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Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

ABSTRACT: Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

SUBMITTER: Dempwolff F

PROVIDER: S-EPMC3206058 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

Dempwolff Felix F Reimold Christian C Reth Michael M Graumann Peter L PL

PloS one 20111101 11

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is ...[more]

PMID: 22069484

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Project description:Background:Whole-cell biosensors are a powerful and easy-to-use screening tool for the fast and sensitive detection of chemical compounds, such as antibiotics. ?-Lactams still represent one of the most important antibiotic groups in therapeutic use. They interfere with late stages of the bacterial cell wall biosynthesis and result in irreversible perturbations of cell division and growth, ultimately leading to cell lysis. In order to simplify the detection of these antibiotics from solutions, solid media or directly from producing organisms, we aimed at developing a novel heterologous whole-cell biosensor in Bacillus subtilis, based on the ?-lactam-induced regulatory system BlaR1/BlaI from Staphylococcus aureus. Results:The BlaR1/BlaI system was heterologously expressed in B. subtilis and combined with the luxABCDE operon of Photorhabdus luminescens under control of the BlaR1/BlaI target promoter to measure the output of the biosensor. A combination of codon adaptation, constitutive expression of blaR1 and blaI and the allelic replacement of penP increased the inducer spectrum and dynamic range of the biosensor. ?-Lactams from all four classes induced the target promoter P blaZ in a concentration-dependent manner, with a dynamic range of 7- to 53-fold. We applied our biosensor to a set of Streptomycetes soil isolates and demonstrated its potential to screen for the production of ?-lactams. In addition to the successful implementation of a highly sensitive ?-lactam biosensor, our results also provide the first experimental evidence to support previous suggestions that PenP functions as a ?-lactamase in B. subtilis. Conclusion:We have successfully established a novel heterologous whole-cell biosensor in B. subtilis that is highly sensitive for a broad spectrum of ?-lactams from all four chemical classes. Therefore, it increases the detectable spectrum of compounds with respect to previous biosensor designs. Our biosensor can readily be applied for identifying ?-lactams in liquid or on solid media, as well as for identifying potential ?-lactam producers.

Project description:BackgroundSubtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot.ResultsIn this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed "online" reporter strains to efficiently monitor the dynamics of subtilin biosynthesis.ConclusionsWithin this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

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Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

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Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

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