Pyruvate and lactate metabolism by Shewanella oneidensis MR-1 under fermentation, oxygen limitation, and fumarate respiration conditions

Abstract

Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O(2), lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O(2) limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.

Fig. 1.

Metabolic pathways implicated in pyruvate fermentation by S. oneidensis MR-1. FDH stands for one or more formate dehydrogenases encoded in the MR-1 genome (SO_0101 to SO_0103, SO_4508 to SO_4511, and SO_4512 to SO_4515). LdhA (SO_0968), fermentative lactate dehydrogenase; PDH (SO_0424 to SO_0426), pyruvate dehydrogenase complex; PflB (SO_2912), pyruvate formate-lyase; Pta (SO_2916), phosphotransacetylase; AckA (SO_2915), acetate kinase; Hyd (SO_3920 and SO_3921) and Hya (SO_2098 and SO_2099), [Fe-Fe] and [Ni-Fe] hydrogenases, respectively. The scheme reflects the predicted localizations of the corresponding enzymes.

Fig. 2.

Growth dynamics of S. oneidensis MR-1 and selected mutants in anaerobic M1 medium supplemented with 20 mM lactate and 30 mM fumarate. Some error bars are smaller than the symbols.

Fig. 3.

Effects of gene deletions on S. oneidensis MR-1 growth (A) and acetate accumulation (B) under O₂-limited conditions in M1 medium supplemented with 90 mM sodium lactate as the sole source of carbon. Experiments were run in controlled bioreactors (see Materials and Methods for details). Samples for data presented here were taken at 16.5 to 17 h after the O₂ limitation phase started. Growth was measured as the optical density at 600 nm. The results are representative of a typical experiment. Experiments were repeated twice, and standard deviations did not exceed 8%.

Fig. 4.

Schematic representation of the proposed spatial localization of enzymes involved in pyruvate reduction. According to the proposed scheme, protons resulting from formate oxidation remain in the periplasm, whereas electrons through the electron transport chain are moved toward Dld-II and used in a reaction of pyruvate reduction, with the latter accompanied by the consumption of two protons in the cytoplasm. This process may therefore lead to PMF generation. Dld-II, oxidative d-lactate dehydrogenase; FDH, formate dehydrogenase.