Transcriptional regulation of the citrate gene cluster of Enterococcus faecalis Involves the GntR family transcriptional activator CitO

Abstract

The genome of the gram-positive bacterium Enterococcus faecalis contains the genes that encode the citrate lyase complex. This complex splits citrate into oxaloacetate and acetate and is involved in all the known anaerobic bacterial citrate fermentation pathways. Although citrate fermentation in E. faecalis has been investigated before, the regulation and transcriptional pattern of the cit locus has still not been fully explored. To fill this gap, in this paper we demonstrate that the GntR transcriptional regulator CitO is a novel positive regulator involved in the expression of the cit operons. The transcriptional analysis of the cit clusters revealed two divergent operons: citHO, which codes for the transporter (citH) and the regulatory protein (citO), and upstream from it and in the opposite direction the oadHDB-citCDEFX-oadA-citMG operon, which includes the citrate lyase subunits (citD, citE, and citF), the soluble oxaloacetate decarboxylase (citM), and also the genes encoding a putative oxaloacetate decarboxylase complex (oadB, oadA, oadD and oadH). This analysis also showed that both operons are specifically activated by the addition of citrate to the medium. In order to study the functional role of CitO, a mutant strain with an interrupted citO gene was constructed, causing a total loss of the ability to degrade citrate. Reintroduction of a functional copy of citO to the citO-deficient strain restored the response to citrate and the Cit(+) phenotype. Furthermore, we present evidence that CitO binds to the cis-acting sequences O(1) and O(2), located in the cit intergenic region, increasing its affinity for these binding sites when citrate is present and allowing the induction of both cit promoters.

FIG. 1.

(A) Genetic organization of cit genes involved in citrate utilization in E. faecalis, S. mutans, L. sakei, L. casei, and S. pyogenes. The shaded box indicates the organization of the highly conserved citDEF genes in the different cit clusters. (B) Phylogenetic tree corresponding to proteins of the GntR family. A multiple sequence alignment was computed using MEGA3. GntR-like regulators were classified in seven subfamilies according to the clusters of branches that emerged from the constructed tree. The shaded ellipse shows the new subfamily constituted by citO homologs.

FIG. 2.

Transcriptional analysis of the cit operons in E. faecalis. (A) Schematic representation of the citHO and oadHDB-citCDEFX-oadA-citMG operons. PcitHO and PcitCL indicate the promoters of the cit operons. The secondary structures T1 and T2 represent putative Rho-independent transcriptional terminators. (B) Northern blot analysis of E. faecalis ATCC 29212 strain. Cells were cultivated in LB or LBC (C) medium. Total RNA was hybridized against specific probes: citH (probe I), citEF (probe II), and citM (probe III). The major RNA species observed in the Northern blot are indicated by arrows.

FIG. 3.

Primer extension analysis of the transcriptional start sites of citHO (A) and oadHDB-citCDEFX-oadA-citMG operons (B). The images show primer extension experiments performed with RNA extracted from strain ATCC 29212 grown in LBC medium. Lanes A, C, G, and T show unrelated sequencing ladders. The length of the fragments is indicated on the left side of the sequence ladder. (A) Lane 1 corresponds to detection of the 5′ end of citHO mRNA using the pecitH oligonucleotide. (B) Lane 1 corresponds to detection of the 5′ end of oadHDB-citCDEFX-oadA-citMG mRNA using the peoadH2 oligonucleotide. (C) Nucleotide sequence of the citH-oadH promoter regions. Transcriptional start sites are indicated (+1); −10 and −35 regions are shown by boxes. The directions of transcription and translation are indicated by horizontal arrows. The coding sequences for CitH and OadH are shown in bold. The sequences protected by CitO in DNase I footprinting experiments on the top and bottom DNA strands are underlined. The direct repeat sequence, which is possibly involved in CitO binding, is in boldface.

FIG. 4.

Schematic representation of the pTCV-lac-derived plasmids. The promoter regions of the citHO (A) and oadHDB-citCDEFX-oadA-citMG (B) operons are depicted. The CitO binding sites identified in vitro (O₁ and O₂) are represented by boxes. The end of each DNA fragment is labeled relative to the transcriptional start site. The levels of accumulated β-galactosidase activity were measured in cell extracts from cultures grown in LBC medium for 8 h.

FIG. 5.

Construction and characterization of an E. faecalis citO-defective strain (JHB1). (A) Schematic representation of citO inactivation by insertion of the pmCitO plasmid. The fragments labeled citO′ are parts of the citO gene; ery and ori(Ts) are the erythromycin cassette and the thermosensitive replication origin of pGh9 plasmid, respectively. (B) Northern blot analysis in citO⁺ (JH2-2) and citO mutant (JHB1) strains. Cells were grown in LB (lanes 1 and 3) or LBC (lanes 2 and 4) medium. The assay was performed as described in the legend of Fig. 2 but using only probe II. Transcripts were detected only for strain JH2-2 grown in the presence of citrate (lane 2) and not in the mutant strain (lane 4) or in the absence of citrate (lanes 1 and 3). (C) Growth curves in LBC medium of E. faecalis JH2-2 and derivative strains, citO⁺ (JH2-2; •), citO mutant(JHB1; ▾), citO mutant harboring the plasmid pCitO (JHB11; ▪) and citO mutant harboring the plasmid pBM02 (JHB12; ▴). (D) Voges-Proskauer assays. Strains JH2-2, JHB1, and JHB11 were grown in LBC medium. A positive reaction was obtained in the wild type (JH2-2) and in the complemented strain (JHB11).

FIG. 6.

Binding of CitO to the citHO-oadHDB-citCDEFX-oadA-citMG IR. (A) Schematic representation of the cit operon IR. PcitHO and PcitCL promoters are indicated. The thick line on the top indicates the location of the 427-bp DNA probe (IR amplicon) used in the gel shift experiments. (B and C) Image of gel shift assay performed with 0.5 nM IR amplicon and increasing concentrations of CitO without (B) and with (C) 1.1 mM citrate. Arrows indicate the positions of the retarded complex C1 and free DNA.

FIG. 7.

DNase I protection experiments of the operons promoters by CitO. A 427-bp DNA fragment including the citHO-oadHDB-citCDEFX-oadA-citMG operon promoter regions was end labeled with [γ-³²P]ATP and used as target DNA in the assays. Each strand was labeled in separate experiments. Unrelated sequencing reactions were run at the side with the DNase I footprinting reactions. Protected regions (O₁ and O₂) are indicated at the sides of the gel lanes.

FIG. 8.

(A) Effect of diverse citrate analogs on DNase I protection of the cit promoters by CitO. The oadHDB-citCDEFX-oadA-citMG coding strand was employed as target DNA. The CitO protein was used at a concentration of 1.5 μM. The different citrate analogs were used at a final concentration of 1.1 mM each. The presence (+) or absence (−) of the compounds and of CitO is shown in the top panel. The boxes indicate the extent of the protected regions. Influence of diverse citrate analogs on the expression of PcitHO-lacZ (B) and PcitCL-lacZ (C) fusions. Strain JHB15 (JH2-2 citO mutant harboring plasmids pCitO/pTCV-PcitHO) and strain JHB16 (JH2-2 citO mutant harboring plasmids pCitO/pTCV-PcitCL) were grown in LB medium and LB medium supplemented with initial concentrations of 30 mM citrate, malate, succinate, α-ketoglutarate, fumarate, acetate, or pyruvate or 10 mM isocitrate. The levels of accumulated β-galactosidase activity were measured 8 h after inoculation. Error bars represents the standard deviation of triplicate measurements.