The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis

Abstract

The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.Shewanella oneidensis strain MR-1 (MR-1) is a facultative anaerobe capable of respiring a variety of substrates, including various metals and metal oxides, a phenotype that is important for bioremediation and metal cycling in natural environments (22,53). At near-neutral pH, Fe(III) and Mn(IV) often are present as insoluble oxide minerals. Dissimilatory metal-reducing bacteria such as MR-1 have developed pathways to transfer electrons from the interior of the cell to these external terminal electron acceptors. In some bacteria, these pathways also can transfer electrons to electrodes, which can be harnessed for renewable energy and remote biosensor applications (23,26,27). Beyond increasing our understanding of this unusual process, applying anaerobic microbial extracellular respiration to new technologies requires a thorough understanding of the molecular dynamics and cellular physiology of electron source utilization (substrate oxidation) and the reduction of insoluble terminal electron acceptor(s). There are four proposed mechanisms to explain how insoluble substrates are reduced by Shewanella: (i) direct contact, (ii) electron shuttling, (iii) chelation, and (iv) electrically conductive appendages (reviewed in reference 18). We will focus on the first three strategies here.Flavins recently ...

Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals

“…Electrodes were not reduced by MR-1 mutants that lacked MtrC. However, in cell suspension assays, MtrClacking strains reduced flavins at 50% the rate observed with the wild-type MR-1 (28). Using the proteoliposome system, we demonstrated that electrons can be transferred directly from MtrCAB to solid-phase Fe(III) oxides only when the entire MtrCAB complex is inserted.…”

“…In vivo, bioelectrochemical experiments have shown the entire Mtr pathway is required to enable S. oneidensis MR-1 biofilms to donate electrons to and accept electrons from extracellular electrodes (28,29). Electrodes were not reduced by MR-1 mutants that lacked MtrC.…”

IRMPD spectroscopy of metalated flavins: structure and bonding of Mq+–lumichrome complexes (Mq+ = Li+–Cs+, Ag+, Mg2+)

“…of insoluble Fe(III) oxide minerals with different electrodes, in which flavins, most notably riboflavin, play a decisive role. 9 The interaction of (alkali) metal ions with lumichrome (LC) forming a metal flavoquinone chelate, the smallest member of the flavins, can serve as a benchmark system for elucidating such processes at the molecular level. Such metal chelates are transient species in aqueous solution.…”

Controlling electron transfer at the microbe–mineral interface

“…Of the Shewanella OMMCs, the S. oneidensis MtrC and OmcA cytochromes that are the subject of the Okamato et al paper (1) have been the most widely studied. They are exported to the extracellular environment by the type II secretion system (10), and omcA mtrC mutants are severely compromised for respiratory mineral Fe(III) reduction and electron transfer to anodes in microbial fuel cells (11,12). The OmcA protein may form a 2:1 complex with MtrCAB, resulting in a system with 40 hemes that is referred to as the "OmcA-MtrCAB" complex in the paper of Okamoto et al (1).…”