Phosphate-Enhanced Stationary-Phase Fitness of Escherichia coli Is Related to Inorganic Polyphosphate Level

We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness.Polyphosphate (poly-P) is a long-chain polymer composed of many orthophosphates linked together by high-energy ATP-like bonds. Studied mainly in prokaryotes, poly-P plays an important role as an energy source, as a regulator of gene expression, as a store of inorganic phosphate, and as a chelator of heavy metals (8, 10). The main enzymes associated with poly-P metabolism in bacteria are the polyphosphate kinase (PPK, encoded by ppk) and the exopolyphosphatase (PPX, encoded by ppx) (1, 2). The ppk ppx double mutant exhibits greatly reduced synthesis of poly-P, is deficient in stationary-phase functions, and lacks resistance to different stresses (6, 17).Previously, we found that expression of several respiratory (ndh, sdhC, ubiC, nuoAB, and cydA) and defense (katG and ahpC) genes was maintained in late stationary phase when the medium''s phosphate concentration was above 37 mM (24, 25). Furthermore, Escherichia coli cells grown in medium containing this critical phosphate concentration had high viability, low oxidative damage, and elevated resistance to external H₂O₂ stress in late stationary phase (25).We examined the relationship between the medium''s phosphate concentration and intracellular poly-P levels in the wild-type and ppk ppx mutant strains to see if the previously observed effects on gene expression, enzyme activity, and tolerance to H₂O₂ were correlated with elevated poly-P levels.     

Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response

Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (P_i), in media deficient in both P_i and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid, phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during P_i starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.     

Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

The biological process for phosphate (P_i) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P_i regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P_i from the medium than the wild-type strain removed.     

Polyphosphate binding and chain length recognition of Escherichia coli exopolyphosphatase

Exopolyphosphatase of Escherichia coli (PPX) is a highly processive enzyme demonstrating the ability to recognize polyphosphates of specific lengths. The mechanisms responsible for the processivity and polymer length recognition of the enzyme were investigated in relation to the manner in which polyphosphate is bound to the enzyme. Multiple polyphosphate binding sites were identified on distant portions of the enzyme and were determined to be responsible for the polymer length recognition of the enzyme. In addition, two independently folded domains were identified. The N-terminal domain contained a quasi-processive polyphosphatase active site belonging to the sugar kinase/actin/hsp70 superfamily. The C-terminal domain contained a single polyphosphate binding site and was responsible for nearly all of the PPX affinity for polyphosphate. This domain was also found to confer a highly processive mode of action to PPX. Collectively, these results were used to describe the interaction of polyphosphate with PPX.     

Pathways of disulfide bond formation in Escherichia coli

Disulfide bond formation is required for the correct folding of many secreted proteins. Cells possess protein-folding catalysts to ensure that the correct pairs of cysteine residues are joined during the folding process. These enzymatic systems are located in the endoplasmic reticulum of eukaryotes or in the periplasm of Gram-negative bacteria. This review focuses on the pathways of disulfide bond formation and isomerization in bacteria, taking Escherichia coli as a model.     

A Novel Assay Reveals Hygrotactic Behavior in Drosophila

Humidity is one of the most important factors that determines the geographical distribution and survival of terrestrial animals. The ability to detect variation in humidity is conserved across many species. Here, we established a novel behavioral assay that revealed the thirsty Drosophila exhibits strong hygrotactic behavior, and it can locate water by detecting humidity gradient. In addition, exposure to high levels of moisture was sufficient to elicit proboscis extension reflex behavior in thirsty flies. Furthermore, we found that the third antennal segment was necessary for hygrotactic behavior in thirsty flies, while arista was required for the avoidance of moist air in hydrated flies. These results indicated that two types of hygroreceptor cells exist in Drosophila: one located in the third antennal segment that mediates hygrotactic behavior in thirst status, and the other located in arista which is responsible for the aversive behavior toward moist air in hydration status. Using a neural silencing screen, we demonstrated that synaptic output from the mushroom body α/β surface and posterior neurons was required for both hygrotactic behavior and moisture-aversive behavior.     

Inhibition of Inorganic Pyrophosphatase from Escherichia coli with Inorganic Phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (P _i) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with P _i, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when P _i is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.     

