Substrate-Level Phosphorylation Is the Primary Source of Energy Conservation during Anaerobic Respiration of Shewanella oneidensis Strain MR-1

Abstract

It is well established that respiratory organisms use proton motive force to produce ATP via F-type ATP synthase aerobically and that this process may reverse during anaerobiosis to produce proton motive force. Here, we show that Shewanella oneidensis strain MR-1, a nonfermentative, facultative anaerobe known to respire exogenous electron acceptors, generates ATP primarily from substrate-level phosphorylation under anaerobic conditions. Mutant strains lacking ackA (SO2915) and pta (SO2916), genes required for acetate production and a significant portion of substrate-level ATP produced anaerobically, were tested for growth. These mutant strains were unable to grow anaerobically with lactate and fumarate as the electron acceptor, consistent with substrate-level phosphorylation yielding a significant amount of ATP. Mutant strains lacking ackA and pta were also shown to grow slowly using N-acetylglucosamine as the carbon source and fumarate as the electron acceptor, consistent with some ATP generation deriving from the Entner-Doudoroff pathway with this substrate. A deletion strain lacking the sole F-type ATP synthase (SO4746 to SO4754) demonstrated enhanced growth on N-acetylglucosamine and a minor defect with lactate under anaerobic conditions. ATP synthase mutants grown anaerobically on lactate while expressing proteorhodopsin, a light-dependent proton pump, exhibited restored growth when exposed to light, consistent with a proton-pumping role for ATP synthase under anaerobic conditions. Although S. oneidensis requires external electron acceptors to balance redox reactions and is not fermentative, we find that substrate-level phosphorylation is its primary anaerobic energy conservation strategy. Phenotypic characterization of an ackA deletion in Shewanella sp. strain MR-4 and genomic analysis of other sequenced strains suggest that this strategy is a common feature of Shewanella.Shewanella oneidensis strain MR-1 is a nonfermentative, facultative anaerobe which respires various substrates, including oxygen, soluble metals, insoluble iron and manganese oxide minerals, electrodes, and organic compounds (8,12,18,22). Other bacteria with the ability to respire electrodes and oxide minerals, such as Geobacter and Geothrix, oxidize acetate to carbon dioxide (4, 7, 9), consistent with these organisms generating ATP primarily from oxidative phosphorylation rather than substrate-level phosphorylation. Yet, an examination of metabolic end products and a variety of central metabolism and flux analyses of MR-1 show that acetate is the major product under anaerobic conditions (18,27,29,31). The general anaerobic metabolism model for MR-1, as depicted in Fig.

Modeling Substrate Utilization, Metabolite Production, and Uranium Immobilization in Shewanella oneidensis Biofilms

“…The parameters derived from the experimental data and the values from the literature were not significantly different from each other, except the f Ac/ED value, which was significantly lower than the value obtained from a similar experiment by Cao et al (2012) (Table 3). The maximum specific growth rate calculated from the model cell yield and maximum specific substrate utilization rate was 0.08 h −1 , which is close to the reported value of 0.087-0.125 h −1 (Tang et al, 2007a;Hunt et al, 2010). The maximum specific growth rate of bacteria in biofilm is usually close to or identical to that found in suspension cultures (Characklis, 1990;Okabe et al, 1994;Nielsen et al, 1997).…”

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

“…PRs have also been heterologously expressed in non-photosynthetic hosts (5,(21)(22)(23)(24)(25). For example, the introduction of a PR in Shewanella oneidensis increased the pmf, which resulted in increases in electrical current generation, lactate uptake, and survival under starvation conditions (23,24).…”

“…For example, the introduction of a PR in Shewanella oneidensis increased the pmf, which resulted in increases in electrical current generation, lactate uptake, and survival under starvation conditions (23,24). In Escherichia coli, the pmf generated by a PR resulted in ATP synthesis (22), was able to drive the flagellar motor (21), and could be used to significantly increase the production of hydrogen by a co-introduced hydrogenase (25,26).…”

The Mtr Pathway of Shewanella oneidensis MR‐1 Couples Substrate Utilization to Current Production in Escherichia coli

“…[8] When grown under conditions for anaerobic metabolism of lactate, wild-type E. coli cannot ferment and is limited in its ability for anaerobic respiration. Under these conditions it is likely that E. coli generates ATP and maintains redox balance using an alternative metabolism, such as overflow metabolism.…”

“…In S. oneidensis, the anaerobic metabolism of lactate co-evolved with the Mtr pathway and combines elements of both respiratory and fermentative pathways. [8] This metabolism requires a terminal electron…”