Phenotypic rescue of a Bacilysin-silenced strain of Bacillus Subtilis with Bacilysin supplementation

Ertekin, Ozan
Bacillus subtilis, the model organism for the gram positive bacteria, is distinguished for its secondary metabolite synthesis. Remarkably, most of these metabolites are small peptides which are generally synthesized non-ribosomally with multifunctional enzyme complexes. Bacilysin, a dipeptide composed of L-alanine and L-anticapsin, is the smallest peptide antibiotic which is produced by several enzymatical steps, however, its biosynthesis does not fully fit the general mechanisms of NRPSs. This small molecule acts specifically as an glucosamine-6-phosphate synthase inhibitor, thus interfering with cell wall formation in target microbes. Previously, it was found that the synthesis of bacilysin is governed by bacA operon and regulation of its synthesis is related with sporulation and quorum-sensing pathways. However, the effect of this dipeptide on its producer is obscure. To elucidate this effect, our research group constructed a bacilysin non-producer strain of B. subtilis named as OGU1. By the use of proteomic methods of 2DE MALDI-TOF MS/MS and nanoLC-MS/MS the differentially expressed proteins of OGU1 in comparison to parental strain PY79 was identified. The number of differentialy expressed proteins was rather high, being generaly underrepresented, and these proteins were mostly related with sporulation and germination pathways. In the present study, the results of comparative proteomics were validated with RT-qPCR analysis of selected 20 genes. Certain phenotypic deficiencies/inadequacies regarding spore pigmentation, lysozyme and chloroform resistance of spores, spore coat protein profile as well as spore germination kinetics were next observed in OGU1. Protein-free and bacilysin containing culture broth concentrate supplementation to growing OGU1 cultures partially or entirely rescued all of these deficient phenotypes, indicating a pleiotropic role of bacilysin on sporulation and germination of its producer.


Stability of a recombinant plasmid carrying alpha-amylase gene in Bacillus subtilis
Gültekin, Güzin Candan; Bayındırlı, Alev (1998-03-01)
Plasmid pSB6 is a streptococcal recombinant plasmid carrying the alpha-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher ...
Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus
Tunca, S; Yilmaz, EI; Piret, J; Liras, P; Özcengiz, Gülay (Elsevier BV, 2004-09-01)
Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum P-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete...
Comparative transcriptomics of bacillus subtilisbacilysin-negative genotype with particular reference to bacilysin formation
Kocabay, Naz; Özcengiz, Gülay; Department of Molecular Biology and Genetics (2021-2-15)
The Gram-positive model prokaryote Bacillus subtilisis a non-pathogenic organism that is known for producing a wide variety of secondary metabolites with pharmacological andantimicrobial activities.The smallestdipeptideantibiotic bacilysin is among the many secondary metabolites that are produced by B. subtilis, and it is composed of L-alanine and L-anticapsin.As shown earlier by our research group, the synthesis of bacilysin is regulated by bacAoperon under the control of quorum sensing and s...
Expression and activity analyses of industrially important extracellular enzymes produced by a bacilysin knock-out mutant of Bacillus Subtilis
Aytekin, Samet; Özcengiz, Gülay; Okay, Sezer; Department of Molecular Biology and Genetics (2018)
Bacilysin, the smallest peptide antibiotic known to date, is produced non-ribosomally by Bacillus subtilis by the collective actions of seven proteins transcribed from bacABCDEF operon and bacG gene. Bacilysin is a two-amino acid peptide composed of L-alanine and a modified amino acid, L-anticapsin. Bacilysin biosynthesis was shown to be strongly regulated by quorum sensing through the actions of global regulator proteins including Spo0K, Spo0H, Spo0A, ComQ/ComX, ComP/ComA as well as several Phr proteins, O...
Proteome-wide analysis of the functional roles of bacilysin biosynthesis in Bacillus subtilis
Özcengiz, Gülay; Demir, Mustafa; Karatas, Ayten Yazgan (2014-07-01)
The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. Most of these metabolites are small peptides that have unusual components and chemical bonds and synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is one of the simplest peptide antibiotics known. It is a dipeptide with an N-terminal Lalanine and an unusual ...
Citation Formats
O. Ertekin, “Phenotypic rescue of a Bacilysin-silenced strain of Bacillus Subtilis with Bacilysin supplementation,” M.S. - Master of Science, Middle East Technical University, 2020.