Anne Farewell

Negative regulation by RpoS: a case of sigma factor competition

A mutation in the Escherichia coli gene encoding the stationary phase‐inducible sigma factor (σs, RpoS) not only abolishes transcription of some genes in stationary phase, but also causes superinduction of other stationary phase‐induced genes. We have examined this phenomenon of repression by σs using as a model system the divergently transcribed stationary phase‐inducible genes, uspA and uspB. uspA is transcribed by σ70‐programmed RNA polymerase and is superinduced in an rpoS mutant, while uspB induction is σs dependent. The data suggest that the superinduction of uspA is caused by an increased amount of σ70 bound to RNA polymerase in the absence of the competing σs. Increasing the ability of σ70 to compete against σs by overproducing σ70 mimics the effect of an rpoS mutation by causing superinduction of σ70‐dependent stationary phase‐inducible genes (uspA and fadD), silencing of σs‐dependent genes (uspB, bolAp1 and fadL) and inhibiting the development of σs‐dependent phenotypes, such as hydrogen peroxide resistance in stationary phase. In addition, overproduction of σs markedly reduced stationary phase expression of a σ70‐dependent promoter. Thus, we conclude that sigma factors compete for a limiting amount of RNA polymerase during stationary phase. The implications of this competition in the passive control of promoter activity is discussed.

Identical, Independent, and Opposing Roles of ppGpp and DksA in Escherichia coli

The recent discovery that the protein DksA acts as a coregulator of genes controlled by ppGpp led us to investigate the similarities and differences between the relaxed phenotype of a ppGpp-deficient mutant and the phenotype of a strain lacking DksA. We demonstrate that the absence of DksA and ppGpp has similar effects on many of the observed phenotypes but that DksA and ppGpp also have independent and sometimes opposing roles in the cell. Specifically, we show that overexpression of DksA can compensate for the loss of ppGpp with respect to transcription of the promoters P(uspA), P(livJ), and P(rrnBP1) as well as amino acid auxotrophy, cell-cell aggregation, motility, filamentation, and stationary phase morphology, suggesting that DksA can function without ppGpp in regulating gene expression. In addition, ppGpp and DksA have opposing effects on adhesion. In the course of our analysis, we also discovered new features of the relaxed mutant, namely, defects in cell-cell aggregation and motility.

Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

ABSTRACT A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ32, was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.

The cadmium-stress stimulon of Escherichia coli K-12

The influence of cadmium on stress protein production in Escherichia coli K-12 (strain MG1655) was analysed using two-dimensional polyacrylamide gel electrophoresis and the gene-protein database of E. coli K-12. Cadmium (273 microM) caused complete but transient inhibition of growth accompanied by the synthesis of cadmium-induced proteins (CDPs). It was found that some CDPs induced during the growth-arrested phase belong to the heat-shock, oxidation stress, SOS and stringent response regulons, while others are general stress inducible proteins (e.g. H-NS, UspA). In addition, trigger factor, adenylate kinase, W-protein, the cold shock protein G041.2, and seven unknown proteins whose synthesis is not known to be controlled by a global regulator, were identified as immediate responders to cadmium exposure. The rate of synthesis of most of the immediate responders to cadmium exposure decreased when the growth of the cells resumed. However, seven CDPs, including those encoded by argI, tyrA and xthA, maintained a high production rate during growth in the presence of cadmium. Two of the unidentified proteins were N-terminally sequenced by Edman degradation. The N-terminal amino acid sequence of one of these proteins (designated F023.3) matches the E. coli open reading frame o216. This ORF is similar to the N-terminal third of the copper-binding protein amine oxidases (encoded by maoA) of both E. coli and Klebsiella pneumoniae (K. aerogenes). The other N-terminally sequenced protein (designated C044.6) matches perfectly the product of the metK gene, S-adenosylmethionine synthetase I. In comparison to untreated cells, cadmium-stressed cells were found to recover more rapidly during subsequent stress conditions, such as ethanol, osmotic, heat shock, and nalidixic acid treatment. The role of the CDPs is discussed in view of their physiological assignments in the cell.

Emergency derepression: stringency allows RNA polymerase to override negative control by an active repressor

The uspA promoter, driving production of the universal stress protein A in response to diverse stresses, is demonstrated to be under dual control. One regulatory pathway involves activation of the promoter by the alarmone guanosine 3′,5′‐bisphosphate, via the β‐subunit of RNA polymerase, whereas the other consists of negative control by the FadR repressor. In contrast to canonical dual control by activation and repression circuits, which depends on concomitant activation and derepression for induction to occur, the ppGpp‐dependent activation of the uspA promoter overrides repression by an active FadR under conditions of severe cellular stress (starvation). The ability of RNA polymerase to overcome repression during stringency depends, in part, on the strength of the FadR operator. This emergency derepression is operative on other FadR‐regulated genes induced by starvation and is argued to be an essential regulatory mechanism operating during severe stress.

