Fifty Years Fused to Lac

Beckwith, Jonathan

doi:10.1146/annurev-micro-092412-155732

Cited by 16 publications

(13 citation statements)

References 60 publications

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“…We analyzed an historically important lethal stress originally identified by Jon Beckwith and his colleagues during their seminal genetic experiments that defined the highly conserved SecY-dependent protein translocation system (19). We show the lethality caused by the expression of a chimeric MalE-LacZ protein consisting of the NH 2 -terminal sequences of MalE (periplasmic maltose-binding protein) joined to a modestly NH 2 -terminally truncated LacZ (β-galactosidase) (20) shares attributes with the oxidative component of antibiotic lethality.…”

Section: Significancementioning

confidence: 99%

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Takahashi

Gruber

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROSdependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ 72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.A gents or conditions that inhibit important biological processes can kill cells. However, physiological processes metabolically downstream of the initial inhibition can also contribute to cell death (1). For example, β-lactam antibiotics not only inhibit penicillin-binding proteins leading to lysis; they also induce a futile cycle of cell wall synthesis and degradation that contributes to their killing (2). In another example, lethal attacks on Escherichia coli mediated by the type VI secretion system P1vir phage and the antimicrobial peptide polymyxin B elicit the production of reactive oxygen species (ROS) that contribute to cell death (3). In bacteria, ROS production has been associated with the lethal effects of diverse stresses (4). In most cases, the detailed mechanisms responsible for ROS production are poorly understood; however, a variety of futile metabolic cycles can elicit H 2 O 2 production, illustrating the breadth of possible metabolic perturbations that could potentially induce oxidative stress (5).Despite widespread evidence that endogenous ROS produced as a consequence of metabolic stress can be lethal to bacteria and eukaryotes, the application of this concept to antibiotic lethality has been complicated. Evidence from multiple investiga...

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Section: Significancementioning

confidence: 99%

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Takahashi

Gruber

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…For this approach, the authors applied a specific agar, multi-well test in combination with E. coli expressing a genetically modified beta galactosidase MalF-β-Gal fusion. The hybrid protein MalF-β-Gal102, containing β–Gal fused to the large periplasmic domain of MalF, does not show β-Gal activity in weight cells due to the introduction of disulfide bonds, but it is active in cells that are defective in disulfide bond formation (Beckwith 2013). Among more than 50,000 screened compounds derived from two collections, six that inhibited EcDsbB and possess similar piridazinone ring structures were selected.…”

Section: Dsb Proteins As Targets Of New Antibacterial Drugsmentioning

confidence: 99%

Bacterial thiol oxidoreductases — from basic research to new antibacterial strategies

Bocian-Ostrzycka

Grzeszczuk

Banaś

et al. 2017

Appl Microbiol Biotechnol

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The recent, rapid increase in bacterial antimicrobial resistance has become a major public health concern. One approach to generate new classes of antibacterials is targeting virulence rather than the viability of bacteria. Proteins of the Dsb system, which play a key role in the virulence of many pathogenic microorganisms, represent potential new drug targets. The first part of the article presents current knowledge of how the Dsb system impacts function of various protein secretion systems that influence the virulence of many pathogenic bacteria. Next, the review describes methods used to study the structure, biochemistry, and microbiology of the Dsb proteins and shows how these experiments broaden our knowledge about their function. The lessons gained from basic research have led to a specific search for inhibitors blocking the Dsb networks.

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“…I felt very fortunate to have seen Jon Beckwith in person on this occasion. I had been impressed by the way Jon was moving from the analysis of the lac operon regulation to genetic dissection of more complex cellular processes, such as protein localization (9). His interest in the downstream principles of gene expression was going to lead to a flourishing period in cell biology.…”

Section: Crossing the Pacific And The Continentmentioning

confidence: 99%

Strolling Toward New Concepts

Ito

2016

Annu. Rev. Microbiol.

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For more than four decades now, I have been studying how genetic information is transformed into protein-based cellular functions. This has included investigations into the mechanisms supporting cellular localization of proteins, disulfide bond formation, quality control of membranes, and translation. I tried to extract new principles and concepts that are universal among living organisms from our observations of Escherichia coli. While I wanted to distill complex phenomena into basic principles, I also tried not to overlook any serendipitous observations. In the first part of this article, I describe personal experiences during my studies of the Sec pathway, which have centered on the SecY translocon. In the second part, I summarize my views of the recent revival of translation studies, which has given rise to the concept that nonuniform polypeptide chain elongation is relevant for the subsequent fates of newly synthesized proteins. Our studies of a class of regulatory nascent polypeptides advance this concept by showing that the dynamic behaviors of the extraribosomal part of the nascent chain affect the ongoing translation process. Vibrant and regulated molecular interactions involving the ribosome, mRNA, and nascent polypeptidyl-tRNA are based, at least partly, on their autonomously interacting properties.

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Fifty Years Fused to Lac

Cited by 16 publications

References 60 publications

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Bacterial thiol oxidoreductases — from basic research to new antibacterial strategies

Strolling Toward New Concepts

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