Exploring The Biosynthesis And Regulation Of Fatty Acids In Shewanella Oneidensis

Shewanella species are well known for their versatile respiration capabilities and wide distribution in environments. Fatty acid (FA), especially unsaturated fatty acid (UFA) and branched-chain fatty acid (BCFA) have crucial roles in membrane biology in organisms ranging from bacteria to humans, but there are few investigations into them in Shewanella. As the membrane composition of Shewanella, at least in part, accounts for their unique physiological characteristics, in this study we take on exploring the FA (mainly UFA) biosynthesis and regulation in Shewanella oneidensis MR-1, the representative strain for the genus.IPTG-inducible protein expression and GFP reporter systems in S. oneidensisWe first construct IPTG-inducible protein expression and GFP reporter systems for S. oneidensis. These systems are verified to be practicable for a series of research subjects in S. oneidensis, including the work in fatty acid biosynthesis and regulation, and used in chapters three, four and five of this thesis.FabR and FadR regulate both aerobic and anaerobic pathways for UFA biosynthesis in S. oneidensisS. oneidensis possesses both aerobic and anaerobic UFA synthesis pathways. The anaerobic pathway introduces a double bond into the growing acyl chain by two specialized proteins, FabA (SO1856) and FabB (SO3072). In the aerobic pathway, fatty acids are desaturated by the only desaturase DesA (SO0197). Both pathways are repressed by FabR (SO0198) directly. The anaerobic pathway is activated by FadR (SO2885) directly, while the aerobic pathway is repressed by FadR indirectly because of the lack of a distinguishable FadR-binding-site in the promoter region of desA. Our results also indicate the cross-talk between the anaerobic and aerobic UFA synthetic pathways in S. oneidensis.Fatty acid composition of S. oneidensis wild type and mutant strains were measured by Gas chromatography-mass spectroscopy (GC-MS). Deletion of fabA or fadR shows different impacts on fatty acid composition. Consistently, growth defect of AfabA under aerobic conditions is weak, significantly differing from that AfadR, which is more severe. Despite this, growth defects of both AfabA and AfadR can be recovered when supplemented with oleate in LB medium.Exploring characteristics of the function and regulation of S. oneidensis FabBTo explore the function and regulation of S. oneidensis FabB, we assessed growth of AfabB, which is as slow as AfadR and much slower than AfabA. Deletion of fabB leads to accumulation of C14, suggesting the insufficient elongation of C14 to C16. AfabB produces more UFA (C14:1), presumably as a means to increase membrane fluidity, while in AfadR, instead, the enhanced fluidity seems to be achieved by elevating the proportion of BCFA (iso/antiso-C15:0). Overexpression of FabB is toxic to S. oneidensis, which deserves further study.FadR regulates transcription of the fabB gene while FabR does not. Nevertheless, there is no distinguishable FadR binding site in the promoter region of fabB. Though fabB promoter activity decreases obviously in AfabA, increased FabA expression doesn not improve fabB promoter activity. Therefore, it seems unlikely that FadR affects the transcription of fabB through FabA. How FadR regulates FabB expression needs further study.Loss of both anaerobic and aerobic pathways (△fabA△desA and AfabB△desA) results in a growth failure, which can be restored by oleate supplementation. Intriguingly, colonies of these double mutants are light-colored. Heme staining demonstrates the low heme contents in the double mutants, implicating an impaired biogenesis of cytochrome c.Exploring reasons for growth defect of S. oneidensis AfadR in minimal mediumUnder aerobic conditions, △fabB and △fadR grow significantly slower than the wild type strain in LB broth and minimal medium (M1). Supplementation of oleate recovers the growth defect of both AfabB and AfadR in LB, but fails to do so with AfadR in M1. pFAC transposon mutagenesis of AfadR in minimal medium containing 0.005% oleate identify prpB (SO0345) and prpF (SO0342) as genes associated with growth defect of AfadR, suggesting an involvement of the methylcitrate cycle. In parallel, isoleucine, a substrate for BCFA, recovers 90% growth defect of AfadR in Ml medium. Therefore, growth defect of AfadR in Ml medium is related to branched chain amino acids metabolism and the methylcitrate cycle.