Secreted Flavin Cofactors for Anaerobic Respiration of Fumarate and Urocanate by Shewanella oneidensis: Cost and Role

Abstract

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by Shewanella. IMPORTANCE Shewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA. KEYWORDS Shewanella, anaerobic respiration, fitness, flavin S hewanella oneidensis strain MR-1 is a gammaproteobacterium found in a variety of aquatic and marine environments, ranging from sediments to aquatic multicellular organisms (1, 2). As a model organism for dissimilatory metal-reducing bac-

Extracellular electron transfer powers flavinylated extracellular reductases in Gram-positive bacteria

“…These enzymes resemble the Gram-positive system in requiring extracytoplasmic flavins and in receiving electrons from quinones in the cytoplasmic membrane. Despite these similarities, the Shewanella system depends upon a distinct flavin transporter (to provide the noncovalently bound flavin adenine dinucleotide cofactor) and quinol oxidase (47)(48)(49)(50). Convergent evolutionary processes thus seem to have resulted in a similar repertoire of extracytoplasmic activities in Gram-negative and Gram-positive lineages.…”

Post-translational flavinylation is associated with diverse extracytosolic redox functionalities throughout bacterial life

“…The Listeria monocytogenes fumarate reductase and the S. oneidensis urocanate reductase have been shown to be flavinylated 11,12 . In both cases, the flavinylation motif is thought to facilitate electron transfer from electron transport chain components encoded elsewhere on the genome to the enzyme active site 11,13 . These observations thus suggest that FMN-binding domains mediate electron transfer from membrane components to a prevalent class of extracytosolic reductases and highlight another connection between flavinylation and respiration.…”

“…Redox-active extracytosolic flavinylated FMN-binding domains have been identified within five characterized electron transfer systems -- including the cation pumping NQR and RNF complexes ( Figure 1A and 1B ), nitrous oxide and organohalide respiratory complexes ( Figure 1C and 1D ), and a Gram-positive extracellular electron transfer system ( Figure 1E ) 6–10 . In addition, ApbE-flavinylated motifs in homologous extracytosolic fumarate and urocanate reductases facilitate transfer from respiratory electron transport chains 11–13 .…”

Developing a population-state decision system for intelligently reprogramming extracellular electron transfer in Shewanella oneidensis

“…To intelligently reprogram the EET pathways, four decision implementation modules were designed and constructed to reprogram the Mtr conduit (OmcA-MtrCAB) ( 25 ), the tetraheme menaquinol dehydrogenase CymA ( 26 ), the electron shuttle flavin synthesis pathway ( 27 ), and the periplasmic flavin adenine dinucleotide hydrolase UshA, respectively ( Fig. 4 A ) ( 28 ). These modules were subsequently subjected to EET ability detection using a WO 3 probe ( 29 ).…”