life survival times were 17 to 24 days. At 2,950 to 10,500 lx, the survival times decreased to 1.5 to 5.5 days. The kinetics of cell death correlated well with the rate of loss of cell mass of starving cells. However, the cause of death could not be attributed to degradation of any specific cell component.     

A quantitative method for the analysis of mammalian cell proliferation in culture in terms of dividing and non-dividing cells

The application of the exponential growth equation is the standard method employed in the quantitative analyses of mammalian cell proliferation in culture. This method is based on the implicit assumption that, within a cell population under study, all division events give rise to daughter cells that always divide. When a cell population does not adhere to this assumption, use of the exponential growth equation leads to errors in the determination of both population doubling time and cell generation time. We have derived a more general growth equation that defines cell growth in terms of the dividing fraction of daughter cells. This equation can account for population growth kinetics that derive from the generation of both dividing and non-dividing cells. As such, it provides a sensitive method for detecting non-exponential division dynamics. In addition, this equation can be used to determine when it is appropriate to use the standard exponential growth equation for the estimation of doubling time and generation time.     

Exponential decay kinetics in "downhill" protein folding

The observation of single-exponential kinetic phases in early stages of protein folding is often interpreted as evidence that these phases are rate limited by significant energy or entropy barriers. However, although the existence of large barriers reliably implies exponential kinetics, the reverse is not necessarily true. A simple model for the hydrophobic collapse of a chain molecule demonstrates that a barrierless or "downhill" diffusional relaxation can give rise to kinetics that are practically indistinguishable from a pure exponential. Within this model, even a highly nonlinear experimental probe such as resonance energy transfer (F?rster transfer) could exhibit a large amplitude decay (greater than 90% in fluorescence) that deviates from a simple exponential by less than 0.5%. Only a detailed analysis of the dynamics is likely to reveal that a free energy barrier is absent.     

Effect of amino acid starvation on UV sensitivity ofLactobacillus acidophilus cells

InLactobacillus acidophilus cultures UV irradiated in the exponential phase of growth, the dosesurvival curve was of the simple exponential type, without any shoulder. If the bacteria were subjected to amino acid starvation prior to irradiation, an shoulder corresponding to a quasi-treshold dose (D_q) of about 780 ergs/mm² appeared in the curve. The administration of protein or RNA-synthesia inhibitors prior to irradiation had the same effect. The effect of pre-irradiation amino acid starvation was abolished by simultaneous thymidine starvation. It was likewise abolished if amino acid starvation was followed by incubation in the presence of amino acids (without thymidine) and then by irradiation of the cells. Post-irradiation amino acid starvation did not lead to the formation of an shoulder but if combined with thymidine starvation it did. It can be concluded from the results that post-irradiation repair processes are facilitated or promoted if, during the post-irradiation interval DNA synthesis is delayed. This delay represents a compensation of the pre-irradiation increase of cellular DNA-content, taking place during inhibition of proteosynthesis. The postirradiation administration of caffeine did not abolish the formation of the shoulder induced by pre-irradiation amino acid starvation; on the contrary, it induced its formation even in exponentially growing, irradiated control bacteria.     

Density- and Size-Dependent Winter Mortality and Growth of Late Chaoborus flavicans Larvae

Winter processes such as overwinter survival and growth of individuals can have wide-ranging consequences for population dynamics and communities within and across seasons. In freshwater organisms winter processes have been mainly studied in fish despite that invertebrates also have substantial impacts on lake and pond food webs. One of the major invertebrate consumers in lake and ponds is the planktonic larvae of the dipteran insect Chaoborus spec. However, while much is known about Chaoborus feeding ecology, behaviour and structuring role in food webs, its winter ecology and how it affects its populations are poorly understood. Here size- and density-dependent winter mortality and body growth of late Chaoborus flavicans larvae were quantified over naturally occurring size and density ranges in autumn and under natural winter conditions using two field enclosure experiments. Winter mortality increased with autumn density but decreased with autumn body size while winter growth rates decreased with autumn density and body sizes. There was also a density- and size-independent background mortality component. The proportion of pupae found in spring decreased strongly and exponentially with autumn density. These results may explain the commonly observed univoltine life cycle and multi-annual density fluctuations in northern Chaoborus populations. They further demonstrate the relevance of winter processes and conditions for freshwater invertebrates and ecosystems.     

