C4-dicarboxylates as growth substrates and signaling molecules for commensal and pathogenic enteric bacteria in mammalian intestine

The C4-dicarboxylates (C4-DC) L-aspartate and L-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella Typhimurium, and succinate as a signaling molecule for host–enteric bacteria interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. L-aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation. During nitrogen assimilation, DcuA catalyzes an L-aspartate/fumarate antiport and serves as a nitrogen shuttle for the net uptake of ammonium only, whereas DcuB acts as a redox shuttle that catalyzes the L-malate/succinate antiport during fumarate respiration. The C4-DC two-component system DcuS-DcuR is active in the intestine and responds to intestinal C4-DC levels. Moreover, in macrophages and in mice, succinate is a signal that promotes virulence and survival of S . Tm and pathogenic E. coli . On the other hand, intestinal succinate is an important signaling molecule for the host and activates response and protective programs. Therefore, C4-DCs play a major role in supporting colonization of enteric bacteria and as signaling molecules for the adaptation of host physiology.

C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes and regulators

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here the interaction of enzymes fumarase FumB and aspartase AspA with the C4-DC transporters DcuA and DcuB of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography (mSPINE). DcuB interacted strongly with FumB and AspA, and DcuA with AspA. The fumB-dcuB and the dcuA-aspA genes encoding the respective proteins are known for their colocalization on the genome and the production of co-transcripts. The data consistently suggest the formation of DcuB/FumB, DcuB/AspA and DcuA/AspA metabolons in fumarate respiration for the uptake of L-malate, or L-aspartate, conversion to fumarate and excretion of succinate after reduction. The DcuA/AspA metabolon catalyzes L-Asp uptake and fumarate excretion in concerted action also to provide ammonia for nitrogen assimilation. The aerobic C4-DC transporter DctA interacted with the regulator EIIAGlc of the E. coli glucose phosphotransferase system. It is suggested that EIIAGlc inhibits C4-DC uptake by DctA in the presence of the preferred substrate glucose.

Identification of a Novel Fumarate Reductase Potentially Involved in Electron Bifurcation

In the succinic acid-producing bacterium Pseudoclostridium thermosuccinogenes the fumarate reductase (FRD) genes reside in an operon together with those encoding an electron bifurcating complex (FlxABCD-HdrABC) that shuttles electrons from NADH to ferredoxin and a disulfide bond. Based on phylogeny and genomic co-occurrence we propose two hypothetical mechanisms via which the FRD is involved in electron bifurcation: (I) A disulfide bond from a hitherto unknown cofactor is reduced by the electron-bifurcating FlxABCD-HdrABC complex, using NADH to generate two thiol groups, while facilitating the unfavourable reduction of ferredoxin by NADH. The disulfide bond is subsequently regenerated via the reduction of fumarate by the FRD using the previously formed thiol groups. (II) The FRD forms an integral part of the FlxABCD-HdrABC complex, and NADH is used to reduce ferredoxin and fumarate directly, without an intermediate disulfide-forming cofactor. Either way enables the conservation of additional energy by a soluble FRD, analogous to fumarate respiration.

Regulation of tartrate metabolism by TtdR and relation to the DcuS–DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli

Escherichia coli catabolizes l-tartrate under anaerobic conditions to oxaloacetate by the use of l-tartrate/succinate antiporter TtdT and l-tartrate dehydratase TtdAB. Subsequently, l-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS–DcuR two-component system. Expression of the l-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of l- and meso-tartrate, and repressed by O2 and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O2, nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR–ttdA promoter region. TtdR was also required for full expression of the DcuS–DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to l-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (l- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C4-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.

Succinate dehydrogenase functioning by a reverse redox loop mechanism and fumarate reductase in sulphate-reducing bacteria

Sulphate- or sulphur-reducing bacteria with known or draft genome sequences (Desulfovibrio vulgaris, Desulfovibrio desulfuricans G20, Desulfobacterium autotrophicum [draft], Desulfotalea psychrophila and Geobacter sulfurreducens) all contain sdhCAB or frdCAB gene clusters encoding succinate : quinone oxidoreductases. frdD or sdhD genes are missing. The presence and function of succinate dehydrogenase versus fumarate reductase was studied. Desulfovibrio desulfuricans (strain Essex 6) grew by fumarate respiration or by fumarate disproportionation, and contained fumarate reductase activity. Desulfovibrio vulgaris lacked fumarate respiration and contained succinate dehydrogenase activity. Succinate oxidation by the menaquinone analogue 2,3-dimethyl-1,4-naphthoquinone depended on a proton potential, and the activity was lost after degradation of the proton potential. The membrane anchor SdhC contains four conserved His residues which are known as the ligands for two haem B residues. The properties are very similar to succinate dehydrogenase of the Gram-positive (menaquinone-containing) Bacillus subtilis, which uses a reverse redox loop mechanism in succinate : menaquinone reduction. It is concluded that succinate dehydrogenases from menaquinone-containing bacteria generally require a proton potential to drive the endergonic succinate oxidation. Sequence comparison shows that the SdhC subunit of this type lacks a Glu residue in transmembrane helix IV, which is part of the uncoupling E-pathway in most non-electrogenic FrdABC enzymes.

Salipiger mucescens gen. nov., sp. nov., a moderately halophilic, exopolysaccharide-producing bacterium isolated from hypersaline soil, belonging to the α-Proteobacteria

Salipiger mucescens gen. nov., sp. nov. is a moderately halophilic, exopolysaccharide-producing, Gram-negative rod isolated from a hypersaline habitat in Murcia in south-eastern Spain. The bacterium is chemoheterotrophic and strictly aerobic (i.e. unable to grow under anaerobic conditions either by fermentation or by nitrate or fumarate respiration). It does not synthesize bacteriochlorophyll a. Catalase and phosphatase are positive. It does not produce acids from carbohydrates. It cannot grow with carbohydrates or amino acids as sole sources of carbon and energy. It grows best at 9–10 % w/v NaCl and requires the presence of Na+ but not Mg2+ or K+, although they do stimulate its growth somewhat when present. Its major fatty-acid component is 18 : 1ω7c (78·0 %). The predominant respiratory lipoquinone found in strain A3T is ubiquinone with ten isoprene units. The G+C content is 64·5 mol%. Phylogenetic analyses strongly indicate that this strain forms a distinct line within a clade containing the genus Roseivivax in the subclass α-Proteobacteria. The similarity value with Roseivivax halodurans and Roseivivax halotolerans is 94 %. In the light of the polyphasic evidence gathered in this study it is proposed that the isolate be classified as representing a new genus and species, Salipiger mucescens gen. nov., sp. nov. The proposed type strain is strain A3T (=CECT 5855T=LMG 22090T=DSM 16094T).