Dataset Information

Screen for inducers of autolysis in Bacillus subtilis.

ABSTRACT: We describe a primary high-throughput screen that uses the reporter strain Bacillus subtilis BAU-102 to identify antibiotics that induce autolysis. The screen measures autolysis in terms of the incipient release of recombinant Escherichia coli beta-galactosidase (beta-Gal) from the periplasmic space of B. subtilis owing to a loss of integrity of the cell wall. In a model screen, beta-Gal release values for 79 members of a library consisting of antibiotics and related compounds were collected, sorted, and plotted as a function of rank. Inducers of autolysis, which included compounds that inhibit cell wall synthesis and those that do not, were readily differentiated from other members of the library on the basis of their elevated beta-galactosidase release responses. The results of the BAU-102 model screen called attention to the antibacterial activity of drugs normally used in other applications, describable as "repurposed." Thus, the screen independently identified the potential antibacterial properties of the antifungal drug miconazole and of the antileishmaniasis drug miltefosine. Daptomycin-induced release of beta-Gal was also detected and occurred in a Ca(2+)-dependent manner.

SUBMITTER: Falk SP

PROVIDER: S-EPMC2935000 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Screen for inducers of autolysis in Bacillus subtilis.

Falk Shaun P SP Noah James W JW Weisblum Bernard B

Antimicrobial agents and chemotherapy 20100706 9

We describe a primary high-throughput screen that uses the reporter strain Bacillus subtilis BAU-102 to identify antibiotics that induce autolysis. The screen measures autolysis in terms of the incipient release of recombinant Escherichia coli beta-galactosidase (beta-Gal) from the periplasmic space of B. subtilis owing to a loss of integrity of the cell wall. In a model screen, beta-Gal release values for 79 members of a library consisting of antibiotics and related compounds were collected, so ...[more]

PMID: 20606070

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Project description:BackgroundSurfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear.ResultsIn this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of L-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of L-leucine and L-valine, not only improved surfactin production but also increased the proportion of the C14 isoform.ConclusionsThis study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.

Dataset Information

Screen for inducers of autolysis in Bacillus subtilis.

Publications

Screen for inducers of autolysis in Bacillus subtilis.

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