Complex Oxidation of Apocytochromes c during Bacterial Cytochrome c Maturation

Abstract

c-Type cytochromes (cyts c) are proteins that contain covalently bound heme and that thus require posttranslational modification for activity, a process carried out by the cytochrome c (cyt c) maturation system (referred to as the Ccm system) in many Gram-negative bacteria. It has been established that during cyt c maturation (CCM), two cysteine thiols of the heme binding motif (CXXCH) within apocytochromes c (apocyts c) are first oxidized largely by DsbA to form a disulfide bond, which is later reduced through a thio-reductive pathway involving DsbD. However, the physiological impacts of DsbA proteins on CCM in fact vary significantly among bacteria. In this work, we used the cyt c-rich Gram-negative bacterium Shewanella oneidensis as the research model to clarify the roles of DsbA proteins in CCM. We show that in terms of the oxidation of apocyts c, DsbA proteins are an important but not critical factor, and, strikingly, oxygen is not either. By exploiting the DsbD-independent pathway, we identify DsbA1, DsbA2, and DsbA3 as oxidants contributing to the oxidation of apocyts c and reductants, such as cysteine, to be an effective antagonist against DsbA-independent oxidation. We further show that DsbB proteins are partially responsible for the reoxidization of reduced DsbA proteins. Overall, our results indicate that the DsbA-DsbB redox pair has a limited role in CCM, challenging the established notion that it is the main oxidant for apocyts c. IMPORTANCE DsbA is a powerful oxidase that functions in the bacterial periplasm to introduce disulfide bonds in many proteins, including apocytochromes c. It has been well established that although DsbA is not essential, it plays a primary role in cytochrome c maturation, based on studies in bacteria hosting several cyts c. Here, with cyt c-rich S. oneidensis as a research model, we show that this is not always the case. Moreover, we demonstrate that DsbB is also not essential for cytochrome c maturation. These results underscore the need to identify oxidants other than DsbA/DsbB that are crucial in the oxidation of apocyts c in bacteria.

Identification of an extracytoplasmic function sigma factor that facilitates c‐type cytochrome maturation and current generation under electrolyte‐flow conditions in Shewanella oneidensisMR‐1

“…3B). These growth characteristics of ∆ dsbD , together with results reported previously (Guo et al ., 2019), support the idea that DsbD is essential for anaerobic respiration by MR‐1.…”

“…Expression of genes encoding the thiol‐disulfide interchange proteins DsbA and DsbC and protein‐disulfide reductase DsbD were also upregulated under the electrolyte‐flow condition (Table 2). A previous study has reported that these proteins are involved in c ‐type cytochrome ( c ‐Cyt) maturation (CCM) and DsbD is required for the growth of MR‐1 under anaerobic fumarate‐ and trimethylamine N ‐oxide‐reducing conditions (Guo et al ., 2019). Given that the extracellular electron transfer (EET) pathway in MR‐1 comprises a number of c ‐Cyts, including an inner membrane‐anchored c ‐Cyt (CymA) and an outer membrane (OM) c ‐Cyt complex (comprised of MtrA, MtrB, OmcA and MtrC) (Kouzuma et al ., 2015; Shi et al ., 2016), it is reasonable to consider that the upregulation of the CCM‐related genes facilitates EET in MR‐1 cells.…”

“…5), demonstrating that DsbD is critical for EET in MR‐1. This idea is reasonable because it has been reported that the loss of DsbD completely prevents c ‐Cyts from maturation (Guo et al ., 2019). CLSM images of WE‐attached cells showed that when compared to MR‐1 AFP and ∆SO_3096 AFP , ∆ dsbD AFP formed sparser biofilm containing a limited number of bacterial cells (Fig.…”

Promoting Extracellular Electron Transfer of Shewanella oneidensis MR-1 by Optimizing the Periplasmic Cytochrome c Network

“…It could be noticed that the coulomb efficiencies obtained in this and early studies (Li et al, 2018) are low, implying the presence of other EAs. Possible candidates would be small redox-active molecules generated during cellular metabolism, such as fumarate, cysteine, and even inorganic sulfur species and nitrogen species (Flynn et al, 2014;Guo et al, 2019). In addition, the output voltage peak of our terminal engineered strain is lagging behind the WT.…”

Structural bioinformatic analysis of DsbA proteins and their pathogenicity associated substrates

“…Bioinformatic and functional studies have revealed that many bacteria encode more than one DsbA [4] , [5] , [19] . Examples include uropathogenic E. coli (UPEC) strain CFT073 that possesses a canonical DsbA and an accessory DsbA homologue DsbL [19] , [54] or the widely distributed facultative anaerobe Shewanella oneidensis , which has a complex DSB machinery with four DsbA (DsbA1-4) and two DsbB (DsbB1-2) homologues [59] . Similarly, Salmonella enterica ( Se ) encodes four DsbA-like proteins [41] , namely DsbA, DsbL, SrgA [42] , a virulence plasmid-encoded DsbA-like protein, and ScsC [60] , [61] , a DsbA-like protein that protects against copper toxicity [57] , [62] .…”