Alkaline phosphatase as a reporter of σS levels and rpoS polymorphisms in different E. coli strains (original) (raw)
Related papers
A differential effect of {sigma} S on the expression of the PHO regulon genes of Escherichia coli
Microbiology, 2004
The RNA polymerase core associated with s S transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by s D -associated RNA polymerase. A mutation in the gene that encodes s S , rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of s S inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by s S . In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by s S . The effect of s S on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position "13 is important for the positive effect of s S on pst. The interpretation of these observations is based on the competition between s S and s D for the binding to the core RNA polymerase.
Journal of Bacteriology, 2002
The general stress resistance of Escherichia coli is controlled by the RpoS sigma factor ( S ), but mutations in rpoS are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing rpoS was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by rpoS mutants in both glucose-and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of rpoS null mutations and loss of general stress resistance. The rate of takeover by rpoS mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher S levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on 70 and involved in the hunger response to nutrient limitation. Indeed, the mutation of rpoS led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence, rpoS polymorphism in E. coli populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than S for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of rpoS mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of rpoS mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.
Microbial Physiology, 2008
It has been demonstrated that about 10% of the Escherichia coli genes are under direct or indirect control of RpoS. Therefore, Weber et al. [2005] proposed that this sigma subunit should be considered a second vegetative sigma factor under non-optimal growth conditions. In this report we demonstrate that in the phosphoenolpyruvate:carbohydrate phosphotransferase system-deficient (PTS–) derivatives, PB11 and PB12 of strain JM101 that permanently grow slowly on glucose, the inactivation of rpoS resulted in decreased growth rates of 50 and 10%, respectively. Real-time PCR (RT-PCR) analysis confirmed the important role of this sigma factor in the PTS– strains and allowed the identification of 19 genes including almost all the glycolytic genes, not previously reported, to be at least partially dependent on RpoS. The transcription level of gpp, spoT, ppa and ndk whose products are involved in ppGpp metabolism was upregulated in strain PB12 as compared to the parental strains PB11 and JM10...
The effect of the rpoSam allele on gene expression and stress resistance in Escherichia coli
The RNA polymerase associated with RpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DNA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS ORF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and partially functional RpoS protein. Here, we present new evidence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length RpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for RpoS level, stress resistance and for the expression of RpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermediate phenotype regarding stress resistance and the expression of σ(S)-dependent genes when compared to the wild-type rpoS(+) strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the σ(70)-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and β-galactosidase. The former was enhanced while the latter was inhibited by high levels of RpoSam.
Anticipating an alkaline stress through the Tor phosphorelay system in Escherichia coli
Molecular Microbiology, 2003
The torCAD operon encoding the TMAO reductase respiratory system is induced in the presence of TMAO by the two-component regulatory system TorS/ TorR. The TorS sensor detects TMAO and transphosphorylates the TorR response regulator via a four-step phosphorelay. Once phosphorylated, TorR activates expression of the torCAD structural operon. In order to identify new genes regulated by the Tor regulatory system, we performed a genome-wide transcriptional analysis by using the DNA array technology. We identified seven new transcriptional units whose expression is modulated by the TorS/TorR phosphorelay system. One unit, tnaLAB , is positively regulated whereas the other six, gadA , gadBC , hdeAB , hdeD , yhiE and yhiM , are negatively regulated by this system. Interestingly, the products of some of these units seem to play a role in the survival of E. coli in conditions of extreme pH. The TnaA tryptophanase has been proposed to counteract alkaline stress, whereas the GadA and GadB glutamate decarboxylases and the HdeA and HdeB proteins are involved in the defence against acid stress. Our hypothesis is that the TorS/TorR phosphorelay triggers alkaline-stress defence to limit alkalinization resulting from the reduction of TMAO in alkaline TMA by the Tor respiratory system. The fact that a D D D D tnaLAB mutant showed a dramatic decrease in survival as a result of TMAO respiration is in agreement with such a model. As regulation of these genes by the TorS/TorR system does not depend on pH modification but rather on the presence of TMAO, we propose that E. coli anticipates alkalinization of the medium due to TMA production by base-resistance gene activation and acidresistance gene repression.
