Investigations of the influence of carbon starvation on the carbohydrate storage compounds (trehalose, glycogen), viability, adenylate pool, and adenylate energy charge in Cellulomonas sp. (DSM20108)

During conditions of energy and carbon excess Cellulomonas sp. accumulates intracellularly two different carbohydrate storage products in different relative concentrations: trehalose and glycogen. During carbon starvation these compounds are degraded at different rates and are therefore characterized metabolically by different half-life periods (glycogen 1.6 h, trehalose 34 h). Other parameters which bear some relation to viability during conditions of stress are compared with these half-life periods. The half-life period of the adenylate energy charge EC_A (52 h) is similar to the trehalose half-life period, and it is concluded that it is trehalose which is essential for long-term survival while glycogen is used in the very early stages of carbon starvation to produce energy for metabolism under these conditions. Evidence is presented that two mechanisms are active for the stabilization of the intracellular adenylate energy charge: specific excretion and adenylate degradation.     

Aerobic and Anaerobic Starvation Metabolism in Methanotrophic Bacteria

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

Starvation of marine flounder, squid and laboratory mice and its effect on the intestinal microbiota

Abstract Starvation processes of microorganisms in natural ecosystems were studied, both in vivo and in vitro, using marine animals (flounder and squid) and laboratory mice. In flounder, starvation resulted in the mixed intestinal microbiota maintaining its viability but decreasing its cell volume. It was also observed that during starvation there was an increased liability for adhesion of th microbiota to both the oil-water interface in the hexadecane water separation technique, and to Sepharose beads with either exposed hydrophobic or cationic charge groups. The population also exhibited the capacity to respond immediately to the addition of nutrients. When the flounder microbiota was starved in vitro the ratio of bacteria cultured on high nutrient: low nutrient media decreased with time of starvation. A similar effect on the intestinal microbiota of squid was observed in vivo when the animals were starved. The in vivo starvation of the mouse also produced a decrease in the mean bacterial cell volume which was concurrrent with a promotion of coliform bacteria. A coliform isolate exhibited similar starvation survival characteristics in vitro. From the data obtained from the flounder, squid and mice, it was concluded that components of the large intestinal microbiota exhibited the starvation survival characteristics previously reported for laboratory studies of planktonic bacteria, when exposed to energy- or nutrient-limited conditions.     

Poly-3-hydroxybutyratc production and changes of bacterial community in the soil

Changes in the number of bacteria and relative distribution of strains producing poly-3-hydroxybutyrate (PHB) in soil were investigated. Samples of chernozem soil were cul tivated with glucose in the presence of a mineral nitrogen source (diammonium hydrogen phos phate) or in its absence, either in a batch or a heterocontinuous cultivation system. In both cultivation systems the addition of glucose resulted in a roughly ten-fold increase of bacteria concentration and an increase in the proportion of strains able to produce PHB granules. When the nitrogen source was added simultaneously with glucose, the concentration of bacteria increased by two orders of magnitude in both cultivation systems. In the batch system changes in the concentration of strains capable of PHB production were very small under these con ditions whereas in the heterocontinuous system their number decreased by almost 50 %. The survival of bacteria in the soil suspension after 57-d starvation was associated with PHB production which differed, depending on the previous treatment of the soil samples. The concentration of bacteria decreased least pronouncedly in the control with water and most significantly during cultivation with glucose and a nitrogen source, where the initial PHB content was very low in spite of high numbers of bacteria.     

