Date

2017

Author

Tekin İşlerel, Elif

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The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. Bacilysin, being produced and excreted by certain strains of B. subtilis, is a dipeptide antibiotic composed of L-alanine and L-anticapsin which is synthesized enzymatically. As shown earlier by our research group, the expression of bacilysin biosynthetic operon (bacA) is relatively constant during the exponential growth, but increases during the transition between exponential and stationary phases and reaches to its maximal level upon entry into stationary phase (16th hour). We also reported for the first time that the expression of bacA operon is under the control of several elements of quorum sensing global regulatory pathway of sporulation initiation and competence development as well as those of some essential two-component pleiotropic regulator proteins that play the critical role in a variety of cellular functions, including the adaptation to nutrient-limiting conditions, motility, chemotaxis and biofilm formation. More recently, we performed comparative cytosolic proteome analysis in the bacilysin producer B. subtilis PY79 and its bacilysin-knock out derivative, OGU1, by using a combination of 2DE MALDI-TOF/MS and GeLC-MS/MS approaches with the aim of identifying functional roles of bacilysin biosynthesis in its producer. Yet, these studies did not cover extracellular proteins of the organisms which could point to some other important physiological alterations in response to the bacilysin knock out mutation. The secretome of B. subtilis is known to change at the beginning of the sporulation phase to include new membrane- and wall-binding proteins as well as those secreted outside, like new degradative enzymes that may provide the cells with new nutrients and those engaged in detoxification, communication, defense and many more. As a Gram-positive organism, the absence of an outer membrane in Bacillus subtilis simplifies protein secretion pathways and allow the organism to secrete high levels of extracellular proteins. The present study aims at an investigation of dynamic changes in the secretome of B. subtilis in the absence of bacilysin production by employing both 2 DE gel-based and gelfree approaches, to improve protein coverage as a function of time and space. At different time points of 12, 16 and 24 hours of cultivation in glucose-based synthetic medium, the secretome components were analyzed, identified and compared in the parental strain PY79 and its and bacilysin knock out mutant OGU1. LC-MS/MS analyses identified a total of 2075 distinct proteins. Of these, a total of 166 proteins were found to be differentially expressed between two strains. As expected, the numbers of total and differentially expressed proteins identified were much lower in 2DE MALDI-TOF/MS approach, still its results well supported the findings from LC-MS/MS analyses. Overall, the differentially expressed proteins mainly belonged the functional categories of transport and metabolism, genetic processes, lifestyle, coping with stress, sporulation and germination and those unknown. The alterations should mainly be regarded to represent a defense or stress-coping mechanism assuming that the absence of bacilysin is sensed by the mutant as a type of stress condition. The present study greatly aided in a comprehensive understanding of whole proteomic alterations in the bacilysin knock-out mutant that remains to be supported by further functional analyses.

Subject Keywords

Proteomics., Bacillus subtilis., Gene expression., Molecular biology.

URI

http://etd.lib.metu.edu.tr/upload/12621675/index.pdf
https://hdl.handle.net/11511/26982

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Graduate School of Natural and Applied Sciences, Thesis