Title |
Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis |
Author |
Ozan Ertekin1, Meltem Kutnu1, Aslı Aras Taşkın1,3, Mustafa Demir1,4, Ayten Yazgan Karataş2, and Gülay Özcengiz1* |
Address |
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, Turkey |
Bibliography |
Journal of Microbiology, 58(4),297-313, 2020,
|
DOI |
10.1007/s12275-020-9064-0
|
Key Words |
Bacillus subtilis, bacilysin, bac operon, sporulation,
chemical complementation, pleiotropy, comparative
proteomics, RT-qPCR |
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. |