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

Ozan Ertekin, Meltem Kutnu, Aslı Aras Taşkin, Mustafa Demir, Ayten Yazgan Karataş, Gülay Özcengiz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

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 nanoLC-MS/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 ScoC-regulated proteins.

Original languageEnglish
Pages (from-to)297-313
Number of pages17
JournalJournal of Microbiology
Volume58
Issue number4
DOIs
Publication statusPublished - 1 Apr 2020

Bibliographical note

Publisher Copyright:
© 2020, The Microbiological Society of Korea.

Funding

This work was supported by the Turkish Scientific and Technical Research Council [the projects TBAG-106T535 and KBAG 116Z351] and partly by Middle East Technical University Scientific Research Funds. We would like to acknowledge Prof. Dörte Becher and Dr. Dirk Albrecht of the Institute of Microbiology, Ernst-Moritz-Arndt-University, Griefswald, Germany and Assoc. Prof. Ömür Çelikbiçak and Dr. Melis Ş. Ekiz of HUNITECH Center of Hacettepe University, Ankara for providing excellent services for MS analyses. We thank our former and present students who provided assistance in certain experiments, and Dr. Sezer Okay for his help in interpretation of comparative genome sequences. This work was supported by the Turkish Scientific and Technical Research Council [the projects TBAG-106T535 and KBAG 116Z351] and partly by Middle East Technical University Scientific Research Funds. We would like to acknowledge Prof. D?rte Becher and Dr. Dirk Albrecht of the Institute of Microbiology, Ernst-Moritz-Arndt-University, Griefswald, Germany and Assoc. Prof. ?m?r ?elikbi?ak and Dr. Melis ?. Ekiz of HUNITECH Center of Hacettepe University, Ankara for providing excellent services for MS analyses. We thank our former and present students who provided assistance in certain experiments, and Dr. Sezer Okay for his help in interpretation of comparative genome sequences.

FundersFunder number
Turkish Scientific and Technical Research CouncilKBAG 116Z351, TBAG-106T535
Consejo Nacional de Investigaciones Científicas y Técnicas
Orta Doğu Teknik Üniversitesi
Hacettepe Üniversitesi

    Keywords

    • bac operon
    • Bacillus subtilis
    • bacilysin
    • chemical complementation
    • comparative proteomics
    • pleiotropy
    • RT-qPCR
    • sporulation

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