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Spohn M.,Interfaculty Institute of Microbiology and Infection Medicine Tuebingen | Wohlleben W.,Microbiology Biotechnology University of Tuebingen 72076 Tuebingen Germany | Stegmann E.,Microbiology Biotechnology University of Tuebingen 72076 Tuebingen Germany
Environmental Microbiology | Year: 2016

Summary: The actinomycete Amycolatopsis japonicum produces the complexing agent ethylenediamine-disuccinate ([S,S]-EDDS), which is an isomer of the widely industrially applied ethylenediamine-tetraacetate (EDTA). In contrast to EDTA, [S,S]-EDDS is readily biodegradable and is therefore an alternative with a favourable environmental profile. Biotechnological production of [S,S]-EDDS, however, is not currently possible because its biosynthesis is inhibited by low-micromolar zinc concentrations. Here we illustrate the development of a new strategy for identifying a biosynthetic pathway that is based on the elucidation of transcriptional regulation and the screening for binding sites of the respective regulator that controls the [S,S]-EDDS biosynthesis genes. To achieve this, we identified the zinc uptake regulator Zur in A.japonicum and showed that it mediates the repression of the zinc uptake system ZnuABCAj. The Zur-binding motif, recognized by the zinc-bound Zur protein in the upstream region of znuABCAj, was used to screen the genome, leading to the identification of the aes genes. Transcriptional analysis and shift assays reveal specific zinc-responsive regulation of the aes genes by Zur, and gene inactivation shows their involvement in [S,S]-EDDS biosynthesis. Zur-mediated zinc repression of the [S,S]-EDDS biosynthesis genes is abolished in a Δzur mutant, which offers now the opportunity to develop a biotechnological process. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Pozzi R.,Interfaculty Institute of Microbiology and Infection Medicine Tuebingen | Coles M.,Max Planck Institute for Developmental Biology | Linke D.,University of Oslo | Kulik A.,Interfaculty Institute of Microbiology and Infection Medicine Tuebingen | And 3 more authors.
Environmental Microbiology | Year: 2015

The investigation of self-resistance in antibiotic producers is important to understand the emergence of antibiotic resistance in pathogens and to improve antibiotic production. Lantibiotics are ribosomally synthesized antibiotics that mostly target peptidoglycan biosynthesis. The actinomycete MicrobisporaATCC PTA-5024 produces the lantibiotic NAI-107, which interferes with peptidoglycan biosynthesis by binding bactoprenol-pyrophosphate-coupled peptidoglycan precursors. In order to understand how Microbispora counteracts the action of its own antibiotic, its peptidoglycan composition was analysed in detail. Microbispora peptidoglycan consists of muropeptides with D-Ala and Gly in similar proportion at the fourth position of the peptide stems and alternative 3-3 cross-links besides the classical 4-3 cross-links. In addition, the NAI-107 biosynthetic gene cluster (mlb) was analysed for the expression of immunity proteins. We show that distinct immunity determinants are encoded in the mlb cluster: the ABC transporter MlbYZ acting cooperatively with the transmembrane protein MlbJ and the lipoprotein MlbQ. NMR structural analysis of MlbQ revealed a hydrophobic surface patch, which is proposed to bind the cognate lantibiotic. This study demonstrates that immunity in Microbispora is not only based on one determinant but on the action of the distinct immunity proteins MlbQ, MlbYZ and MlbJ. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

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