Mesa Redonda: Citrate metabolism is a diverse and highly regulated trait in lactic acid bacteria

BLANCATO, VS

San Miguel de Tucumán

Simposio; V Simposio Internacional de Bacterias Lácticas (SIBAL); 2016

CERELA

In lactic Acid Bacteria (LAB) citrate fermentation is a strain-dependent trait directly involved in cheese flavour and quality. This metabolism involves the intracellular conversion of citrate to oxaloacetate (OAA) and acetate by citrate lyase. OAA is then decarboxylated to pyruvate by oxaloacetate decarboxylase (OAD), which is further metabolized towards aroma compounds (diacetyl, acetoin and butanediol). Our studies on this metabolism reflect a fine-tuned regulation at transcriptional level.In the starter culture Lactococcus lactis biovar. diacetylactis the genes associated with citrate metabolism (cit cluster) are organized in two operons, one involved in citrate transport (citQRP plasmidic operon) and the other in its conversion to pyruvate (citM-citI-CDEFXG operon). Both operons are transcriptionally induced at low pH, by a still unknown mechanism. Whereas, in Weissella paramesenteroides (non-starter LAB, NS-LAB) CitI was identified as the transcriptional factor (DeoR family) that induce the cit cluster by binding to two AT-rich operator sites located between citI and citMCDEFGP operons; no repression by PTS sugars was detected in this pathway.The molecular mechanism of induction of the cit divergent operons (citHO and oadHDBcitCDEFoadAcitXG) in Enterococcus faecalis (NS-LAB) relies on the activator CitO (GntR superfamily). In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing their expression. Structure-based site-directed mutagenesis analysis allowed to obtain clues on the ligand-protein interaction, Arg97A and His191A changes resulted in decreased binding affinities for citrate. Further studies indicated that the binding of Ni2+ or Zn2+ cations to the C-terminal domain of the protein improved CitO-citrate interaction. Furthermore, we found that PTS-sugars exerts a transcriptional repression through CcpA-dependent (via three active cre sites) and CcpA-independent mechanisms which allows to control the expression of the citHO operon as well as the catabolic operon oadHDBcitCDEFoadAcitXG.The cit clusters of E. faecium strains (NS-LAB) can be classified as type I and II according to the genes present in the operons. Type I encodes CitI regulator, CitM cytoplasmic soluble OAD and CitP citrate transporter; whereas type II encodes CitO, membrane OAD complex and CitH citrate transporter. In type I microorganisms, induction of the pathway seems to be mediated by CitI in the presence of citrate and repressed by PTS sugars.