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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.

SUBMITTER: Blancato VS

PROVIDER: S-EPMC2576652 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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

Blancato Víctor S VS Repizo Guillermo D GD Suárez Cristian A CA Magni Christian C

Journal of bacteriology 20080919 22

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 trans ...[more]

PMID: 18805984

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Project description:BACKGROUND: Iron-sulfur clusters are ubiquitous and evolutionarily ancient inorganic prosthetic groups, the biosynthesis of which depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in Gram-positive bacteria. Within the Firmicutes phylum, the Enterococcus spp. genus have recently assumed importance in clinical microbiology being considered as emerging pathogens for humans, wherein Enterococcus faecalis represents the major species associated with nosocomial infections. The aim of this study was to carry out a phylogenetic analysis in Enterococcus faecalis V583 and a structural and conformational characterisation of it SufU protein. RESULTS: BLAST searches of the Enterococcus genome revealed a series of genes with sequence similarity to the Escherichia coli SUF machinery of [Fe-S] cluster biosynthesis, namely sufB, sufC, sufD and SufS. In addition, the E. coli IscU ortholog SufU was found to be the scaffold protein of Enterococcus spp., containing all features considered essential for its biological activity, including conserved amino acid residues involved in substrate and/or co-factor binding (Cys50,76,138 and Asp52) and, phylogenetic analyses showed a close relationship with orthologues from other Gram-positive bacteria. Molecular dynamics for structural determinations and molecular modeling using E. faecalis SufU primary sequence protein over the PDB:1su0 crystallographic model from Streptococcus pyogenes were carried out with a subsequent 50 ns molecular dynamic trajectory. This presented a stable model, showing secondary structure modifications near the active site and conserved cysteine residues. Molecular modeling using Haemophilus influenzae IscU primary sequence over the PDB:1su0 crystal followed by a MD trajectory was performed to analyse differences in the C-terminus region of Gram-positive SufU and Gram-negative orthologous proteins, in which several modifications in secondary structure were observed. CONCLUSION: The data describe the identification of the SUF machinery for [Fe-S] cluster biosynthesis present in the Firmicutes genome, showing conserved sufB, sufC, sufD and sufS genes and the presence of the sufU gene coding for scaffold protein, instead of sufA; neither sufE nor sufR are present. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU nearby on the ISC machinery. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes denoting the Gram positive region (GPR), suggested as a possible candidate for interaction with other factors and/or regulators.

Dataset Information

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

Publications

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

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