Physiological responses of Lactococcus lactis ML3 to alternating conditions of growth and starvation

Lactococcus lactis species can survive periods of carbohydrate starvation for relatively long periods of time. In the first hours of starvation, however, the maximal glycolytic and arginine deiminase (ADI) pathway activities decline rapidly. The rate of decrease of the pathway activities diminishes as soon as the cells become depleted of energy-rich intermediates. Loss of glycolytic activity is associated with loss of glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase and pyruvate kinase activities. Upon addition of sugar to starved cultures these enzymatic, and thus the glycolytic, activities can be restored to 100% values. The recovery of enzymatic activities is inhibited by chloramphenicol, indicating that protein synthesis is involved. In contrast, restoration of ADI pathway activity does not require de novo synthesis of proteins. General protein degradation and synthesis have been studied in growing and starving cells using [³⁵S]methionine-labeling of proteins and two-dimensional gel analysis. The breakdown of bulk proteins in exponentially growing cells shows first-order rate kinetics (t1/2 of approximately 5 h). Following an initial breakdown of proteins with a t1/2 of 5 h during the first hour(s) of starvation, bulk proteins are degraded very slowly in starving energy-depleted cells. The breakdown of proteins during starvation appears to be (largely) nonspecific. The rate of synthesis of proteins decreases rapidly in the first hour(s) of starvation. From the onset of starvation on at least 45 proteins are no longer synthesized. During starvation relative induction of fourteen to fifteen proteins can be observed.Abbreviations ADI Arginine deiminase - ATP adenosine triphosphate - PEP phosphoenolpyruvate - membrane potential - pH pH gradient - PTS sugar phosphotransferase system - CDM chemically defined medium - TCA trichloro-acetic acid     

Interactions between density-dependent processes, population dynamics and control of an invasive plant species, Tripleurospermum perforatum (scentless chamomile)

Tripleurospermum perforatum is an invasive weedy species which exhibits strong over-compensating density dependence. Interactions between density-dependent survival, probability of flowering and fecundity were modelled and their impact on the population dynamics were examined. When only fecundity was density-dependent, the dynamics were similar to those observed in the model containing all three density-dependent terms. Density-dependent survival was a stabilizing process when acting in combination with density-dependent fecundity and probability of flowering; removing density-dependent survival from the model produced two-point cycles. The addition of a seed bank was also stabilizing. Simulations of control strategies at different life-history stages indicated that full control would be difficult due to the strong over-compensating density dependence, with severe reductions in fecundity and late season survival necessary in order to reduce equilibrium seed density and biomass.     

Evolution of density-dependent dispersal in a structured metapopulation

We study the evolution of density-dependent dispersal in a structured metapopulation subject to local catastrophes that eradicate local populations. To this end we use the theory of structured metapopulation dynamics and the theory of adaptive dynamics.The set of evolutionarily possible dispersal functions (i.e., emigration rates as a function of the local population density) is derived mechanistically from an underlying resource-consumer model. The local resource dynamics is of a flow-culture type and consumers leave a local population with a constant probability per unit of time κ when searching for resources but not when handling resources (i.e., eating and digesting). The time an individual spends searching (as opposed to handling) depends on the local resource density, which in turn depends on the local consumer density, and so the average per capita emigration rate depends on the local consumer density as well.The derived emigration rates are sigmoid functions of local consumer population density. The parameters of the local resource-consumer dynamics are subject to evolution. In particular, we find that there exists a unique evolutionarily stable and attracting dispersal rate κ^∗ for searching consumers. The κ^∗ increases with local resource productivity and decreases with resource decay rate. The κ^∗ also increases with the survival probability during dispersal, but as a function of the catastrophe rate it reaches a maximum before dropping off to zero again.     

Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (cytochrome c, azurin, myoglobin) reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. Rates of Fe²⁺→Ru³⁺ ET reactions in cytochrome c decay exponentially with tunneling-pathway length (decay constant 0.73?Å^–1); these rates also decay exponentially with Ru-Fe distance (decay constant 1.1?Å^–1). In azurin, a β-sheet protein, Cu⁺→Ru³⁺ rates exhibit an exponential Cu-Ru distance dependence with a decay constant of 1.1?Å^–1. Comparison of distant couplings in azurin and myoglobin suggests that hydrogen bonds are better mediators across β sheets than through α helices.     

The specific phosphorylation of a 40S ribosomal protein in growth-arrested tetrahymena is induced by sodium

In the past few years, in vivo phosphorylation of ribosomal proteins has been the subject of extensive studies and the results have shown that reversible phosphorylation of small subunit ribosomal protein S6, ubiquitous in eukaryotic cells, is apparently related to regulation of protein synthesis initiation. Thus the level of protein synthesis under various conditions is correlated with the level of S6 phosphorylation. In exponentially growing Tetrahymena, however, such phosphorylation does not occur, but when these cells are transferred to starvation buffers, the rate of protein synthesis is drastically reduced and a 40S ribosomal protein analogous to S6 of higher eukaryotic cells is fully and rapidly phosphorylated in all the ribosomes. We have studied the conditions which lead to this phosphorylation in growth-arrested Tetrahymena, in order to understand the physiological significance of this process. Our results show that there is no obvious correlation between this phosphorylation and starvation. Moreover, it is not a developmentally regulated process related to the conjugation cycle, but a modification induced by the presence of sodium ions or high concentration of Tris in the starvation buffer. The physiological significance of this process is discussed in terms of accumulation of negative charge density probably required for initiation of protein synthesis in the growth-arrested cells starving in Na+-containing buffers.     