Intracellular cAMP in Dictyostelium discoideum: Characterization of an aggregation-deficient mutant with elevated cAMP pools

Forty aggregation-deficient mutants of Dictyostelium discoideum were screened for changes in intracellular cAMP during the first 10 hr of starvation. The pools in 39 of the mutants remained low and relatively static during this period. However, amoebae of one mutant, strain HC151, exhibited significantly elevated levels of intracellular cAMP during vegetative growth and for several hours after starvation. A more detailed analysis of this mutant indicated that the elevated cAMP pools in these cells are a consequence of the premature appearance and partial activation of an adenylate cyclase. The mutation(s) altering adenylate cyclase regulation in this strain appears to map in linkage group IV. Complementation tests between strain HC151 and another mutant, HH201, which has recently been shown to produce an adenylate cyclase activity precociously [1], indicated that the mutations affecting adenylate cyclase activity in these strains map at different loci. Although both of these mutations behave recessively in heterozygous diploids with respect to gross development, an examination of early cAMP metabolism and terminal spore differentiation in these diploids suggest that these mutations are at least partially expressed during some stage(s) of the developmental cycle.     

Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia

The carbon that rhizobia in root nodules receive from their host powers both N₂ fixation, which mainly benefits the host, and rhizobium reproduction. Rhizobia also store energy in the lipid poly-3-hydroxybutyrate (PHB), which may enhance rhizobium survival when they are carbon limited, either in nodules or in the soil between hosts. There can be a conflict of interest between rhizobia and legumes over the rate of PHB accumulation, due to a metabolic tradeoff between N₂ fixation and PHB accumulation. To quantify the benefits of PHB to carbon-limited rhizobia, populations of genetically uniform rhizobia with high vs. low PHB (confirmed by flow cytometry) were generated by fractionating Sinorhizobium meliloti via density gradient centrifugation, and also by harvesting cells at early vs. late stationary phase. These rhizobia were starved for 165 days. PHB use during starvation was highly predictive of both initial reproduction and long-term population maintenance. Cultured S. meliloti accumulated enough PHB to triple their initial population size when starved, and to persist for c . 150 days before the population fell below its initial value. During the first 21 days of nodule growth, undifferentiated S. meliloti within alfalfa nodules accumulated enough PHB to support significant increases in reproduction and survival during starvation.     

Poly-β-hydroxybutyrate metabolism in Azospirillum brasilense and the ecological role of PHB in the rhizosphere

Abstract Azospirillum brasilense is a rhizosphere microorganism which has potential use for promoting plant growth in economically important crops. Its ability to survive the adverse conditions imposed by nutrient starvation and competition in the rhizosphere is of great importance. A. brasilense accumulates up to 70% of its cell dry weight with poly-β-hydroxybutyrate (PHB). In the presence of stress factors such as ultraviolet radiation, desiccation and osmotic stress, PHB-rich cells survived better than PHB-poor cells. Polymer-rich cells of Azospirillum fixed N₂ in the absence of exogenous carbon and combined nitrogen. The enzymes of the PHB cycle in both the synthesis and degradation processes as well as during starvation were more active in PHB-rich cells. After 24 h of starvation there was a peak of activity of d (−)β-hydroxybutyrate dehydrogenase, β-ketothiolase and thiophorase due to PHB degradation. Additionally, acetoacetyl-CoA reductase dropped to a minimum level because PHB could not be synthesized. The possible utilization of PHB as a sole carbon and energy source by A. brasilense and other bacteria during establishment, proliferation and survival in the rhizosphere will be discussed.     

Chemical changes in cell envelope and poly-β-hydroxybutyrate during short term starvation of a marine bacterial isolate

Qualitative and quantitative changes were observed in lipids, poly--hydroxybutyrate (PHB), and a cell wall peptidoglycan consitutent in a marine bacterial isolate during starvation for 24 h in an energy and nutrient-free medium. While the amount and composition of the membrane fatty acids fluctuated within the first hours of starvation, the total amount of fatty acids decreased during the starvation period. Furthermore, the ratio of monounsaturated to saturated fatty acids decreased and the proportion of short chain fatty acids increased. In the very early phase of starvation the bacteria contained PHB, which had been accumulated during the growth phase, but after 3 h no PHB was detected. Cells starved for phosphorus showed a different pattern as PHB was initially accumulated and did not decrease until 5 h of starvation. Synthesis of the cell wall amino acid d-alanine was initiated during the first phase of starvation. The effects of these changes on membrane fluidity and uptake of substrates as well as the use of fatty acids and PHB as energy resources during starvation are discussed.Non-common abbreviations FID flame ionization detector - GC gas chromatography - HFBA heptafluorobutyric anhydride - MS mass spectrometry - NSS nine salt solution - PHB poly--hydroxybutyrate - PFB pentafluorobenzylbromide     

Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium.

