Differential mechanisms of binding of anti-sigma factors Escherichia coli Rsd and bacteriophage T4 Asia to E. coli RNA polymerase lead to diverse physiological consequences

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70

Antimicrobial activity of Mycobacteriophage D29 Lysin B during Mycobacterium ulcerans infection

Buruli Ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Although the current antibiotic treatment for BU is effective, daily administrations for a prolonged period of time, combined with potential risk of severe side effects, negatively impact on patient adherence. In that sense, we tested the efficacy of an alternative strategy based on Lysin B (LysB), a phage encoded lipolytic enzyme that degrades the mycolylarabinogalactan-peptidoglycan complex present in the mycobacterial cell wall. In this study, we show that LysB not only displays lytic activity against M. ulcerans isolates in vitro, but also leads to a decrease of M. ulcerans proliferation in infected mouse footpads. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.The projectwas developed withinthescopeof the projectsNORTE-01-0145-FEDER-000013and NORTE-01-0145-FEDER-000023,supported by the Northern Portugal Regional Operational Programme (NORTE2020),under the Portugal2020 Partnership Agreement through FEDER.This work was also supported by BioTecNorte operation (NORTE-01-0145-FEDER -000004) funded by the European Regional Development Fund under the scope of NORTE2020.This study was supportedby the Portuguese Foundation for Scienceand Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit; the Competitiveness Factors Operational Programme (COMPETE 2020) projectsPOCI-01-0145-FEDER-006684 and POCI-01-0145-FEDER-007038; and the project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). This study was also supported by Infect-ERA grant Infect-ERA/0002/2015 :BU_SPONT_HEAL. AGF,GT, and HO wouldlike to acknowledge FCT for the individual fellowships SFRH/BPD/112903/2015, SFRH/BPD/64032/2009,and SFRH/BPD/111653/2015,respectively. CMG received an individual QRENfellowship (UMINHO/BPD/15/2014). GangaGen acknowledges CSIR/ OSDD,Govt of India,for funding this project.The funders had no role in study design,data collection and analysis, decision to publish, or preparation of the manuscriptinfo:eu-repo/semantics/publishedVersio

Transcriptional switching in Escherichia coli during stress and starvation by modulation of Sigma 70 activity

During active growth of Escherichia coli, majority of the transcriptional activity is carried out by the housekeeping sigma factor (Sigma 70), whose association with core RNAP is generally favoured because of its higher intracellular level and higher affinity to core RNAP. In order to facilitate transcription by alternative sigma factors during nutrient starvation, the bacterial cell uses multiple strategies by which the transcriptional ability of Sigma 70 is diminished in a reversible manner. The facilitators of shifting the balance in favour of alternative sigma factors happen to be as diverse as a small molecule (p)ppGpp (represents ppGpp or pppGpp), proteins (DksA, Rsd) and a species of RNA (6S RNA). Although 6S RNA and (p)ppGpp were known in literature for a long time, their role in transcriptional switching has been understood only in recent years. With themelucidation of function of DksA, a new dimension has been added to the phenomenon of stringent response. As the final outcome of actions of (p)ppGpp, DksA, 6S RNA and Rsd is similar, there is a need to analyse hese mechanisms in a collective manner. We review the recent trends in understanding the regulation of Sigma 70 by (p)ppGpp, DksA, Rsd and 6S RNA and present a case for evolving a unified model of RNAP redistribution during starvation by modulation of Sigma 70 activity in E. coli

Both regions 4.1 and 4.2 of E. coli σ70{\sigma}{^7{^0}} are together required for binding to bacteriophage T4 AsiA in vivo

The T4 AsiA is an anti-sigma factor encoded by one of the early genes of Bacteriophage T4. It has been shown that AsiA inhibits transcription from promoters containing -10 and -35 consensus sequence by binding to sigma(70) of E. coli. Binding of AsiA to sigma(70) in vivo, in E. coli, leads to inhibition of transcription of essential genes resulting in killing of the organism. By using various in vitro methods, the region of sigma(70) binding to AsiA have been mapped to domain 4.2. Additionally, mutational analysis of sigma(70) has also identified amino acid residues in domain 4.1 which are critical for interaction with AsiA. Based on NMR studies it has been suggested that either of these regions can bind to AsiA, a conclusion which was supported by high degree of amino acid homology between domain 4.1 and 4.2. However, it is not clear whether under in vivo conditions, AsiA exerts its transcription inhibitory effect by binding to one of these regions or both the regions together. In order to understand the mechanism of AsiA mediated inhibition of E. coli transcription in vivo, in terms of specific binding requirements to region 4.1 and/or 4,2, we have studied the interaction of these sub-domains with AsiA by Yeast two hybrid system as well as by co-expressing and affinity purification of the interacting partners in vivo in E. coli. It was observed that minimum fragment of sigma(70) showing observable binding to AsiA, must possess sub-domains 4.1 and 4.2 together. No binding could be detected in sigma(70) fragments lacking a part of either domain 4.1 or 4.2, in any of the assays. This data was also supported by in vitro binding studies wherein only sigma(70) fragments carrying both region 4.1 and 4.2 showed binding to AsiA. Co-expression of region 4.1 and 4.2 fragments together also did not show any interaction with AsiA. The results presented here suggest that binding of AsiA to sigma(70) in vivo, requires the presence of both sub-domains of region 4 of sigma(70)

Rv1218c, an ABC Transporter of Mycobacterium tuberculosis with Implications in Drug Discovery▿ †

Efflux systems are important in determining the efficacy of antibiotics used in the treatment of bacterial infections. In the last decade much attention has been paid to studying the efflux pumps of mycobacteria. New classes of compounds are under investigation for development into potential candidate drugs for the treatment of tuberculosis. Quite often, these have poor bactericidal activities but exhibit excellent target (biochemical) inhibition. Microarray studies conducted in our laboratories for deciphering the mode of action of experimental drugs revealed the presence of putative ABC transporters. Among these transporters, Rv1218c was chosen for studying its physiological relevance in mediating efflux in Mycobacterium tuberculosis. A ΔRv1218c mutant of M. tuberculosis displayed a 4- to 8-fold increase in the inhibitory and bactericidal potency for different classes of compounds. The MICs and MBCs were reversed to wild-type values when the full-length Rv1218c gene was reintroduced into the ΔRv1218c mutant on a multicopy plasmid. Most of the compound classes had significantly better bactericidal activity in the ΔRv1218c mutant than in the wild-type H37Rv, suggesting the involvement of Rv1218c gene product in effluxing these compounds from M. tuberculosis. The implication of these findings on tuberculosis drug discovery is discussed

Identification of carbohydrate structures as receptors for localised adherent enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli strains of diffused adherent (DA) and localised adherent (LA) phenotypes were tested for their ability to bind to glycolipids. DA strains did not bind to the glycolipids tested, while LA strains bound to asialo GM1, asialo GM2, globoside and lacto-N-neotetraose in decreasing order of avidity. The minimum common sequence among the four glycolipids could be delineated as GalNac β 1–4 Gal as the binding epitope with GalNac β 1–3 Gal and GlcNac β 1–3 Gal serving as relatively weaker binders. The binding was not inhibited by a variety of free oligosaccharides or by the neoglycoproteins tested. Adhesion-negative mutants of an enteropathogenic LA strain showed a markedly reduced binding to asialo GM1 indicating that the recognition of GalNac β 1–4 Gal was correlated with the ability to adhere to HeLa cells. Thus recognition and binding to glycolipids could play an important role in colonisation through adherence to intestinal surfaces