Studies On The Physiological Roles Of Periplasmic Cytochromes C In Sulfite Oxidation And Extracellular Electron Transport In Shewanella

Cytochrome c（c-Cyt）,the hemoproteins with covalently attached heme cofactors,as electron carriers and oxidoreductases are widely involved in energy transfer processes,such as respiration and photosynthesis in organisms.Shewanella,a large group of Gram-negative facultative anaerobes,are renowned for their unparallel respiratory（reduction of electron acceptors）diversity,and have shown great application potential in bioenergy and bioremediation.These characteristics are mainly attributed to a large number of c-Cyts,which vary in sequence,structure and functionality;for instance,Shewanella oneidensis,the research model of the genus,possesses 42 different c-Cyts in total.Nearly half of S.oneidensis c-Cyts have been investigated and understandings of their function and physiological impacts are more or less established.However,we still know little about the remaining half,especially those located in the periplasm with low molecular mass and lacking catalytic activity,called small periplasmic function-unknown c-Cyt（SPFU c-Cyt）This dissertation focused on these SPFU c-Cyts,and made attempts to uncover the metabolic pathways in which certain SPFU c-Cyts are involved,and to explore the effects of a periplasmic network composed of the SPFU c-Cyts on extracellular electron transfer（EET）,the best recognized characteristics of S.oneidensis.In silicon analysis of the SPFU c-Cyts encoded in S.oneidensis revealed that three of their encoding genes are clustered on the genome and are all annotated for proteins as heme subunits of sulfite dehydrogenase.Sulfite is a chemically active sulfur-containing inorganic compound,which can be utilized as an electron acceptor by a variety of microorganisms.The ability of S.oneidensis to reduce sulfite has been known for more than 3 decades,but whether it can oxidize sulfite remains unaddressed.In this study,we discovered that sulfite oxidation in S.oneidensis is catalyzed by an atypical sulfite dehydrogenase in the periplasm,and sulfite is preferentially oxidized by cells in the presence of oxygen rather than reduced to produce H₂S.This atypical sulfite dehydrogenase consists of a highly conserved catalytic subunit and three single heme SPFU c-Cyts.These three SPFU c-Cyts transfer the electrons extracted from the sulfite oxidation to oxygen through cyt cbb₃oxidase（the enzyme dictating oxygen respiration under normal condition）,a process which is essential for sulfite oxidation.Genomic analysis suggests that such sulfite dehydrogenases exist scarcely in bacteria,only in five genuses such as Shewanella and Vibrio.An interesting finding is that cyt bd oxidase（the enzyme secondary to cyt cbb₃for oxygen respiration）of S.oneidensis is sensitive to sulfite,providing an additional dimension to the understanding of the role of cyt bd oxidase in combating environmental stresses.EET is a well-known signature of S.oneidensis,but its low efficiency has been a major bottleneck for S.oneidensis to be exploited as an electroactive biocatalyst in bioelectrochemical systems,such as microbial fuel cell（MFC）.Although it is well established that a SPFU c-Cyts network plays a critical role in regulating EET efficiency,the understanding of the network in terms of structure and electron transfer activity is obscure and partial.In this work,we attempted to systematically investigate the impacts of the network components on EET in their absence and overproduction individually in MFCs.We found that overexpression of cct A leads to accelerated electron transfer between Cym A and the Mtr system,which function as the Inner-membrane primary quinol oxidase and the outer membrane（OM）electron hub in EET.In contrast,nap B,fcc A,and tsd B in excess severely impair EET,reducing EET capacity in MFCs by more than 50%.Based on the results from both strategies,a series of engineered strains lacking fcc A,nap B,and tsd B in combination while overproducing cct A were tested for a maximally optimized c-Cyt network.A strain depleted of all nap B,fcc A,and tsd B with cct A overproduction achieved the highest maximum power density in MFC（436.5 m W/m²）,～3.62-fold higher than that of wild type（WT）.By revealing that optimization of periplasmic c-Cyt composition is a practical strategy for improving EET efficiency,our work underscores the importance in understanding physiological and electrochemical characteristics of c-Cyts involved in EET.In conclusion,we firstly illustrated the physiological role of three SPFU c-Cyts in this study,and found that they could act as electron transfer subunits in sulfite oxidation pathway of S.oneidensis.Secondly,we confirmed that a SPFU c-Cyt network is able to regulate EET efficiency of S.oneidensis.The results obtained,on the one hand,expand the current understanding of the functions and physiological roles of specific c-Cyts,and on the other hand,by revealing the promotion and competition relationship in function between the same type of c-Cyts,provide a reference for proper optimization of periplasmic c-Cyt composition to enhance EET efficiency.