FNR/ArcA-2,3-butanediol regulatory axis enhances glucose uptake and adhE expression in high alcohol-producing Klebsiella pneumoniae.

Previously, we found that Klebsiella pneumoniae is a high-alcohol producer, which contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, the molecular mechanisms underlying its high-alcohol production remain unclear. By comparing biological phenotypes of K. pneumoniae with those of Escherichia coli, we found that the former is better at metabolizing high concentrations of glucose, exhibits faster growth rates, and produces higher levels of alcohol, with the 2,3-butanediol (2,3-BD) pathway being the key determinant underlying these differences. More importantly, species possessing the 2,3-BD pathway, including Klebsiella aerogenes, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter hormaechei, have all demonstrated high-alcohol production capabilities similar to that of K. pneumoniae, suggesting a conserved mechanism for alcohol production among 2,3-BD-positive Enterobacteriaceae. Deletion of the 2,3-BD pathway reduced the expression of the phosphotransferase system (PTS) and the alcohol dehydrogenase gene adhE, which normally confer enhanced glucose metabolism and alcohol production. Pathway deletion also downregulated multiple virulence determinants, including capsular polysaccharides (CPS), lipopolysaccharides (LPS), and type 1 and 3 fimbriae, thereby attenuating virulence in a murine pneumonia model. In addition, the regulator FNR enhanced alcohol production by upregulating adhE and 2,3-BD pathway genes, whereas ArcA activated the 2,3-BD pathway in an ArcB-independent manner, revealing a non-canonical signaling mechanism that couples metabolic adaptation with virulence. Collectively, our findings identify the "FNR/ArcA-2,3-BD pathway axis" as a key molecular mechanism driving high alcohol production in K. pneumoniae, highlighting the 2,3-BD pathway as a key link between bacterial metabolism and virulence and a potential therapeutic target for controlling K. pneumoniae infections.IMPORTANCEKlebsiella pneumoniae has emerged as a novel cause of NAFLD due to its high alcohol-producing capacity. However, the molecular basis of this phenotype remains unclear. In this study, we demonstrate that the FNR/ArcA-2,3-BD pathway axis drives high levels of alcohol production by enhancing glucose-PTS system and adhE expression, and that this pathway is conserved among Enterobacteriaceae with strong alcohol-producing abilities. Moreover, loss of the 2,3-BD pathway reduces the expression of multiple virulence factors and attenuates pathogenicity in a murine pneumonia model. These findings identify the FNR/ArcA-2,3-BD axis as a potential target for mitigating excessive alcohol production and virulence in bacteria.