CitAB Two-Component System-Regulated Citrate Utilization Contributes toVibrio choleraeCompetitiveness with the Gut Microbiota

ABSTRACTCitrate is a ubiquitous compound and can be utilized by many bacterial species, including enteric pathogens, as a carbon and energy source. Genes involved in citrate utilization have been extensively studied in some enteric bacteria, such asKlebsiella pneumoniae; however, their role in pathogenesis is still not clear. In this study, we investigated citrate utilization and regulation inVibrio cholerae, the causative agent of cholera. The putative anaerobic citrate fermentation genes inV. cholerae, consisting ofcitCDEFXG,citS-oadGAB, and the two-component system (TCS) genescitAB, are highly homologous to those inK. pneumoniae. Deletion analysis shows that thesecitgenes are essential forV. choleraegrowth when citrate is the sole carbon source. The expression ofcitCandcitSoperons was dependent on citrate and CitAB, whose transcription was autorepressed and regulated by another TCS regulator, ArcA. In addition, citrate fermentation was under the control of catabolite repression. Mouse colonization experiments showed thatV. choleraecan utilize citratein vivousing the citrate fermentation pathway and thatV. choleraelikely needs to compete with other members of the gut microbiota to access citrate in the gut.

Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

ABSTRACTOxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm ofLactococcus lactisILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049–4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases ofL. lactisaccept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transaminationin vivowas further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.