Mutations in two global regulators lower individual mortality in Escherichia coli

There has been considerable investigation into the survival of bacterial cells under stress conditions, but little is known about the causes of mortality in the absence of exogenous stress. That there is a basal frequency of cell death in such populations may reflect that it is either impossible to avoid all lethal events, or alternatively, that it is too costly. Here, through a genetic screen in the model organism Escherichia coli, we identify two mutants with lower frequencies of mortality: rssB and fliA. Intriguingly, these two genes both affect the levels of different sigma factors within the cell. The rssB mutant displays enhanced resistance to multiple external stresses, possibly indicating that the cell gains its increased vitality through elevated resistance to spontaneous, endogenous stresses. The loss of fliA does not result in elevated stress resistance; rather, its survival is apparently due to a decreased physical stress linked to the insertion of the flagellum through the membrane and energy saved through the loss of the motor proteins. The identification of these two mutants implies that reducing mortality is not impossible; rather, due to its cost, it is subject to trade-offs with other traits that contribute to the competitive success of the organism

The E. coli Anti-Sigma Factor Rsd: Studies on the Specificity and Regulation of Its Expression

Background: Among the seven different sigma factors in E. coli s 70 has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting s 70-dependent transcription at the onset of stationary growth. Although binding of Rsd to s 70 has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown. Methodology/Principal Findings: We have performed studies to unravel the function and regulation of Rsd expression in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with s 70, with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with s 38, the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and s 38-ors 70-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of s 70-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most notably, the dam-dependent methylation of a cluster of GATC sites turned ou

LKB1 as the ghostwriter of crypt history

Familial cancer syndromes present rare insights into malignant tumor development. The molecular background of polyp formation and the cancer prone state in Peutz-Jeghers syndrome remain enigmatic to this day. Previously, we proposed that Peutz-Jeghers polyps are not pre-malignant lesions, but an epiphenomenon to the malignant condition. However, Peutz-Jeghers polyp formation and the cancer-prone state must both be accounted for by the same molecular mechanism. Our contribution focuses on the histopathology of the characteristic Peutz-Jeghers polyp and recent research on stem cell dynamics and how these concepts relate to Peutz-Jeghers polyposis. We discuss a protracted clonal evolution scenario in Peutz-Jeghers syndrome due to a germline LKB1 mutation. Peutz-Jeghers polyp formation and malignant transformation are separately mediated through the same molecular mechanism played out on different timescales. Thus, a single mechanism accounts for the development of benign Peutz-Jeghers polyps and for malignant transformation in Peutz-Jeghers syndrome

TraR, a Homolog of a RNAP Secondary Channel Interactor, Modulates Transcription

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed

In Vivo Transcription Dynamics of the Galactose Operon: A Study on the Promoter Transition from P1 to P2 at Onset of Stationary Phase

Quantitative analyses of the 5′ end of gal transcripts indicate that transcription from the galactose operon P1 promoter is higher during cell division. When cells are no longer dividing, however, transcription is initiated more often from the P2 promoter. Escherichia coli cells divide six times before the onset of the stationary phase when grown in LB containing 0.5% galactose at 37°C. Transcription from the two promoters increases, although at different rates, during early exponential phase (until the third cell division, OD600 0.4), and then reaches a plateau. The steady-state transcription from P1 continues in late exponential phase (the next three cell divisions, OD600 3.0), after which transcription from this promoter decreases. However, steady-state transcription from P2 continues 1 h longer into the stationary phase, before decreasing. This longer steady-state P2 transcription constitutes the promoter transition from P1 to P2 at the onset of the stationary phase. The intracellular cAMP concentration dictates P1 transcription dynamics; therefore, promoter transition may result from a lack of cAMP-CRP complex binding to the gal operon. The decay rate of gal-specific transcripts is constant through the six consecutive cell divisions that comprise the exponential growth phase, increases at the onset of the stationary phase, and is too low to be measured during the stationary phase. These data suggest that a regulatory mechanism coordinates the synthesis and decay of gal mRNAs to maintain the observed gal transcription. Our analysis indicates that the increase in P1 transcription is the result of cAMP-CRP binding to increasing numbers of galactose operons in the cell population

Appropriate DevR (DosR)-Mediated Signaling Determines Transcriptional Response, Hypoxic Viability and Virulence of Mycobacterium tuberculosis

Background: The DevR(DosR) regulon is implicated in hypoxic adaptation and virulence of Mycobacterium tuberculosis. The present study was designed to decipher the impact of perturbation in DevR-mediated signaling on these properties. Methodology/Principal Findings: M. tb complemented (Comp) strains expressing different levels of DevR were constructed in Mut1 * background (expressing DevR N-terminal domain in fusion with AphI (DevRN-Kan) and in Mut2DdevR background (deletion mutant). They were compared for their hypoxia adaptation and virulence properties. Diverse phenotypes were noted; basal level expression (,5.362.3 mM) when induced to levels equivalent to WT levels (,25.869.3 mM) was associated with robust DevR regulon induction and hypoxic adaptation (Comp 9 * and 10*), whereas low-level expression (detectable at transcript level) as in Comp 11 * and Comp15 was associated with an adaptation defect. Intermediate-level expression (,3.361.2 mM) partially restored hypoxic adaptation functions in Comp2, but not in Comp1 * bacteria that coexpressed DevRN-Kan. Comp * strains in Mut1 * background also exhibited diverse virulence phenotypes; high/very low-level DevR expression was associated with virulence whereas intermediate-level expression was associated with low virulence. Transcription profiling and gene expression analysis revealed up-regulation of the phosphate starvation response (PSR) in Mut1 * and Comp11 * bacteria, but not in WT/Mut2DdevR/other Comp strains, indicating a plasticity in expression pathways that is determined by the magnitude of signaling perturbation through DevRN-Kan

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

Stringent response of Escherichia coli: revisiting the bibliome using literature mining

Understanding the mechanisms responsible for cellular responses depends on the systematic collection and analysis of information on the main biological concepts involved. Indeed, the identification of biologically relevant concepts in free text, namely genes, tRNAs, mRNAs, gene products and small molecules, is crucial to capture the structure and functioning of different responses. Results In this work, we review literature reports on the study of the stringent response in Escherichia coli. Rather than undertaking the development of a highly specialised literature mining approach, we investigate the suitability of concept recognition and statistical analysis of concept occurrence as means to highlight the concepts that are most likely to be biologically engaged during this response. The co-occurrence analysis of core concepts in this stringent response, i.e. the (p)ppGpp nucleotides with gene products was also inspected and suggest that besides the enzymes RelA and SpoT that control the basal levels of (p)ppGpp nucleotides, many other proteins have a key role in this response. Functional enrichment analysis revealed that basic cellular processes such as metabolism, transcriptional and translational regulation are central, but other stress-associated responses might be elicited during the stringent response. In addition, the identification of less annotated concepts revealed that some (p)ppGpp-induced functional activities are still overlooked in most reviews. Conclusions In this paper we applied a literature mining approach that offers a more comprehensive analysis of the stringent response in E. coli. The compilation of relevant biological entities to this stress response and the assessment of their functional roles provided a more systematic understanding of this cellular response. Overlooked regulatory entities, such as transcriptional regulators, were found to play a role in this stress response. Moreover, the involvement of other stress-associated concepts demonstrates the complexity of this cellular response