Soil bacterial DNA and biovolume profiles measured by flow-cytometry

Abstract Flow-cytometry was used to measure cell volumes and DNA contents of single cells in cultures of soil bacteria during exponential growth and starvation conditions. DNA was measured after staining with mitramycin/ethidium bromide. The measurement of DNA was calibrated with rifampicin-treated cells of E. coli containing even numbers of genomes per cell. Cell volumes were assessed by scatter light measurements. Constant DNA to biovolume relations over a range of cell sizes were found for each of the bacteria at exponential growth, and DNA contents per cell varied over a range equivalent to 1–4 genomes per cell. At generation times of 1.0–1.5 h, two genomes were registered as a mean. After starvation of washed cells in a salt solution (24 hrs), a fraction of the cells in each culture had DNA contents equivalent to 1 genome, but significant fractions retained DNA contents equivalent to 2–4 genomes. Attempts to create cells with even numbers of genomes per cell by treatment with rifampicin was successful on an Acinetobacter sp. In contrast, the response to rifampicin was less clear for Pseudomonas fluorescens and P. chlororaphis , and unclear for the gram positive bacteria isolated from soil. The mean decrease in biovolume upon starvation was 4.1 times (range 1.3–8.1 times) and larger than the mean decrease in DNA content of 1.8 (range 1.3–2.7 times). Cell volume determinations by measurements of scatter light was compared with volume determinations by fluorescence microscopy. The amounts of scatter light per volumes was variable, not only did we find large differences between bacterial types, but also between starving and exponentially growing cells of the same isolate. In order to use light scatter as a measure of biovolume, internal standards has to be chosen of comparable size and surface properties as to soil bacteria.     

Cell division after inhibition of chromosome replication in Escherichia coli

The residual cell divisions after thymine starvation of exponential cultures of TJK16, a thymine-requiring derivative of Escherichia coli B/r, were evaluated. The results indicate that under the conditions used (glucose minimal medium 37 °), (1) only cells that had terminated a round of replication divided; (2) once termination had occurred, thymine starvation and replication no longer affected the time of cell division; (3) synchronously terminating subpopulations of cells began to divide about 17 min after termination; after that time, the rate of division decreased exponentially. The results confirm the previously inferred asymmetric distribution of D-periods in an exponential population of E. coli bacteria and suggest that an event associated with termination of replication is required for cell division. The method of data evaluation presented can be used to determine the duration of the D-period and to find the parameter values (halflife and onset) of the stochastic phase of the D-period in exponential cultures, eliminating the need for synchronization procedures.     

Prediction of X-ray induced mitotic delay and recovery of G2 cells

Abstract. A mathematical model is presented that predicts the delay of mitosis caused by X-irradiation of an asynchronous, exponentially growing cell culture (data of Schneiderman & Schneiderman, 1984). In the model, based on Gompertz kinetics, the driving function to generate the curves is a simple exponential decay expression. For the delayed mitotic progress curves, this function characterizes the distribution of the time required for cells to enter mitosis.     

Prediction of X-ray induced mitotic delay and recovery of G2 cells

A mathematical model is presented that predicts the delay of mitosis caused by X-irradiation of an asynchronous, exponentially growing cell culture (data of Schneiderman & Schneiderman, 1984). In the model, based on Gompertz kinetics, the driving function to generate the curves is a simple exponential decay expression. For the delayed mitotic progress curves, this function characterizes the distribution of the time required for cells to enter mitosis.     

Phospholipase D controls Dictyostelium development by regulating G protein signaling

Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2βγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gβγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling.     

Density-dependent effects in a parasitic nematode,Elaphostrongylus rangiferi,in the snnail intermediate host

Summary Density-dependent effects in Elaphostrongylus rangiferi, a parasitic nematode in the CNS and muscular system of reindeer, were studied in a laboratory population of the snail intermediate host, Arianta arbustorum. The rates in parasite growth, development and mortality were all affected by parasite density. The effects on growth and development were, however, much more marked, than the effect on mortality.All density-dependent rates were intensified by decreasing snail size, and by snail starvation. The snail host showed marked tissue reactions against infection, and the intensity of these reactions increased with increasing parasite density. The mechanism behind the observed density-dependent rates is discussed, and is tentatively concluded to be competition for nutritive substances in the host tissue.The importance of a density-dependent developmental rate in natural populations of this parasite is discussed, and it is hypothesized that this effect may counteract the strong temperature-dependent developmental rate of E. rangiferi In a more general context it is pointed out that density-dependent developmental rates, although common amongst animal populations, has been neglected in models of population dynamics. Developmental rates are usually represented by a constant time lag in such models, but should be treated as a density-dependent variable.