The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli.

KatF is required for the expression of some 32 carbon starvation proteins in Escherichia coli including 6 previously identified as Pex. Mutants with the katF gene survive carbon and nitrogen starvation poorly. Many of the KatF-regulated starvation proteins are common to those induced by other stresses, and the mutant failed to develop starvation-mediated cross protection to osmotic, oxidative, and heat stresses. Furthermore, thermal resistance was not induced in the mutant by heat preadaptation, and it exhibited an altered pattern of protein synthesis at elevated temperature. Thus, KatF is a major switch that controls the starvation-mediated resistant state in E. coli.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.     

Effect of DnaK and DnaJ proteins deprivation on Escherichia coli response to starvation

E. coli defects in response to nutritional starvation caused by DnaK and DnaJ proteins deprivation are examined. The ability of delta dnaKdnaJ mutant to survive carbon, nitrogen and phosphorus starvation is highly impaired while delta dnaJ mutant is characterized by the diminished survival of phosphorus starvation only. delta dnaKdnaJ mutant grows slowly utilizing maltose and glycerol and delta dnaJ mutant utilizes glycerol inefficiently. The growth on alternate nitrogen sources is comparable to wild-type strain.     

The heat shock gene, htpG, and thermotolerance in the cyanobacterium, Synechocystis sp. PCC 6803

The htpG null mutant was obtained by inserting a chloramphenicol resistance cassette (Cm(r)) in the htpG coding sequence. The htpG null mutant (delta htpG), delta hsp16.6, and the double mutant, delta htpG::hsp16.6 cells showed little growth disadvantage at 30 degrees C and 37 degrees C, but not at 40 degrees C. This suggests that HtpG and HSP16.6 proteins do not have an essential role during growth at normal and mildly elevated temperatures. Cell growth, cell survival rate, and oxygen electrode measurements demonstrated that delta htpG, delta hsp16.6, and delta htpG::hsp16.6 cells were sensitive to heat stress. Decreased basal and acquired thermotolerance was observed when mutants were heat shocked, with delta htpG::hsp16.6 being the most sensitive. A comparison of mutants showed that delta hsp16.6 was more sensitive to heat shock than delta htpG.     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).     

The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival

Proteins of the glucose-starvation stimulon were identified by using two-dimensional gel electrophoresis and the gene–protein database of Escherichia coli. Members of this stimulon Included enzymes of the Embden–Meyerhof–Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetytase. The synthesis of these enzymes was found to be Induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle. Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, In part, similar to the response of cells shifted to anaerobiosis. These modulations suggest that the glucose-starved cell increases the relative flow of carbon through the Pta–AckA pathway. Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose-starved cells as pta and pta–ackA double mutants were found to be impaired in their ability to survive glucose starvation. The survival characteristics of ackA mutants and the wild-type parent were indistinguishable. Moreover, the pta mutant failed to induce several proteins of the glucose-starvation stimulon.     

Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content.

The physiology of Pseudomonas putida KT2442 with respect to growth and carbon starvation was studied. During the transition from growth to nongrowth, the cell shape changes from cylindrical to spheric, a change which is accompanied by reductions in cell size, DNA and ribosome content, and the rate of total protein synthesis. In addition, a pattern of general cross-protection develops, which enables the cells to survive environmental stresses such as high and low temperatures, elevated osmolarity, solvents, and oxidative agents. Cultures are almost fully viable during 1 month of carbon, nitrogen, and multiple-nutrient starvation and are considered to be in an active nondormant state. In contrast, strain KT2442 does not survive well under conditions of sulfate and phosphate starvation.     

A heat shock-resistant mutant of Saccharomyces cerevisiae shows constitutive synthesis of two heat shock proteins and altered growth

A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10(-7) from a culture treated with ethyl methane sulfonate. Cells of the mutant are approximately 1,000-fold more resistant to lethal heat shock than those of the parental strain. Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio 2+:2- from heterozygotes constructed with the wild-type strain of the opposite mating type, and are, therefore, attributed to a single nuclear mutation. The mutated gene in the mutant was herein designated hsr1 (heat shock response). The hsr1 allele is recessive to the HSR1+ allele of the wild-type strain. Exponentially growing cells of hsr1 mutant were found to constitutively synthesize six proteins that are not synthesized or are synthesized at reduced rates in HSR1+ cells unless appropriately induced. These proteins include one hsp/G0-protein (hsp48A), one hsp (hsp48B), and two G0-proteins (p73, p56). Heterozygous diploid (hsr1/HSR1+) cells do not synthesize the proteins constitutively induced in hsr1 cells, which suggests that the product of the HSR1 gene might negatively regulate the synthesis of these proteins. The hsr1 mutation also led to altered growth of the mutant cells. The mutation elongated the duration of G1 period in the cell cycle and affected both growth arrest by sulfur starvation and growth recovery from it. We discuss the problem of which protein(s) among those constitutively expressed in growing cells of the hsr1 mutant is responsible for heat shock resistance and alterations in the growth control.     

Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Starvation proteins in Escherichia coli: kinetics of synthesis and role in starvation survival

Starvation proteins synthesized by Escherichia coli at the onset of carbon starvation (R. G. Groat and A. Matin, J. Indust. Microbiol. 1:69-73, 1986) exhibited four temporal classes of synthesis in response to glucose or succinate starvation, indicating sequential expression of carbon starvation response (cst) genes. A cst mutant of E. coli showed greatly impaired carbon starvation survival. Thus, it appears that E. coli undergoes a significant molecular realignment in response to starvation, which increases its resistance to this stress. New polypeptides were also synthesized by E. coli in response to phosphate or nitrogen starvation. Some of these polypeptides were unique to a given starvation regimen, but at least 13 appeared to be synthesized regardless of the nutrient deprivation causing the starvation.     