When an Escherichia coli K-12 culture was starved for glucose, 50% of the cells lost viability in about 6 days. When a K-12 mutant lacking five distinct peptidase activities, CM89, was starved in the same manner, viability was lost much more rapidly; 50% of the cells lost viability in about 2 days, whereas a parent strain lacking only one peptidase activity lost 50% viability in about 4 days. Compared with the wild-type strain and with its parent strain CM17, CM89 was defective in both protein degradation and protein synthesis during carbon starvation. Similar results were obtained with glucose-starved Salmonella typhimurium LT2 and LT2-derived mutants lacking various peptidase activities. An S. typhimurium mutant lacking four peptidases, TN852, which was deficient in both protein degradation and synthesis during carbon starvation (Yen et al., J. Mol. Biol. 143:21-33, 1980), was roughly one-third as stable as the isogenic wild type. Isogenic S. typhimurium strains that lacked various combinations of three of four peptidases and that displayed protein degradation and synthesis rates intermediate between those of LT2 and TN852 (Yen et al., J. Mol. Biol. 143:21-33, 1980) displayed corresponding stabilities during carbon starvation. These results point to a role for protein degradation in the survival of bacteria during starvation for carbon.     

Poly-3-Hydroxybutyrate in Legionella pneumophila, an Energy Source for Survival in Low-Nutrient Environments

Chloroform-soluble material was extracted from two strains of L. pneumophila serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited culture and was inversely related to the concentration of iron metabolized. Chemostat-grown cells survived in a culturable state for at least 600 days when incubated at 24°C in a low-nutrient tap water environment. Nile red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PHB utilization, as revealed by the decline in mean cellular fluorescence and cell complexity, correlated with loss of culturability. Fluorescence microscopy provided visual evidence of PHB utilization, with a marked reduction in the number of Nile red-stained granules during starvation. Heat shock treatment failed to resuscitate nonculturable cells. This study demonstrates that L. pneumophila accumulates significant intracellular reserves of PHB, which promote its long-term survival under conditions of starvation.     

富养罗尔斯通氏菌菌株PHB"泄漏"机理的初探

建立了一种分离纯化聚羟基丁酸(Polyhydroxybutyrate,PHB)颗粒的改良方法。采用这种方法从Ralstonia eutropha菌株H16(野生型)、SK1489(Tn5诱变的PHB泄漏菌株)、JMP222(野生的PHB泄漏菌株)分离了PHB颗粒。进一步比较研究了不同菌株的PHB解聚酶和3-羟基丁酸脱氢酶的活性。研究结果表明,菌株SK1489的PHB解聚酶活性(48h培养后达1．82U／mg)明显高于野生型菌株H16(48h培养后达0．37U／mg),菌株JMP222的3-羟基丁酸脱氢酶活性(培养96h后达165．9U／mg)比菌株H16培养(96h后达64．0U／mg)高许多。这些结果显示,不同菌株PHB的泄漏有不同的原因,突变株SK1489导致PHB泄漏的原因是解聚酶活性高,而野生型JMP222PHB泄漏的原因主要是3-羟基丁酸脱氢酶活性高。     

The stringent response plays a key role in Bacillus subtilis survival of fatty acid starvation

The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram‐negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram‐positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp‐synthetases (Rel_Bs, RelP and RelQ) or bearing a Rel_Bs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.     

Adenylate Energy Charge in Escherichia coli During Growth and Starvation

The value of the adenylate energy charge, [(adenosine triphosphate) + (1/2) (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types.     

