L-Aspartate as a high-quality nitrogen source in Escherichia coli: Regulation of L-aspartase by the nitrogen regulatory system and interaction of L-aspartase with GlnB.

Escherichia coli uses the C4-dicarboxylate transporter DcuA for L-aspartate/fumarate antiport, which results in the exploitation of L-aspartate for fumarate respiration under anaerobic conditions and for nitrogen assimilation under aerobic and anaerobic conditions. L-Aspartate represents a high-quality nitrogen source for assimilation. Nitrogen assimilation from L-aspartate required DcuA, and aspartase AspA to release ammonia. Ammonia is able to provide by established pathways the complete set of intracellular precursors (ammonia, L-aspartate, L-glutamate, and L-glutamine) for synthesizing amino acids, nucleotides, and amino sugars. AspA was regulated by a central regulator of nitrogen metabolism, GlnB. GlnB interacted with AspA and stimulated its L-aspartate deaminase activity (NH3 -forming), but not the reverse amination reaction. GlnB stimulation required 2-oxoglutarate and ATP, or uridylylated GlnB-UMP, consistent with the activation of nitrogen assimilation under nitrogen limitation. Binding to AspA was lost in the GlnB(Y51F) mutant of the uridylylation site. AspA, therefore, represents a new type of GlnB target that binds GlnB (with ATP and 2-oxoglutarate), or GlnB-UMP (with or without effectors), and both situations stimulate AspA deamination activity. Thus, AspA represents the central enzyme for nitrogen assimilation from L-aspartate, and AspA is integrated into the nitrogen assimilation network by the regulator GlnB.

DcuA of aerobically grown Escherichia coli serves as a nitrogen shuttle (L-aspartate/fumarate) for nitrogen uptake.

DcuA of Escherichia coli is known as an alternative C4 -dicarboxylate transporter for the main anaerobic C4 -dicarboxylate transporter DcuB. Since dcuA is expressed constitutively under aerobic and anaerobic conditions, DcuA was suggested to serve aerobically as a backup for the aerobic (DctA) transporter, or for the anabolic uptake of C4 -dicarboxylates. In this work, it is shown that DcuA is required for aerobic growth with L-aspartate as a nitrogen source, whereas for growth with L-aspartate as a carbon source, DctA was needed. Strains with DcuA catalyzed L-aspartate and C4 -dicarboxylate uptake (like DctA), or an L-aspartate/C4 -dicarboxylate antiport (unlike DctA). DcuA preferred L-aspartate to succinate in transport (KM = 43 and 844 µM, respectively), whereas DctA has higher affinity for C4 -dicarboxylates like succinate compared to L-aspartate. When L-aspartate was supplied as the sole nitrogen source together with glycerol as the carbon source, L-aspartate was taken up by the bacteria and fumarate (or L-malate) was excreted in equimolar amounts. Both reactions depended on DcuA. L-Aspartate was taken up in amounts required for nitrogen metabolism but not for carbon metabolism. Therefore, DcuA catalyzes an L-aspartate/C4 -dicarboxylate antiport serving as a nitrogen shuttle for nitrogen supply without net carbon supply.

The E. coli dicarboxylic acid transporters DauA act as a signal transducer by interacting with the DctA uptake system.

The Slc26A/SulP family of ions transporter is ubiquitous and widpsread in all kingdon of life. In E. coli, we have demonstrated that the Slc26 protein DauA is a C4-dicarboxilic acids (C4-diC) transporter active at acidic pH. The main C4-diC transporter active at pH7 is DctA and is induced by C4-diC via the DcuS/R two component system. DctA interacts with DcuS, the membrane embedded histidine kinase, to transfers DcuS to the responsive state, i.e. in the absence of DctA, DcuS is permanently "on", but its activity is C4-diC-dependent when in complex with DctA. Using phenotypic characterization, transport assays and protein expression studies, we show that at pH7 full DctA production depends on the presence of DauA. A Bacterial Two Hybrid system indicates that DauA and the sensor complex DctA/DcuS physically interact at the membrane. Pull down experiments completed by co-purification study prove that DauA and DctA interact physically at the membrane. These data open a completely new aspect of the C4-diC metabolism in E. coli and reveals how the bacterial Slc26A uptake systems participate in multiple cellular functions. This constitutes a new example of a bacterial transporter that acts as a processor in a transduction pathway.

C4-Dicarboxylate Utilization in Aerobic and Anaerobic Growth.