An essential role for the Escherichia coli DnaK protein in starvation-induced thermotolerance, H2O2 resistance, and reductive division.

During a 3-day period, glucose starvation of wild-type Escherichia coli produced thermotolerant, H2O2-resistant, small cells with a round morphology. These cells contained elevated levels of the DnaK protein, adjusted either for total protein or on a per-cell basis. Immunoprecipitation of [35S]methionine-labeled protein produced by such starving cells demonstrated that DnaK underwent continuous synthesis but at decreasing rates throughout this time. Glucose resupplementation of starving cells resulted in rapid loss of thermotolerance, H2O2 resistance, and the elevated DnaK levels. A dnaK deletion mutant, but not an otherwise isogenic wild-type strain, failed to develop starvation-induced thermotolerance or H2O2 resistance. The filamentous phenotype associated with DnaK deficiency was suppressed by cultivation in a defined glucose medium. When starved for glucose, the nonfilamentous and rod-shaped dnaK mutant strain failed to convert into the small spherical form typical of starving wild-type cells. The dnaK mutant retained the ability to develop adaptive H2O2 resistance during growth but not adaptive resistance to heat. Complementation of DnaK deficiency by using Ptac-regulated dnaK+ and dnaK+J+ expression plasmids confirmed a specific role for the DnaK molecular chaperone in these starvation-induced phenotypes.     

RCB-mediated chlorophagy caused by oversupply of nitrogen suppresses phosphate-starvation stress in plants

Inorganic phosphate (Pi) and nitrogen (N) are essential nutrients for plant growth. We found that a five-fold oversupply of nitrate rescues Arabidopsis (Arabidopsis thaliana) plants from Pi-starvation stress. Analyses of transgenic plants that overexpressed GFP-AUTOPHAGY8 showed that an oversupply of nitrate induced autophagy flux under Pi-depleted conditions. Expression of DIN6 and DIN10, the carbon (C) starvation-responsive genes, was upregulated when nitrate was oversupplied under Pi starvation, which suggested that the plants recognized the oversupply of nitrate as C starvation stress because of the reduction in the C/N ratio. Indeed, formation of Rubisco-containing bodies (RCBs), which contain chloroplast stroma and are induced by C starvation, was enhanced when nitrate was oversupplied under Pi starvation. Moreover, autophagy-deficient mutants did not release Pi (unlike wild-type plants), exhibited no RCB accumulation inside vacuoles, and were hypersensitive to Pi starvation, indicating that RCB-mediated chlorophagy is involved in Pi starvation tolerance. Thus, our results showed that the Arabidopsis response to Pi starvation is closely linked with N and C availability and that autophagy is a key factor that controls plant growth under Pi starvation.

Disturbance of the carbon/nitrogen ratio induces partial chloroplast degradation via autophagy under phosphate starvation and rescues phosphate starvation stress.     

Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli.

The intracellular nucleotide pool of Escherichia coli W3110 reproducibly changes from conditions of growth in phosphate excess to phosphate starvation, with at least two nucleotides appearing under starvation conditions and two nucleotides appearing only under excess phosphate conditions. Strains bearing a deletion of the phoA gene show the same pattern, indicating that dephosphorylation by alkaline phosphatase is not responsible for the changes. Strains with mutations in the phoU gene, which result in constitutive expression of the pho regulon, show the nucleotide pattern of phosphate-starved cells even during phosphate excess growth. These changes in nucleotides are therefore due to phoU mutation but not to alkaline phosphatase constitutivity. In fact, a phoR (phoR68) mutant strain has the patterns of the wild type in spite of being constitutive for alkaline phosphatase. That these nucleotides might be specific signals for pho regulon expression was supported by the fact that the two nucleotides appearing under phosphate starvation induced the synthesis of alkaline phosphatase in repressed permeabilized wild-type cells under conditions of phosphate excess.     

Comparative proteome analyses of phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant reveal root characteristics associated with phosphorus efficiency

SUMMARY: Low phosphorus (P) availability is a major limitation for plant growth. To better understand the molecular mechanism of P efficiency in maize, comparative proteome analyses were performed on the roots of the low-P-tolerant mutant 99038 and wild-type Qi-319 grown under P-sufficient (+P) or P-deficient (-P) conditions. Over 10% of proteins detected on two-dimensional electrophoresis (2-DE) gels showed expression that was altered twofold or more between the genotypes under +P or -P conditions. We identified 73 (+P) and 95 (-P) differentially expressed proteins in response to phosphate (Pi) starvation. These proteins were involved in a large number of cellular and metabolic processes, with an obvious functional skew toward carbon metabolism and regulation of cell proliferation. Further analysis of proteome data, physiological measurements and cell morphological observations showed that, compared to the wild-type, the low-P-tolerant mutant could accumulate and secrete more citrate under Pi starvation, which facilitates solubilization of soil Pi and enhances Pi absorption. The proportion of sucrose in the total soluble sugars of the low-P-tolerant mutant was significantly higher, and cell proliferation in root meristem was accelerated. This resulted in better developed roots and more advantageous root morphology for Pi uptake. These results indicate that differences in citrate secretion, sugar metabolism and root-cell proliferation are the main reasons for higher tolerance to low-P conditions in the mutant compared to the wild-type. Thus, the mutant displayed specialized P-efficient root systems with a higher capacity for mobilization of external Pi and increased cell division in the root meristem under Pi starvation.     

Differential stringent responses of Streptomyces coelicolor M600 to starvation of specific nutrients

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the deltarelA mutant was a bld phenotype forming condensed mycelia, whereas the deltarshA mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the deltarelA mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the deltarshA mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.     

Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.     

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.