Comparative biological network analysis for differentially expressed proteins as a function of bacilysin biosynthesis in Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis produces a diverse range of secondary metabolites with different structures and activities. Among them, bacilysin is an enzymatically synthesized dipeptide that consists of L-alanine and L-anticapsin. Previous research by our group has suggested bacilysin's role as a pleiotropic molecule in its producer, B. subtilis PY79. However, the nature of protein interactions in the absence of bacilysin has not been defined. In the present work, we constructed a protein-protein interaction subnetwork by using Omics Integrator based on our recent comparative proteomics data obtained from a bacilysin-silenced strain, OGU1. Functional enrichment analyses on the resulting networks pointed to certain putatively perturbed pathways such as citrate cycle, quorum sensing and secondary metabolite biosynthesis. Various molecules, which were absent from the experimental data, were included in the final network. We believe that this study can guide further experiments in the identification and confirmation of protein-protein interactions in B. subtilis.

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

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.