C4-dicarboxylates and the C4-dicarboxylic amino acid l-aspartate support aerobic and anaerobic growth of Escherichia coli and related bacteria. In aerobic growth, succinate, fumarate, D- and L-malate, L-aspartate, and L-tartrate are metabolized by the citric acid cycle and associated reactions. Because of the interruption of the citric acid cycle under anaerobic conditions, anaerobic metabolism of C4-dicarboxylates depends on fumarate reduction to succinate (fumarate respiration). In some related bacteria (e.g., Klebsiella), utilization of C4-dicarboxylates, such as tartrate, is independent of fumarate respiration and uses a Na+-dependent membrane-bound oxaloacetate decarboxylase. Uptake of the C4-dicarboxylates into the bacteria (and anaerobic export of succinate) is achieved under aerobic and anaerobic conditions by different sets of secondary transporters. Expression of the genes for C4-dicarboxylate metabolism is induced in the presence of external C4-dicarboxylates by the membrane-bound DcuS-DcuR two-component system. Noncommon C4-dicarboxylates like l-tartrate or D-malate are perceived by cytoplasmic one-component sensors/transcriptional regulators. This article describes the pathways of aerobic and anaerobic C4-dicarboxylate metabolism and their regulation. The citric acid cycle, fumarate respiration, and fumarate reductase are covered in other articles and discussed here only in the context of C4-dicarboxylate metabolism. Recent aspects of C4-dicarboxylate metabolism like transport, sensing, and regulation will be treated in more detail. This article is an updated version of an article published in 2004 in EcoSal Plus. The update includes new literature, but, in particular, the sections on the metabolism of noncommon C4-dicarboxylates and their regulation, on the DcuS-DcuR regulatory system, and on succinate production by engineered E. coli are largely revised or new.

Conversion of the sensor kinase DcuS of Escherichia coli of the DcuB/DcuS sensor complex to the C4 -dicarboxylate responsive form by the transporter DcuB.

The sensor kinase DcuS of Escherichia coli co-operates under aerobic conditions with the C4 -dicarboxylate transporter DctA to form the DctA/DcuS sensor complex. Under anaerobic conditions C4 -dicarboxylate transport in fumarate respiration is catalyzed by C4 -dicarboxylate/fumarate antiporter DcuB. (i) DcuB interacted with DcuS as demonstrated by a bacterial two-hybrid system (BACTH) and by co-chromatography of the solubilized membrane-proteins (mHPINE assay). (ii) In the DcuB/DcuS complex only DcuS served as the sensor since mutations in the substrate site of DcuS changed substrate specificity of sensing, and substrates maleate or 3-nitropropionate induced DcuS response without affecting the fumarate site of DcuB. (iii) The half-maximal concentration for induction of DcuS by fumarate (1 to 2 mM) and the corresponding Km for transport (50 µM) differ by a factor of 20 to 40. Therefore, the fumarate sites are different in transport and sensing. (iv) Increasing levels of DcuB converted DcuS from the permanent ON (DcuB deficient) state to the fumarate responsive form. Overall, the data show that DcuS and DcuB form a DcuB/DcuS complex representing the C4 -dicarboxylate responsive form, and that the sensory site of the complex is located in DcuS whereas DcuB is required for converting DcuS to the sensory competent state.

CitA (citrate) and DcuS (C4-dicarboxylate) sensor kinases in thermophilic Geobacillus kaustophilus and Geobacillus thermodenitrificans.

The thermophilic Geobacillus thermodenitrificans and Geobacillus kaustophilus are able to use citrate or C4-dicarboxylates like fumarate or succinate as the substrates for growth. The genomes of the sequenced Geobacillus strains (nine strains) each encoded a two-component system of the CitA family. The sensor kinase of G. thermodenitrificans (termed CitAGt) was able to replace CitA of Escherichia coli (CitAEc) in a heterologous complementation assay restoring expression of the CitAEc-dependent citC-lacZ reporter gene and anaerobic growth on citrate. Complementation was specific for citrate. The sensor kinase of G. kaustophilus (termed DcuSGk) was able to replace DcuSEc of E. coli. It responded in the heterologous expression system to C4-dicarboxylates and to citrate, suggesting that DcuSGk is, like DcuSEc, a C4-dicarboxylate sensor with a side-activity for citrate. DcuSGk, unlike the homologous DctS from Bacillus subtilis, required no binding protein for function in the complementation assay. Thus, the thermophilic G. thermodenitrificans and G. kaustophilus contain citrate and C4-dicarboxylate sensor kinases of the CitA and DcuS type, respectively, and retain function and substrate specificity under mesophilic growth conditions in E. coli.

Neutralization of the CD95 ligand by APG101 inhibits invasion of glioma cells in vitro.

Glioblastoma is a disease characterized by rapid invasive tumour growth. Studies on the proapoptotic CD95/CD95L signalling pathway recently suggested a significant contribution of CD95 signalling towards the high degree of motility in glioma cells. Apogenix has developed APG101, a clinical phase II compound designed to bind and neutralize CD95L, and thus to interfere with CD95/CD95L-based signalling. APG101 has shown clinical efficacy in a controlled randomized phase II trial in patients with recurrent glioma. Because APG101 is not cytotoxic to tumour cells in vitro, we postulated that the anti-invasive function of APG101 is the main mechanism of action for this compound. Using three-dimensional spheroid invasion assays in vitro and in murine brain tissue cultures, we found that knockdown of endogenous CD95L reduced the invasive phenotype in our two glioblastoma model cell lines U87-MG and U251-MG. Invasion was restored in CD95L knockdown cells upon the addition of soluble recombinant CD95L and this effect was inhibited by APG101. We conclude that CD95L from autocrine and paracrine sources contributes towards the invasive phenotype of glioblastoma cells and that APG101 acts as a suppressor of proinvasive signalling by the CD95/CD95L pathway in glioblastoma.