Genetic analysis of regulatory mutants of alkaline phosphatase of E. coli
Genetics, 1975
A fine structure map of the phoR region of E. coli, mutations of which affect the rate of alkaline phosphatase synthesis, was constructed by Hfr X F- crosses. Mutations causing three different phenotypes (previously reported as phoRa, phoRb, phoRc (Garen and Echols 1962a,b) are clustered in three closely linked genetic loci. PhoR mutants of all three types, including the phoRb type not previously tested, are recessive to wild-type phoR+. In addition, phoRa and phoRc complement each other, while phoRa and phoRb do not. Our results support the hypothesis of Morris et al. (1974) that phoRc mutants represent a cistron (phoB) different from phoR.
Genetics, 1980
The expression of alkaline phosphatase (the product of the phoA gene) in Escherichia coli is believed to be subject to both positive control by the phoB gene product and negative control by the phoR gene product. We have isolated a large number of PhoA- mutants in the phoR- genetic background. Among mutants altered in the positive control of alkaline phosphatase, some were phoB mutants; others had a mutation in a new gene, designated phoM. We believe that the phoM gene codes for a positive regulator that acts together with the phoB gene product in phoA gene expressions.--The phoM phenotype was found to be masked in phoR+ strains. This and other evidence support a positive regulatory role for the phoR gene product as well.--Our experiments demonstrate that phoA is under positive control by three different positive regulators: the product of the phoB, phoM and phoR genes. The phoB gene product is always needed together with either the phoR or phoM gene product. In addition, the phoR g...
Microbial Cell Factories, 2011
Background: It is quite important to understand how the central metabolism is regulated under nitrogen (N)limitation as well as carbon (C)-limitation. In particular, the effect of C/N ratio on the metabolism is of practical interest for the heterologous protein production, PHB production, etc. Although the carbon and nitrogen metabolisms are interconnected and the overall mechanism is complicated, it is strongly desirable to clarify the effects of culture environment on the metabolism from the practical application point of view. Results: The effect of C/N ratio on the metabolism in Escherichia coli was investigated in the aerobic continuous culture at the dilution rate of 0.2 h -1 based on fermentation data, transcriptional RNA level, and enzyme activity data. The glucose concentration was kept at 10 g/l, while ammonium sulfate concentration was varied from 5.94 to 0.594 g/l. The resultant C/N ratios were 1.68 (100%), 2.81(60%), 4.21(40%), 8.42(20%), and 16.84(10%), where the percentage values in brackets indicate the ratio of N-concentration as compared to the case of 5.94 g/l of ammonium sulfate. The mRNA levels of crp and mlc decreased, which caused ptsG transcript expression to be upregulated as C/N ratio increased. As C/N ratio increased cra transcript expression decreased, which caused ptsH, pfkA, and pykF to be up-regulated. At high C/N ratio, transcriptional mRNA level of soxR/S increased, which may be due to the activated respiratory chain as indicated by up-regulations of such genes as cyoA, cydB, ndh as well as the increase in the specific CO 2 production rate. The rpoN transcript expression increased with the increase in C/N ratio, which led glnA, L, G and gltD transcript expression to change in similar fashion. The nac transcript expression showed similar trend as rpoN, while gdhA transcript expression changed in reverse direction. The transcriptional mRNA level of glnB, which codes for P II , glnD and glnK increased as C/N ratio increases. It was shown that GS-GOGAT pathway was activated for gdhA mutant under N-rich condition. In the case of glnL mutant, GOGAT enzyme activity was reduced as compared to the wild type under N-limitation. In the case of gltB, D mutants, GDH and GS enzymes were utilized under both N-rich and N-limited conditions. In this case, the transcriptional mRNA level of gdhA and corresponding GDH enzyme activity was higher under N-limitation as compared to Nrich condition.