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.
Highlights
Within the Enterobacteriaceae family, a distinction is made between mixed-acid (e.g., Escherichia, Salmonella, and Shigella) and 2,3-butanediol fermenters (e.g., Klebsiella, Serratia, and Enterobacter) based on their fermentation end products produced during sugar fermentation
We previously showed that transfer of the S. plymuthica RVH1 budAB operon conveys to Escherichia coli the capacity to produce acetoin, to prevent lethal medium acidification and to reverse acidification (Vivijs et al, 2014a)
The observation that acetoin-producing S. flexneri showed reduced acidification in the exponential growth phase, but did not deacidify the medium during the stationary phase (Figure 1), EFFECT OF HYDROGENASE 3 INACTIVATION ON FERMENTATIVE GROWTH OF ACETOIN-PRODUCING E. coli To characterize in more detail the role of formate disproportionation on the capacity of E. coli to attenuate medium acidification during fermentative growth, we constructed a clean deletion of the hycE gene
Summary
Within the Enterobacteriaceae family, a distinction is made between mixed-acid (e.g., Escherichia, Salmonella, and Shigella) and 2,3-butanediol fermenters (e.g., Klebsiella, Serratia, and Enterobacter) based on their fermentation end products produced during sugar fermentation. Acetoin is reduced to 2,3-butanediol by the 2,3-butanediol dehydrogenase (BDH), which can catalyze the reversed reaction Each of these three reactions consumes an intracellular proton, and this potentially explains the observed stationary-phase deacidification. Our results suggested the involvement of a deacidification mechanism different from proton consumption during acetoin production To identify this mechanism, we performed random transposon mutagenesis in budAB-containing E. coli searching for mutants that lost their stationary-phase deacidification capacity but still produced acetoin. We performed random transposon mutagenesis in budAB-containing E. coli searching for mutants that lost their stationary-phase deacidification capacity but still produced acetoin This led us to identify the FHL complex as the primary deacidification mechanism in 2,3-butanediol-fermenting Enterobacteriaceae
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