Nitrosoguanidine Assay of Episomal Integration in Escherichia coli

Mutations affecting utilization of lactose and resistance to the male-specific phages f1, f2, and Qbeta tend to occur simultaneously more often than expected by chance in Hfr strains whose origin of transfer is close to the genes for lactose utilization, but not in F(+) strains. Strains derived from the Hfr, but exhibiting poor ability to transfer early chromosomal genes, may or may not show this comutation phenomenon. These results support the concept that the F factor is integrated into the Hfr chromosome during vegetative growth, but is autonomous in the F(+) strains and could serve as an assay for episomal localization.     

Polyphosphate Accumulation in Escherichia coli in Response to Defects in DNA Metabolism

Phenol-chloroform extraction of [³²P]orthophosphate-labeled Escherichia coli cells followed by alkaline gel electrophoresis revealed, besides the expected chromosomal DNA, two non-DNA species that we have identified as lipopolysaccharides and polyphosphates by using a combination of biochemical and genetic techniques. We used this serendipitously found straightforward protocol for direct polyphosphate detection to quantify polyphosphate levels in E. coli mutants with diverse defects in the DNA metabolism. We detected increased polyphosphate accumulation in the ligA, ligA recBCD, dut ung, and thyA mutants. Polyphosphate accumulation may thus be an indicator of general DNA stress.DNA replication intermediates, also known as Okazaki fragments, have classically been detected by pulse labeling thymine-limited thyA mutant cells with [³H]thymidine, a DNA-specific label (27). However, when limited for thymidine, thyA mutants are known to undergo thymine-less death (1), a phenomenon during which chromosomal DNA suffers single-strand breaks (24). The products of this nicking could be mistaken for Okazaki fragments, compromising DNA synthesis studies that rely on [³H]thymidine labeling (28, 37). Caveats were also raised against interpreting [³H]thymidine labeling as an accurate reflection of DNA synthesis in cells of higher eukaryotes, on the basis of differences with [³²P]orthophosphate DNA labeling (10, 15, 30).To avoid the possibility of thymine starvation in our experiments, we also attempted to visualize Okazaki fragments by using the [³²P]orthophosphate label which we routinely employ to label chromosomal DNA for pulsed-field gel electrophoresis (17, 36). Since we expected that the bulk of the ³²P label will be deposited into RNA, we removed RNA altogether by separating chromosomal DNA from replication intermediates in alkaline agarose gels. We found, however, that Okazaki pieces cannot be detected using [³²P]orthophosphate even by alkaline agarose because there are other molecules in larger amounts in the cells that take in ³²P-label and mask the replication intermediates. We report on the identification and quantification of two of the “masking species” in wild-type Escherichia coli, as well as in several mutants.     

Polyphosphate kinase is a component of the Escherichia coli RNA degradosome

Xer site-specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes. Two related recombinases, XerC and XerD, mediate this recombination, which 'undoes' the potential damage of homologous recombination. Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers. In contrast, recombination at the Escherichia coli recombination site, dif , occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid. Here the DNA sequence features of a family of core recombination sites in which the XerC- and XerD-binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules. Sequence changes in either the XerC- or XerD-binding site can alter the recombination outcome. Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC-mediated cleavage in vitro , and an apparent increased overall bending in recombinase–core-site complexes.     

Colorimetric Assay for Lysine Decarboxylase in Escherichia coli

A new assay is described for lysine decarboxylase. It is rapid and reproducible in assaying large numbers of samples, a situation in which earlier methods were less convenient. The new method is valuable in the study of peptide fractions and amino acid mixtures which stimulate induction of lysine decarboxylase. It may be useful for work on enzyme structure and modification, genetics, and kinetics.     

Polyphosphate kinase regulates error-prone replication by DNA polymerase IV in Escherichia coli

The ppk gene encodes polyphosphate kinase (Ppk), an enzyme that catalyses the polymerization of inorganic phosphate into long chains of polyphosphate (polyP). An insertion mutation in ppk causes a decrease in adaptive mutation in Escherichia coli strain FC40. Adaptive mutation in FC40 mostly results from error-prone DNA polymerase IV (Pol IV), encoded by dinB; most of the antimutagenic phenotype of the ppk mutant disappears in a dinB mutant strain. In addition, the ppk mutant causes a decrease in growth-dependent mutations produced by overexpressing Pol IV. However, the amount of Pol IV protein is unchanged in the ppk mutant strain, indicating that the activity or fidelity of Pol IV is altered. Adaptive mutation is inhibited both by the absence of Ppk, which results in low amounts of polyP, and by overproduction of Ppk, which results in high amounts of polyP, suggesting that an optimal level of polyP is necessary. Taken together, these results suggest a novel mechanism involving polyP that directly or indirectly regulates DNA polymerase activity or fidelity.