Superimposition of TyrR protein-mediated regulation on osmoresponsive transcription of Escherichia coli proU in vivo

Osmotic regulation of proU expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the σ70-controlled promoter of proU in Escherichia coli, we were able to demonstrate a superposed TyrR-mediated activation by L-phenylalanine (Phe), as well as repression by L-tyrosine, of proU expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of proU at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in E. coli

A dnaC mutation in Escherichia coli that affects copy number of ColE1-like plasmids and the PriA-PriB (but Not Rep-PriC) pathway of chromosomal replication restart

Escherichia coli nusG and rho mutants, which are defective in transcription termination, are killed following transformation with several ColE1-like plasmids that lack the plasmid-encoded copy-number regulator gene rom because of uncontrolled plasmid replication within the cells. In this study, a mutation [dnaC1331(A84T)] in the dnaC gene encoding the replicative helicase-loading protein was characterized as a suppressor of this plasmid-mediated lethality phenotype. The mutation also reduced the copy number of the plasmids in otherwise wild-type strains. In comparison with the isogenic dnaC+ strain, the dnaC mutant was largely unaffected for (i) growth on rich or minimal medium, (ii) tolerance to UV irradiation, or (iii) survival in the absence of the PriA, RecA, or RecB proteins. However, it was moderately SOS-induced and was absolutely dependent on both the Rep helicase and the PriC protein for its viability. A dnaC1331(A84T) dam mutant, but not its mutH derivative, exhibited sensitivity to growth on rich medium, suggestive of a reduced capacity in the dnaC1331(A84T) strains to survive chromosomal double-strand breaks. We propose that DnaC-A84T is proficient in the assembly of replication forks for both initiation of chromosome replication (at oriC) and replication restart via the Rep-PriC pathway, but that it is specifically defective for replication restart via the PriA-PriB pathway (and consequently also for replication of the Rom- ColE1-like plasmids)

Host factor titration by chromosomal R-loops as a mechanism for runaway plasmid replication in transcription termination-defective mutants of Escherichia coli

Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 °C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 °C following overexpression of rnhA+. Mutant combinations rnhA-nusG or recG-rho were synthetically lethal at 21 °C, with the former being suppressed by recG+ overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 °C or 37 °C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA+ or recG+. Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom+, and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and Δ64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication

Why is transcription coupled to translation in bacteria?

Active mechanisms exist to prevent transcription that is uncoupled from translation in the protein-coding genes of bacteria, as exemplified by the phenomenon of nonsense polarity. Bacterial transcription-translation coupling may be viewed as one among several co-transcriptional processes, including those for mRNA processing and export in the eukaryotes, that operate in the various life forms to render the nascent transcript unavailable for formation of otherwise deleterious R-loops in the genome

Trans-acting mutations in loci other than kdpDE that affect kdp operon regulation in Escherichia coli: effects of cytoplasmic thiol oxidation status and nucleoid protein H-NS on kdp expression

Transcription of the K+ transport operon kdp in Escherichia coli is induced during K+-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE. In this study, we screened for new dke (named dke for decreased kdp expression) mutations (in loci other than kdpDE) that led to substantially decreased kdp expression. One dke mutation was shown to be in hns, encoding the nucleoid protein H-NS. Another dke mutation was mapped to trxB (encoding thioredoxin reductase), and an equivalent reduction in kdp expression was demonstrated also for trxA mutants that are deficient in thioredoxin 1. Exogenously provided dithiothreitol rescued the kdp expression defect in trxB but not trxA mutants. Neither trxB nor trxA affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR. Mutations in genes dsbC and dsbD did not affect kdp expression, suggesting that the trx effects on kdp are not mediated by alterations in protein disulfide bond status in the periplasm. Reduced kdp expression was observed even in a trxB strain that harbored a variant KdpD polypeptide bearing no Cys residues. A trxB hns double mutant was even more severely affected for kdp expression than either single mutant. The dke mutations themselves had no effect on strength of the signal controlling kdp expression, and constitutive mutations in kdpDE were epistatic to hns and trxB. These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal for kdp transcription