Physiological and structural effects of phosphorus starvation on the unicellular green alga Scenedesmus

The effects of phosphorus starvation on morphology and intracellular structure and on reactions related to the energy metabolism of the unicellular green alga Scenedesmus obtusiusculus (Chod.) were studied over a period of 96 h by employing transmission electron microscopy and various methods for measurement of physiological reactions. Increase in cell size and shape and in cell wall thickness are dominating features of phosphorus starvation. There is also an increase in number and size of starch granules and lipid globules and the internal structure of the cells appears successively disorganized. Shortage of phosphorus in the medium initially induces an increase of the adenylate pool whereas the energy charge value remains the same as for the controls. The photosynthetic and respiratory activities are high during incipient phosphorus starvation. After 24 h, as shortage of phosphorus becomes critical, the internal phosphorus reaches a low steady-state value, and this is also true for the adenylate energy charge. The total content of adenylates, however, peaks after 24 h of starvation and then decreases with increasing length of phosphorus starvation. Light-induced oxygen evolution appears not to be as much inhibited by a low phosphorus content in the cells as by the concomitant starch accumulation. The data indicate that the strategy for survival of the cells in a phosphorus-poor environment includes morphological and physiological changes that facilitate the transfer and adaption of the cells to environments with a more favourable supply of phosphorus, such as the often oxygen-poor but phosphorus-rich bottom zones.     

Diverse morphological types of dormant cells and conditions for their formation in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

Differences in generation of dormant forms (DF) were revealed between two strains of non-sporeforming gram-negative bacteria Azospirillum brasilense, Sp7 (non-endophytic) and Sp245 (endophytic strain). In post-stationary ageing bacterial cultures grown in a synthetic medium with a fivefold decreased initial nitrogen content, strain Sp7 formed two types of cyst-like resting cells (CRC). Strain Sp245 did not form such types of DF under the same conditions. CRC of the first type were formed in strain Sp245 only under phosphorus deficiency (C > P). The endophytic strain was also shown to form structurally differentiated cells under complete starvation, i.e. at a transfer of early stationary cultures, grown in the media with C > N unbalance, to saline solution (pH 7.2). These DF had a complex structure similar to that of azotobacter cysts. The CRC, which are generated by both azospirilla strains and belong to distinct morphological types, possessed the following major features: absence of division; specific ultrastructural organization; long-term maintenance of viability (for 4 months and more); higher heat resistance (50–60°C, 10 min) as compared with vegetative cells, i.e. the important criteria for dormant prokaryotic forms. However, CRC of non-endophytic strain Sp7 had higher heat resistance (50, 55, 60°C). The viability maintenance and the portion of heat-resistant cells depended on the conditions of maturation and storage of CRC populations. Long-term storage (for 4 months and more) of azospirilla DF populations at ?20°C was optimal for maintenance of their colony-forming ability (57% of the CFU number in stationary cultures), whereas the largest percentage of heat-resistant cells was in CRC suspensions incubated in a spent culture medium (but not in saline solution) at room temperature. The data on the intraspecies diversity of azospirilla DF demonstrate the relation between certain type DF formation to the type of interaction (non-endophytic or endophytic) with the plant partner and provide more insight into the adaptation mechanisms that ensure the survival of gram-negative non-spore-forming bacteria in nature.     

Physiological Properties of Nitrogen-scavenging Bacteria from the Marine Environment

Nine selected strains of marine bacteria from the marine environment, although taxonomically heterogeneous in character, exhibited a capacity for prolonged growth on 'nitrogen-free' media; only one strain, a Klebsiella sp., fixed dinitrogen under any of a considerable range of test conditions. The non-nitrogen-fixing bacteria were able to scavenge low levels of nitrogenous materials, principally ammonia, from the atmosphere. Contrary to previous suggestions, this growth did not have an abnormally low cellular protein and nitrogen content. Some strains with apparently low intracellular protein contents, as determined on a cellular dry weight basis, showed accumulations of carbonaceous storage products which distorted the cytochemical analyses. Representative strains grown in chemostat culture showed a high affinity (i.e. low Ks values—0.35–0.52 μmol/1) for the NH₄+ ion, compared with Escherichia coli (1.75 μmol/1) and preliminary studies of their glutamine synthetases suggested that the affinities ( K_m values—0.25–0.29 μmol/1) of this enzyme for hydroxylamine were similar to the few values reported for other marine bacteria.     

