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This dissertation, Expression Profiling of Bacillus Subtilis Sulfur Responsive Genes Using S-methyl-cysteine (SMeC) as Sole Sulfur Source by Yee-leng, Daniel, Yap, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Expression Profiling Of Bacillus Subtilis Sulfur responsive Genes Using S- Methyl-Cysteine (SMeC) as Sole Sulfur Source Submitted by Daniel Yap Yee Leng for the degree of Doctor of Philosophy at The University of Hong Kong (HKU) in March 2006 Sulfur is an essential metabolite found in amino acids, oligopeptides, coenzymes and various co-factors which play a critical role in combination in the construction of biomass and electrochemical functions. The S-H sulfhydryl group within the cysteine residue in exported proteins also helps to maintain protein structure by forming a disulfide bond via cysteine oxidation. This redox cycle is versatile, and acts to mitigate oxidative stress in all aerobic organisms. Furthermore, all proteins in cells are translated, beginning with methionine. The Gram-Positive bacterium Bacillus subtilis can use a variety of compounds as the sole sulfur source, including methionine (in contrast to Escherichia coli), cysteine, inorganic sulfate, sulfonates, methylthioribose (MTR) and S-methyl- cysteine (SMeC). However, the metabolism of sulfur compounds remains incompletely understood. Many proteins that are induced by an excess or repressed by a shortage of sulfur are of unknown functions or without experimental characterization. To date, the only sulfur-related regulators known in B. subtilis are CysL, YtlI, YrzC (CymR) and Spx. Nor is it entirely clear how availability of sulfur might influence the activities of these regulators. To explore the sulfur metabolism of B. subtilis, we used a genome-wide macroarray to interrogate the changes in gene-transcripts during the growth of the B. subtilis wild-type strain in the presence of four different sulfur sources (including methionine and its closest analog S-methyl-cysteine). All four sulfur sources are known to support bacterial growth, though at varying rates depending on sulfur assimilation efficiency. Consistency of the arrays was explored using the operon criteria, to confirm that genes present in operons were indeed co- varying. The expression data was initially analyzed using two statistical methods to distinguish a coherent set of sulfur responsive genes (G ) whose relative sulfur strengths of induction differed significantly depending on the sulfur source used. The operons assigned to particular gene functional groups (referred to as GATs) were found to be unevenly distributed. This coordinative gene expression level indicated that biologically-related genes were systematically regulated in response to sulfur source perturbations. Many G genes belong to the cell sulfur protective responses during atypical conditions controlled by HrcA, CtsR, SigD (flagellation), Spo0A and SpoIIID (sporulation). In conditions where sulfur- containing amino acids were scarce, one of the three peptide transporting systems was induced to import extra-cellular nutrients. Unexpectedly, a strong transcriptional repression of arginine biosynthesis and transport operons was observed in cells grown in SMeC and MTR. Subsequently, G genes were clustered into co-expressed operon sets sulfur considering their gene co-variations in four sulfur sources. Then, standard motif searching tools was used to search for the conserved nucleotides in the regulatory regions of each co-expressed operon set. Finally, two putative

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