Evidence for transcription attenuation rendering cryptic a sigmaS- dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the σ S- and σ 70-bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S. typhimurium has a cryptic P1 promoter that is recognized by σ S RNA polymerase in vitro and that a 22-bp deletion from +63 to +84 (relative to the start site of transcription) confers σ S-dependent in vivo expression of a reporter gene fusion to P1. Primer extension analysis of RNA isolated from cells carrying the wild-type and mutant S. typhimurium proU constructs indicated that a primer which hybridizes proximal to +60 is able to detect P1-initiated transcripts from both constructs but a primer which hybridizes distal to +85 is able to do so only from the latter. Our results suggest that the σ S-controlled proU P1 promoter in S. typhimurium may be rendered cryptic because of factor-dependent transcription attenuation within a short distance downstream of the promoter start site

Osmoregulation in Enterobacteriaceae: role of proline betaine transport systems

In a wide variety of organisms, L-proIine and glycine betaine are amongst the compounds accumulated intracellularly as compatible solutes to counteract the effects of turgor reduction during growth in water-stressed environments. Two osmoregulatory transport systems, ProP and ProU, have been identified and characterized in the enterobacteria, each of which participates in the active concentration of both L-proline and glycine betaine from the culture medium in response to osmotic stress. The expression of genes encoding the components of the ProU porter is induced 400-fold upon growth in high-osmolarity medium; elucidation of the molecular mechanisms underlying such regulation would possibly enable understanding of the large class of processes that involve transduction of mechanical signals to chemical ones within biological systems. A complete understanding of the molecular basis by which ProP and ProU function in osmoregulation would also provide insight into the mechanisms of similar adaptation at the cellular level in the economically important genera of microbes and higher plants

An unusual suicidal interaction in Escherichia coli involving nucleoid protein H-NS

A conditional-lethal mutation (rpoB364) mapping to the gene that encodes the β -subunit of RNA polymerase was obtained in Escherichia coli. This mutation caused cell filamentation at the restrictive growth temperature and partial derepression of the osmotically regulatedproU operon at the permissive growth temperature. Even under the latter condition, transformants of therpoB364 mutant strain carrying the plasmid vector pACYC184, but not those carrying other polA-dependent multicopy plasmids such as pACYC177 or pBR322, were killed in early stationary phase; one class of suppressor mutants isolated as survivors within these transformant colonies were further derepressed forproU-lac expression, and the mutation in each of several independent clones of this class was mapped tohns, the gene that encodes the protein H-NS of theE. coli nucleoid. Thehns mutations did not suppress the conditional-lethal growth phenotype of therpoB364 mutant itself. On the other hand, intracellular overproduction of guanosine 3', 5'-bispyrophosphate (ppGpp) in therpoB364 strain alleviated both the growth inhibition at the restrictive temperature and the pACYC184-mediated stationary-phase lethality. Upon subcloning into pUC19 or into pACYC177, a 105-bpXbal-HindIII fragment from pACYC184 was shown to be sufficient to confer therpoB364 hns +-dependent lethal phenotype. We suggest that the level in stationary-phase cultures of a gene product(s) that interacts with the pACYC184 DNA fragment is altered in therpoB364 hns+derivative (compared to that inrpoB+ orrpoB364 hns strains) and that this results in cell suicide

Effects of H-NS and potassium glutamate on &#963;<SUP>S</SUP>- and &#963;<SUP>70</SUP>-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the &#963;s and &#963;70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of &#963;s selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS

Employee engagement practices in private hospitals: a cross sectional study in mayiladuthurai

Employee Engagement has become a hot topic in the world of human resources management.Employee Engagement is a state of emotional and intellectual involvement that employees have in an organization. The private hospitals in India have become the happening industry. The corporate culture has attracted many billions from abroad also in the form of medical tourism. The HR practices in these hospitals have a bearing on these revenue generation activities. This study conducted in five major hospitals of Mayiladuthurai town was to find out the levels of employee engagement, the drivers of it, to analyze their impact and to offer suggestions to improve the same. A structured questionnaire with 27 statements was administered to various non-medical employees across the hospitals. Simple mean score calculations are used as a prelude to a more elaborative study in the future. The findings show the cross section of hospitals has ratings at par with current international standards on the drivers of engagement