Changes in adenine nucleotides and energy charge in isolated winter wheat cells during low temperature stress

Adenylate energy charge (AEC) and adenine nucleotide levels of isolated winter wheat (Triticum aestivum L. cv Kharkov 22 MC) cells exposed to various low temperature stresses were determined. During ice encasement at −1°C, nucleotide levels decreased gradually in approximate relation to a decline in cell viability. AEC values remained high even after 5 weeks of icing when cell viability was severely reduced. When isolated cell suspensions were exposed to various cooling and freezing regimes ranging from −10 to −30°C, cell damage was dependent on the minimum temperature imposed and the duration of exposure to the freezing stress. The levels of all three adenine nucleotides declined with increasing severity of the imposed stress, but AEC values remained high even at −30°C when nearly all of the cells were killed. The addition of 10 millimolar Ca²⁺ to cell suspensions enhanced survival during low temperature stresses, but did not influence nucleotide levels other than through its effect on cell viability. These results indicate that impairment of the ion transport system during the early stages of ice encasement prior to a detectable decline in cell viability cannot be attributed to changes in the adenylate energy charge system of the cell.     

Changes in Protein Composition of Three Bacterial Isolates from Marine Waters during Short Periods of Energy and Nutrient Deprivation

Two-dimensional gel electrophoresis analysis of and total cell protein determination for three bacterial isolates from marine waters at the onset and after 24 h of energy and nutrient deprivation demonstrated that the three isolates exhibited different pathways of starvation survival. Two strains appeared to synthesize new proteins during starvation.     

Mutational Analysis of Dark Endogenous Metabolism in the Blue-Green Bacterium Anacystis nidulans

We describe a mutant (strain 704) of the obligate photoautotroph Anacystis nidulans which behaves like the wild type under continuous illumination but which in the dark rapidly loses viability, respires little, and incorporates label into ribonucleic acid and protein at rates considerably less than observed with the darkened wild type. Extracts of this mutant strain show no detectable 6-phosphogluconate dehydrogenase (EC 1.1.1.44) activity. Spontaneous revertants of mutant 704 were selected as survivors of prolonged incubation in darkness. Of 10 such strains examined, none had regained 6-phosphogluconate dehydrogenase activity, and all had lost detectable glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity. Although dark survival of these revertants paralleled that of the wild type, rates of dark endogenous respiration and incorporation of labeled precursors into ribonucleic acid were still very low, comparable to those observed with strain 704. These results are consistent with the following hypotheses concerning dark endogenous metabolism in unicellular blue-green bacteria. (i) Although the oxidative pentose phosphate cycle (hexose monophosphate shunt) may play a major role in endogenous metabolism in A. nidulans, as proposed by others, it is not the only pathway capable of providing energy for maintenance of viability in darkness. (ii) Much of the endogenous metabolic activity (respiration and macromolecular synthesis) observed in darkened cultures of wild-type A. nidulans is not required for survival alone, and must therefore serve other functions.     

Functional capacities and the adenylate energy charge in Escherichia coli under conditions of nutritional stress.

Functional capacities in Escherichia coli cells starved for glucose were examined by comparing protein synthesis, utilization of new substrates, and maintenance of viability with the adenylate energy charge of the culture. When growth ceased because of glucose exhaustion in an E. coli culture, the energy charge dropped from 0.90 to about 0.80. During this time, the viable-cell count and the capacity for protein synthesis and for induction of new enzymes were maintained only if other substrates were available in the medium. The culture could be maintained for many hours without growth or death if glucose was added slowly; the energy charge in this case stabilized at about 0.80. A consistent transient decrease in the energy charge to around 0.80, accompanied by a decrease in protein synthesis, was also observed during the adaptation from glucose to other substrates during diauxic growth on glucose and glycerol or lactose.