New insights in the citrate fermentation of Enterococcus faecium IQ23 and its relationship with aroma production

QUINTANA, I; MARTINO, G; ESPARIZ, M; MAGNI, C; BLANCATO, VS

Rosario

Congreso; IX Congreso Argentino de Microbiología General. SAMIGE; 2013

Sociedad Argentina de Microbiología General

Most enterococci are considered part of the natural flora occurring in the gastrointestinal tract of human and domestic animals. These bacteria emerged as opportunistic pathogens due to its natural and acquired antibiotic resistance. Nevertheless, these microorganisms as well as other members of Lactic Acid Bacteria also play an important role in food industry because they contribute to flavor development. In particular citrate fermentation and production of C-4 compounds (diacetyl and acetoin) are essential in dairy fermented products. This metabolism depends on the presence of citrate transporters and the citrate lyase complex, which split citrate into oxaloacetate and acetate. The oxaloacetate is further metabolized to pyruvate and finally to diacetyl and acetoin, increasing the intracellular pH as consequence. Enterococcus faecium is among the less studied members of the genus and though there are some published biochemical studies, they can be diverse and little is known about genetics and regulation of citrate metabolism itself. Previous experiments performed in our laboratory led us to the isolation of E. faecium IQ23. Through a series of PCR and sequencing experiments we could confirm the presence of citF, citI and citP, coding for the alpha subunit of the citrate lyase, a transcriptional regulator which belongs to the SorC family present in Lactococcus lactis and the citrate transporter CitP, member of the 2-hydroxycarboxylate, respectively. Transport experiments using a fluorescent probe in resting cells suggested that citrate transport and metabolism are induced by citrate and the optimal pH is 5,5. We could also infer that the transporter does not exchange citrate for an organic acid such as malate, succinate, pyruvate, lactate or acetate. Besides transport was inhibited when Mg2+ , Ni2+, Cu2+, Ca2+ and Sr2+ were added to the buffer and was restored in the presence of EDTA. Next, we corroborated the uptake of 14 C-citrate in resting cells. In this case E. faecium IQ23 grown in LB supplemented with citrate could incorporate citrate while nor E. faecium IQ23 grown in LB supplemented with glucose neither E. faecium IQ110 (cit- strain) grown in both conditions could. This results support those obtained with fluorescence experiments. Finally we quantified the production of diacetyl and acetoin, both involved in flavour development. Our results threw evidence that this production is dependent on citrate concentration, but is not necessarily associated to its metabolism since we detected aroma compounds when cells were grown without citrate in media supplemented either with glucose or pyruvate. However, we observed a lowering on the production when glucose was added to media containing citrate, suggesting that catabolite repression was taking place. To confirm this hypothesis we searched for CcpA binding sites (CRE sites) in the cit operon promoter and citI sequences finding one putative regulatory region.