| Summary: | 博士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 94 === Swarming migration of Serratia marcescens requires both flagellar motility and cellular differentiation and is a population-density-dependent behavior. While the flhDC and quorum-sensing systems have been characterized as important factors regulating S. marcescens swarming, the underlying molecular mechanisms are currently far from being understood. Serratia swarming is thermoregulated and is characterized by continuous surface migration on rich swarming agar surfaces at 30 degrees C but not at 37 degrees C. To further elucidate the mechanisms, identification of specific and conserved regulators that govern the initiation of swarming is essential. We performed transposon mutagenesis to screen for S. marcescens strain CH-1 mutants that swarmed at 37 degrees C. Analysis of a "precocious-swarming" mutant revealed that the defect in a conserved dapASm-nlpBSm genetic locus and pirinSm gene which are closely related to the synthesis of bacterial cell wall peptidoglycan and pyruvate dehydrogenase (PDH) enzyme activity, respectively. In dapASm-nlpBSm mutant, further complementation and gene knockout studies showed that nlpBSm, which encodes a membrane lipoprotein, NlpBSm, but not dapASm, is specifically involved in swarming regulation. We also present evidence that DapASm is involved in the determination of cell-envelope-associated phenotypes and that NlpBSm is involved in the regulation of swarming motility. In S. marcescens PirinSm, protein pull-down and bacterial two-hybrid assays followed by SDS-PAGE and electrospray ionization (ESI) MASS-MASS analyses showed pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with PirinSm. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity were inhibited by PirinSm in S. marcescens CH-1. Accordingly, the PDH E1, PDH enzyme complex activities and cellular ATP concentration were increased for up to 250 %, 140 % and 220 % respectively in S. marcescens CH-1 pirinSm mutant. Our results showed that PirinSm plays a regulatory role in the process of pyruvate catabolism to acetyl-CoA through interaction with PDH E1 subunit and inhibiting PDH complex activity in S. marcescens CH-1, suggesting PirinSm be an important protein involved in determining direction of pyruvate metabolism on whether to go towards the tricarboxylic acid (TCA) cycle or the fermentation pathways.
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