Increased RNA polymerase availability directs resources towards growth at the expense of maintenance

Nutritionally induced changes in RNA polymerase availability have been hypothesized to be an evolutionary primeval mechanism for regulation of gene expression and several contrasting models have been proposed to explain how such ‘passive’ regulation might occur. We demonstrate here that ectopically elevating Escherichia coli RNA polymerase (Eσ70) levels causes an increased expression and promoter occupancy of ribosomal genes at the expense of stress‐defense genes and amino acid biosynthetic operons. Phenotypically, cells overproducing Eσ70 favours growth and reproduction at the expense of motility and damage protection; a response reminiscent of cells with no or diminished levels of the alarmone guanosine tetraphosphate (ppGpp). Consistently, we show that cells lacking ppGpp displayed markedly elevated levels of free Eσ70 compared with wild‐type cells and that the repression of ribosomal RNA expression and reduced growth rate of mutants with constitutively elevated levels of ppGpp can be suppressed by overproducing Eσ70. We conclude that ppGpp modulates the levels of free Eσ70 and that this is an integral part of the alarmones means of regulating a trade‐off between growth and maintenance.

Metabolic control of the Escherichia coli universal stress protein response through fructose‐6‐phosphate

The universal stress protein (Usp) superfamily encompasses a conserved group of proteins involved in stress resistance, adaptation to energy deficiency, cell motility and adhesion, and is found in all kingdoms of life. The paradigm usp gene, uspA, of Escherichia coli is transcriptionally activated by a large variety of stresses, and the alarmone ppGpp is required for this activation. Here, we show that the uspA gene is also regulated by an intermediate of the glycolytic/gluconeogenic pathways. Specifically, mutations and conditions resulting in fructose‐6‐phosphate (F‐6‐P) accumulation elicit superinduction of uspA upon carbon starvation, whereas genetic manipulations reducing the pool size of F‐6‐P have the opposite effect. This metabolic control of uspA does not act via ppGpp. Other, but not all, usp genes of the usp superfamily are similarly affected by alterations in F‐6‐P levels. We suggest that alterations in the pool size of phosphorylated sugars of the upper glycolytic pathway may ensure accumulation of required survival proteins preceding the complete depletion of the external carbon source. Indeed, we show that uspA is, in fact, induced before the carbon source is depleted from the medium.

Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli

Bacterial accommodation to moderate concentrations of cadmium is accompanied by transient activation of general stress proteins as well as a sustained induction of other proteins of hitherto unknown functions. One of the latter proteins was previously identified as the product of the Escherichia coli yodA ORF. The yodA ORF encodes 216 aa residues (the YodA protein) and the increased synthesis of YodA during cadmium stress was found probably to be a result of transcriptional activation from one single promoter upstream of the structural yodA gene. Analysis of a transcriptional gene fusion, P(yodA)-lacZ, demonstrated that basal expression of yodA is low during exponential growth and expression is increased greater than 50-fold by addition of cadmium to growing cells. However, challenging cells with additional metals such as zinc, copper, cobalt and nickel did not increase the level of yodA expression. In addition, hydrogen peroxide also increased yodA expression whereas the superoxide-generating agent paraquat failed to do so. Surprisingly, cadmium-induced transcription of yodA is dependent on soxS and fur, but independent of oxyR. Moreover, a double relA spoT mutation abolished induction of yodA during cadmium exposure but ppGpp is not sufficient to induce yodA since expression of the gene is not elevated during stationary phase. After 45 min of cadmium exposure the YodA protein was primarily detected in the cytoplasmic fraction but was later (150 min) found in both the cytoplasmic and periplasmic compartments.

Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli

Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ‐proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ‐proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5′‐untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ‐proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.

The tumor suppressor homolog in fission yeast, myh1+, displays a strong interaction with the checkpoint gene rad1+

The DNA glycosylase MutY is strongly conserved in evolution, and homologs are found in most eukaryotes and prokaryotes examined. This protein is implicated in repair of oxidative DNA damage, in particular adenine mispaired opposite 7,8-dihydro-8-oxoguanine. Previous investigations in Escherichia coli, fission yeast, and mammalian cells show an association of mutations in MutY homologs with a mutator phenotype and carcinogenesis. Eukaryotic MutY homologs physically associate with several proteins with a role in replication, DNA repair, and checkpoint signaling, specifically the trimeric 9-1-1 complex. In a genetic investigation of the fission yeast MutY homolog, myh1(+), we show that the myh1 mutation confers a moderately increased UV sensitivity alone and in combination with mutations in several DNA repair genes. The myh1 rad1, and to a lesser degree myh1 rad9, double mutants display a synthetic interaction resulting in enhanced sensitivity to DNA damaging agents and hydroxyurea. UV irradiation of myh1 rad1 double mutants results in severe chromosome segregation defects and visible DNA fragmentation, and a failure to activate the checkpoint. Additionally, myh1 rad1 double mutants exhibit morphological defects in the absence of DNA damaging agents. We also found a moderate suppression of the slow growth and UV sensitivity of rhp51 mutants by the myh1 mutation. Our results implicate fission yeast Myh1 in repair of a wider range of DNA damage than previously thought, and functionally link it to the checkpoint pathway.