Replication of plasmids derived from Shiga toxinconverting bacteriophages in starved Escherichia coli

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage lambda, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA+ and relA” cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the l PR promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for lambda PR, were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to lambda) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA” hosts). Possible clinical implications of these results are discussed

Genetic response to metabolic fluctuations: correlation between central carbon metabolism and DNA replication in Escherichia coli

BACKGROUND: Until now, the direct link between central carbon metabolism and DNA replication has been demonstrated only in Bacillus. subtilis. Therefore, we asked if this is a specific phenomenon, characteristic for this bacterium and perhaps for its close relatives, or a more general biological rule. RESULTS: We found that temperature-sensitivity of mutants in particular genes coding for replication proteins could be suppressed by deletions of certain genes coding for enzymes of the central carbon metabolism. Namely, the effects of dnaA46(ts) mutation could be suppressed by dysfunction of pta or ackA, effects of dnaB(ts) by dysfunction of pgi or pta, effects of dnaE486(ts) by dysfunction of tktB, effects of dnaG(ts) by dysfunction of gpmA, pta or ackA, and effects of dnaN159(ts) by dysfunction of pta or ackA. The observed suppression effects were not caused by a decrease in bacterial growth rate. CONCLUSIONS: The genetic correlation exists between central carbon metabolism and DNA replication in the model Gram-negative bacterium, E. coli. This link exists at the steps of initiation and elongation of DNA replication, indicating the important global correlation between metabolic status of the cell and the events leading to cell reproduction

Differential inhibition of transcription from σ70- and σ32-dependent promoters by rifampicin

AbstractRifampicin is an antibiotic which binds to the β subunit of prokaryotic RNA polymerases and prevents initiation of transcription. It was found previously that production of heat shock proteins in Escherichia coli cells after a shift from 30°C to 43°C is not completely inhibited by this antibiotic. Here we demonstrate that while activity of a pL-lacZ fusion (pL is a σ70-dependent promoter) in E. coli cells is strongly inhibited by rifampicin, a pgroE-lacZ fusion, whose activity is dependent on the σ32 factor, retains significant residual activity even at relatively high rifampicin concentrations. Differential sensitivity to this antibiotic of RNA polymerase holoenzymes containing either the σ70 or the σ32 subunit was confirmed in vitro. Since the effects of an antibiotic that binds to the β subunit can be modulated by the presence of either the σ70 or the σ32 subunit in the holoenzyme, it is tempting to speculate that binding of various σ factors to the core of RNA polymerase results in different conformations of particular holoenzymes, including changes in the core enzyme

Studies of Streptococcus anginosus Virulence in Dictyostelium discoideum and Galleria mellonella Models

For many years, Streptococcus anginosus has been considered a commensal colonizing the oral cavity, as well as the gastrointestinal and genitourinary tracts. However, recent epidemiological and clinical data designate this bacterium as an emerging opportunistic pathogen. Despite the reported pathogenicity of S. anginosus, the molecular mechanism underpinning its virulence is poorly described. Therefore, our goal was to develop and optimize efficient and simple infection models that can be applied to examine the virulence of S. anginosus and to study host-pathogen interactions. Using 23 S. anginosus isolates collected from different infections, including severe and superficial infections, as well as an attenuated strain devoid of CppA, we demonstrate for the first time that Dictyostelium discoideum is a suitable model for initial, fast, and large-scale screening of virulence. Furthermore, we found that another nonvertebrate animal model, Galleria mellonella, can be used to study the pathogenesis of S. anginosus infection, with an emphasis on the interactions between the pathogen and host innate immunity. Examining the profile of immune defense genes, including antimicrobial peptides, opsonins, regulators of nodulation, and inhibitors of proteases, by quantitative PCR (qPCR) we identified different immune response profiles depending on the S. anginosus strain. Using these models, we show that S. anginosus is resistant to the bactericidal activity of phagocytes, a phenomenon confirmed using human neutrophils. Notably, since we found that the data from these models corresponded to the clinical severity of infection, we propose their further application to studies of the virulence of S. anginosus

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ70-dependent (p1, pB, and p2) and two σS-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models

Properties of Escherichia coli RNA polymerase from a strain devoid of the stringent response alarmone ppGpp

The stringent response alarmone guanosine tetraphosphate (ppGpp) affects transcription from many promoters. ppGpp binds directly to the transcription enzyme of Escherichia coli, RNA polymerase. Analysis of the crystal structure of RNA polymerase with ppGpp suggested that binding of this nucleotide may result in some conformational or post-translational alterations to the enzyme. These changes might affect in vitro performance of the enzyme. Here, a comparison of the in vitro properties of RNA polymerases isolated from wild type and ppGpp-deficient bacteria shows that both enzymes do not differ in i) transcription activity of various promoters (e.g. σ70-rrnB P1, λpL, T7A1), ii) response to ppGpp, iii) promoter-RNA polymerase open complex stability. Thus, it may be concluded that ppGpp present in the bacterial cell prior to purification of the RNA polymerase does not result in the alterations to the enzyme that could be permanent and affect its in vitro transcription capacity