Formate hydrogen lyase mediates stationary-phase deacidification and increases survival during sugar fermentation in acetoin-producing enterobacteria

Abstract

Two fermentation types exist in the Enterobacteriaceae family. Mixed-acid fermenters produce substantial amounts of lactate, formate, acetate, and succinate, resulting in lethal medium acidification. On the other hand, 2,3-butanediol fermenters switch to the production of the neutral compounds acetoin and 2,3-butanediol and even deacidify the environment after an initial acidification phase, thereby avoiding cell death. We equipped three mixed-acid fermenters (Salmonella Typhimurium, S. Enteritidis and Shigella flexneri) with the acetoin pathway from Serratia plymuthica to investigate the mechanisms of deacidification. Acetoin production caused attenuated acidification during exponential growth in all three bacteria, but stationary-phase deacidification was only observed in Escherichia coli and Salmonella, suggesting that it was not due to the consumption of protons accompanying acetoin production. To identify the mechanism, 34 transposon mutants of acetoin-producing E. coli that no longer deacidified the culture medium were isolated. The mutations mapped to 16 genes, all involved in formate metabolism. Formate is an end product of mixed-acid fermentation that can be converted to H2 and CO2 by the formate hydrogen lyase (FHL) complex, a reaction that consumes protons and thus can explain medium deacidification. When hycE, encoding the large subunit of hydrogenase 3 that is part of the FHL complex, was deleted in acetoin-producing E. coli, deacidification capacity was lost. Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduced lactate and acetate production, but increased glucose consumption and formate and ethanol production. Analysis of a hycE mutant in S. plymuthica confirmed that medium deacidification in this organism is also mediated by FHL. These findings improve our understanding of the physiology and function of fermentation pathways in Enterobacteriaceae.

Deacidification by FhlA-dependent hydrogenase is involved in urease activity and urinary stone formation in uropathogenic Proteus mirabilis

“…The deacidification of the medium requires fhlA and hyf. It has been known that formate, a product of mixed-acid fermentation, can be converted to H 2 and CO 2 by the FHL complex, in a process consuming protons and resulting in medium deacidification, thereby increasing bacterial survival 17 . We hypothesized that uropathogenic P. mirabilis could use the FHL system to consume protons, leading to deacidification of the environment.…”

“…medium deacidification during mix acid fermentation 17 and acid resistance in the anaerobic environment 18 . In addition, hydrogenases of FHL might play a role in virulence by neutralizing hydroxyl free radicals (OH•), providing energy and maintaining acid-base homeostasis 19 .…”

Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists

“…The apparent primary role of the enzyme complexes is to dissipate reductant and detoxify formate during persistence under anoxia. However, three independent studies have indicated that FHL complexes are also critical for acid tolerance in E. coli and S. Typhimurium (79,84,322). These complexes mediate the net consumption of protons from the cytosol (HCOO Ϫ ϩ H ϩ ¡ CO 2 ϩ H 2 ) and, hence, may provide a simple but elegant mechanism to regulate internal pH.…”

FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism

“…When the pH in the periplasmic space of the E. coli cell becomes too acidic, FocA working together with FHL-1 alleviates pH stress [8, 23, 64, 78–80]. In this situation, the proton associated with formic acid might not be relayed back to H209, but instead would be translocated directly into the cytoplasm along with the anion (); this might result from the H209/T91 proton-relay system being overwhelmed by the high concentration of formic acid, with FocA adopting an inwardly directed channel-like function (see ).…”