The alternative sigma factor, σS, affects polyhydroxyalkanoate metabolism in Pseudomonas putida

To determine whether the stationary sigma factor, σ^S, influences polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440, an rpoS -negative mutant was constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 ( phaC1 ) and polyhydroxyalkanoate depolymerase ( phaZ ). By comparison with the wild-type, the rpoS mutant showed a higher polyhydroxyalkanoate degradation rate and increased expression of the translational fusion during the stationary growth phase. These results suggest that σ^S might control the genes involved in polyhydroxyalkanoate metabolism, possibly in an indirect manner. In addition, survival and oxidative stress assays performed under polyhydroxyalkanoate- and nonpolyhydroxyalkanoate- accumulating conditions demonstrated that the accumulated polyhydroxyalkanoate increased the survival and stress tolerance of the rpoS mutant. According to this, polyhydroxyalkanoate accumulation would help cells to overcome the adverse conditions encountered during the stationary phase in the strain that lacks RpoS.     

The alternative sigma factor, sigmaS, affects polyhydroxyalkanoate metabolism in Pseudomonas putida

To determine whether the stationary sigma factor, sigma(S), influences polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440, an rpoS-negative mutant was constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 (phaC1) and polyhydroxyalkanoate depolymerase (phaZ). By comparison with the wild-type, the rpoS mutant showed a higher polyhydroxyalkanoate degradation rate and increased expression of the translational fusion during the stationary growth phase. These results suggest that sigma(S) might control the genes involved in polyhydroxyalkanoate metabolism, possibly in an indirect manner. In addition, survival and oxidative stress assays performed under polyhydroxyalkanoate- and nonpolyhydroxyalkanoate- accumulating conditions demonstrated that the accumulated polyhydroxyalkanoate increased the survival and stress tolerance of the rpoS mutant. According to this, polyhydroxyalkanoate accumulation would help cells to overcome the adverse conditions encountered during the stationary phase in the strain that lacks RpoS.     

Molecular analysis of polyphosphate accumulation in bacteria

The dynamic behavior of inorganic polyphosphate (polyP), its accumulation and disappearance, is the most striking aspect of polyP metabolism in bacteria. Imbalance between polyP synthesis and degradation results in fluctuations of polyP by 100- to 1000-fold. We here review recent results with respect to this polyP metabolism in bacteria. PolyP accumulation in response to amino acid starvation, accompanied by increased levels of stringent factors, has been observed in Escherichia coli. Inhibition by stringent factors of polyphosphatase interrupts the dynamic balance between the synthesis and degradation of polyP, accounting for polyP accumulation. Polyphosphate kinase is required for activation of intracellular protein degradation, which is required for adaptation at the onset of amino acid starvation. The adaptation to amino acid starvation is mediated by the network of stringent response and polyP metabolism. PolyP accumulation independent of stringent response has also been observed. Novobiocin, an inhibitor for DNA gyrase, stimulated accumulation of polyP but not that of stringent factors. However, a temperature-sensitive DNA gyrase mutant did not exhibit polyP accumulation at the non-permissive temperature. Antagonistic relationship of polyP to nucleic acid synthesis, explored by Harold, appears to be more complicated. We discuss relationship of Pi regulation to polyP accumulation in E. coli and Klebsiella aerogenes. A function of polyP as an in vivo phosphagen affecting polyP accumulation is also discussed.     

Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells

Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention.     

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

Inorganic polyphosphate (polyP), a linear polymer of hundreds of phosphate residues linked by ATP-like phosphoanhydride bonds, is found in all organisms and performs a wide variety of functions. This study shows that polyP accumulation occurs in Mycobacterium tuberculosis upon exposure to various stress conditions. M. tuberculosis possesses a single homolog of ppk-1, and we have disrupted ppk-1 in the M. tuberculosis genome by allelic replacement. The mutant strain exhibited negligible levels of intracellular polyP, decreased expression of sigF and phoP, and reduced growth in the stationary phase and displayed a survival defect in response to nitrosative stress and in THP-1 macrophages compared to the wild-type strain. We report that reduction in polyP levels is associated with increased susceptibility of M. tuberculosis to certain TB drugs and impairs its ability to cause disease in guinea pigs. These results suggest that polyP contributes to persistence of M. tuberculosis in vitro and plays an important role in the physiology of bacteria residing within guinea pigs.     

Collaborative effects of Photobacterium CuZn superoxide dismutase (SODs) and human AP endonuclease in DNA repair and SOD-deficient Escherichia coli under oxidative stress

The defenses against free radical damage include specialized repair enzymes that correct oxidative damage in DNA and detoxification systems such as superoxide dismutases (SODs). These defenses may be coordinated genetically as global responses. We hypothesized that the expression of SOD and DNA repair genes would inhibit DNA damage under oxidative stress. Therefore, protection of Escherichia coli mutants deficient in SOD and DNA repair genes (sod-, xth-, and nfo-) was demonstrated by transforming the mutant strain with a plasmid pYK9 that encoded Photobacterium leiognathi CuZnSOD and human AP endonuclease. The results show that survival rates were increased in sod+ xth- nfo+ cells compared with sod- xth- ape-, sod- xth- ape-, and sod+ xth- ape- cells under oxidative stress generated with 0.1 mM paraquat or 3 mM H2O2. The data suggest that, at the least, SOD and DNA repair enzymes may collaborate on protection and repair of damaged DNA. Additionally, both enzymes are required for protection against free radicals.     

