Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis

Ozan Ertekin; Meltem Kutnu; Aslı Aras Ta&#; Mustafa Demir; Ayten Yazgan Karata&#; Gülay Özcengiz

doi:10.1007/s12275-020-9064-0

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Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis: Ozan Ertekin ¹, Meltem Kutnu ¹, Aslı Aras Ta&# ^351;kın1,3, Mustafa Demir ^1,4, Ayten Yazgan Karata&# ^351;2, Gülay Özcengiz ¹; Journal of Microbiology 2020;58(4):297-313.
DOI: https://doi.org/10.1007/s12275-020-9064-0
Published online: January 28, 2020

¹Department of Biological Sciences/Molecular Biology and Genetics, Middle East Technical University, Ankara, Turkey, ³Faculty of Biology and Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany, ⁴Biotechnology and Bioengineering, Ecole polytechnique fédérale de Lausanne, Lausanne, Switzerland, ²Molecular Biology-Biotechnology and Genetics Research Center (MOBGAM) and Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey¹Department of Biological Sciences/Molecular Biology and Genetics, Middle East Technical University, Ankara, Turkey, ³Faculty of Biology and Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany, ⁴Biotechnology and Bioengineering, Ecole polytechnique fédérale de Lausanne, Lausanne, Switzerland, ²Molecular Biology-Biotechnology and Genetics Research Center (MOBGAM) and Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey

Corresponding author: Gülay Özcengiz , Tel: +90-31-2210-5170,

Received: 8 February 2019 • Revised: 11 December 2019 • Accepted: 24 December 2019

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Abstract

Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA􍿁::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, no bacilysin activity was detectable in bioassays and inability of the OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry analysis. Phenotypic analyses revealed the deficiencies in OGU1 with respect to colony pigmentation, spore coat proteins, spore resistance and germination, which could be rescued by external addition of bacilysin concentrate into its cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were used as complementary approaches to compare cytosolic proteomes of OGU1. 2-DE identified 159 differentially expressed proteins corresponding to 121 distinct ORFs. In nanoLCMS/ MS, 76 proteins were differentially expressed in OGU1. Quantitative transcript analyses of selected genes validated the proteomic findings. Overall, the results pointed to the impact of bacilysin on expression of certain proteins of sporulation and morphogenesis; the members of mother cell compartment- specific σE and σK regulons in particular, quorum sensing and two component-global regulatory systems, peptide transport, stress response as well as CodY- and ScoCregulated proteins.

Keywords: Bacillus subtilis;bacilysin;bac operon;sporulation;chemical complementation;pleiotropy;comparative proteomics;RT-qPCR

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Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis

J. Microbiol. 2020;58(4):297-313. Published online January 28, 2020

DOI: https://doi.org/10.1007/s12275-020-9064-0

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