Transcriptional Control of the Citrate-Inducible citMCDEFGRP Operon, Encoding Genes Involved in Citrate Fermentation in Leuconostoc paramesenteroides

Abstract

In this study we describe the expression pattern of the Leuconostoc paramesenteroides citMCDEFGRP operon in response to the addition of citrate to the growth medium. An 8.8-kb polycistronic transcript, which includes the citMCDEFGRP genes, was identified; its synthesis was dramatically induced upon addition of citrate to the growth medium. We also found that expression of the cit operon is subjected to posttranscriptional regulation, since processing sites included in four complex secondary structures (I, II, III, and IV) were identified by Northern blot analysis and mapped by primer extension. Upstream of the citMCDEFGRP operon a divergent open reading frame, whose expression was also increased by citrate, was identified by DNA sequencing and designated citI. The start and end sites of transcription of the cit operon and citI gene were mapped. The start sites are separated by a stretch of 188 bp with a very high A؉T content of 77% and are preceded by transcriptional promoters. The end sites of the transcripts are located next to the 3 end of two secondary structures characteristic of -independent transcriptional terminators. The effect of the citI gene on expression of the cit operon was studied in Escherichia coli. The presence of the citI gene in cis and in trans resulted in increased activity of the cit promoter. These data provide the first evidence that citrate fermentation in Leuconostoc is regulated at the transcriptional level by a transcriptional activator rather than by a repressor.Citrate metabolism is carried out by only a few strains of lactic acid bacteria. This metabolic ability is invariable linked to endogenous plasmids that contain the gene encoding the transporter responsible for citrate uptake from the medium. Citrate transporters (CitPs) have been found in strains belonging to the genera Lactococcus and Leuconostoc, bacteria in which the mechanism of citrate fermentation has been studied in detail (1, 14-16). The first step in the breakdown of citrate inside the cell involves its conversion to acetate and oxalacetate by citrate lyase, a three-subunit enzyme (2). In the next step, oxalacetate is decarboxylated by oxalacetate decarboxylase, yielding pyruvate and carbon dioxide (for a review, see reference 9). The pathway generates a proton motive force (PMF) by a secondary mechanism (10). Electrogenic exchange of divalent citrate and monovalent lactate, catalyzed by CitP, efficiently generates a membrane potential, inside negative (17). Moreover a pH gradient (inside alkaline) is formed by the consumption of scalar protons in the decarboxylation of oxalacetate (10). Together, the membrane potential and pH gradient constitute the PMF, which seems to contribute significantly to the growth advantage observed during cometabolism of citrate and glucose in both Lactococcus and Leuconostoc (14,17).Like all the known citrate lyases, the Leuconostoc enzyme forms a functional complex of three proteins: a ␥ subunit (acyl carrier protein [ACP]), a ␤ subunit (citryl-S-ACP lyase), and an ␣ subunit (citrate...

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

“…The answer is provided by the different induction profiles of the citrate metabolic pathways in Leuconostoc and Lactococcus species. In Leuconostoc mesenteroides, the enzymes of the pathway are induced by the presence of citrate in the medium, while in L. lactis, the induction additionally requires a low pH in the medium, suggesting a role in acid stress (40,85,89,92,93,95). The conversion of carbohydrate to lactic acid results in acidification of the medium, which eventually inhibits growth.…”

Characterization of an oxaloacetate decarboxylase that belongs to the malic enzyme family

“…The citM gene from W. paramesenteroides [11] and L. lactis [12] and mae gene from Lc. mesenteroides [22] were described as potentially encoding the oxaloacetate decarboxylase.…”

“…Transcriptional analysis showed that in W. paramesenteroides, the genes encoding the citrate permease (citP), the citrate lyase complex (citDEF) and a putative ME (citM) are transcribed in a polycistronic unit (citMCDEFGRP) thus constituting the cit operon. The expression of this operon is increased in the presence of citrate [11]. In L. lactis CRL264, the citrate utilization genes are organized in two different operons: a chromosomal cit operon including the genes citM-CDEFXG (in a molecular organization similar to W. paramesenteroides) and a plasmidic operon including the citP gene.…”

CitI, a Transcription Factor Involved in Regulation of Citrate Metabolism in Lactic Acid Bacteria

“…Despite that, no regulator involved in the control of the expression of citrate utilization genes has been characterized in LAB. The dairy Weissella paramesenteroides J1 carries the cit operon, which includes the genes encoding the oxaloacetate decarboxylase (citM), the citrate lyase active complex (citCDEFG), and the citrate permease (citP) (14) (Fig. 1A), which are key proteins involved in the citrate fermentation pathway.…”

“…Among them, only DeoR has been purified and has had its interactions with its operators investigated (24). In a previous study, we demonstrated that the transcription of the citI gene and the cit operon is induced when the J1 strain is grown in the presence of citrate (14). Overexpression of CitI in Escherichia coli resulted in trans activation of the Pcit promoter, suggesting that CitI is a transcriptional regulator (14).…”