Polyphosphate accumulation by Pseudomonas putida CA-3 and other medium-chain-length polyhydroxyalkanoate-accumulating bacteria under aerobic growth conditions

Pseudomonas putida CA-3 accumulates polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) concurrently under nitrogen limitation. Five other mclPHA-accumulating Pseudomonas strains are capable of simultaneous polyP and mclPHA biosynthesis. It appears that polyP is not the rate-limiting step for mclPHA accumulation in these Pseudomonas strains.     

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by ³¹P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.     

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes

Mitochondrial dysfunction caused by excessive Ca2+ accumulation is a major contributor to cardiac cell and tissue damage during myocardial infarction and ischemia-reperfusion injury (IRI). At the molecular level, mitochondrial dysfunction is induced by Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, which leads to the dissipation of mitochondrial membrane potential (ΔΨm), disruption of adenosine triphosphate production, and ultimately cell death. Although the role of Ca2+ for induction of mPTP opening is established, the exact molecular mechanism of this process is not understood. The aim of the present study was to test the hypothesis that the adverse effect of mitochondrial Ca2+ accumulation is mediated by its interaction with inorganic polyphosphate (polyP), a polymer of orthophosphates linked by phosphoanhydride bonds. We found that cardiac mitochondria contained significant amounts (280±60 pmol/mg of protein) of short-chain polyP with an average length of 25 orthophosphates. To test the role of polyP for mPTP activity, we investigated kinetics of Ca2+ uptake and release, ΔΨm and Ca2+-induced mPTP opening in polyP-depleted mitochondria. polyP depletion was achieved by mitochondria-targeted expression of a polyP-hydrolyzing enzyme. Depletion of polyP in mitochondria of rabbit ventricular myocytes led to significant inhibition of mPTP opening without affecting mitochondrial Ca2+ concentration by itself. This effect was observed when mitochondrial Ca2+ uptake was stimulated by increasing cytosolic [Ca2+] in permeabilized myocytes mimicking mitochondrial Ca2+ overload observed during IRI. Our findings suggest that inorganic polyP is a previously unrecognized major activator of mPTP. We propose that the adverse effect of polyphosphate might be caused by its ability to form stable complexes with Ca2+ and directly contribute to inner mitochondrial membrane permeabilization.     

The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein.

The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded sigma(s) subunit of RNA polymerase. Sigma(s) is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfq-encoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.     

Ultrastructural changes in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells under stress conditions

Ultrastructural organization of the aerobic yeast Yarrowia lipolytica was studied under conditions of oxidative, heat, and ethanol stresses. It was shown that the following uniform changes in cell ultrastructure did not depend on the type of stress: enlargement of mitochondria, enhanced number and enlargement of peroxisomes, and formation of lipid granules. Similar ultrastructural changes also occurred during the transition of cells to the stationary growth phase. It was shown for the first time that accumulation of polyphosphate granules occurred as a stress response in yeasts. Moreover, numerous globular structures of unknown nature appeared on the cell wall surface under oxidative or heat stress. Under ethanol stress, the cells developed clearly marked deep invaginations of the cytoplasmic membrane. (The same changes in the cytoplasmic membrane were observed in the cells grown on ethanol.) Variations of the cell envelope structure along with the formation of polyphosphate granules were not observed in the stationary growth phase. Ultrastructural changes in the cells under stress conditions are in agreement with the previous data on survival, respiratory activity, and variations of the antioxidant systems.     

Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli.

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase.     

Dynamic polyphosphate metabolism in cyanobacteria responding to phosphorus availability

Despite the crucial role of polyphosphate (polyP) in aquatic environments, its metabolism in cyanobacteria responding to nutrients is poorly understood. We investigate polyP in three cyanobacteria species, specifically unicellular picocyanobacteria, under various nutritional conditions. Our experiments show that the accumulation of polyP in cyanobacteria is strongly dynamic, depending on phosphate levels and growth stages. ‘Overplus’ uptake of phosphorus (P) during the lag phase leads to the rapid accumulation of polyP, followed by lower polyP quotas during the exponential growth stage as a result of competing ‘luxury’ P uptake and polyP utilization to support rapid cell division. Cyanobacteria are capable of P deficiency responses that preferentially maintain polyP. However, preferential utilization of polyP occurs under severe P stress, suggesting the crucial role of polyP as P reserve to support cellular survival. Strong variability was observed among different species of cyanobacteria in their ability to accumulate polyP, and likely in the threshold P levels at which preferential polyP degradation occurs. This suggests that some cyanobacteria may be more adaptive to P-stressed or P-fluctuating conditions. Our results explain and provide important insights into the variability of polyP observed in aquatic environments where picocyanobacteria are the dominant primary producers.     

Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance

The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions. We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance. The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem. For imipenem, the survival was approximately 40 times lower. Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant. Our results indicate that rpoS gene is involved in tolerance to antibiotics in P. aeruginosa during the stationary phase and heat stress. However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene.     

Polyphosphate Accumulation by Pseudomonas putida CA-3 and Other Medium-Chain-Length Polyhydroxyalkanoate-Accumulating Bacteria under Aerobic Growth Conditions

Pseudomonas putida CA-3 accumulates polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) concurrently under nitrogen limitation. Five other mclPHA-accumulating Pseudomonas strains are capable of simultaneous polyP and mclPHA biosynthesis. It appears that polyP is not the rate-limiting step for mclPHA accumulation in these Pseudomonas strains.