NAD+-dependent post-translational modification of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein reported to be a target of several covalent modifications in many organisms. In a previous study, enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains were shown to secrete GAPDH and the protein to bind human plasminogen and fibrinogen. Here we report that GAPDH of these pathogens is ADP-ribosylated either in the cytoplasm or in the extracellular medium. GAPDH catalyzes its own modification, which involves Cys-149 at the active site. ADP-ribosylation of extracellular GAPDH may play an important role in the host-pathogen interaction, as also proposed in other pathogens. [Int Microbiol 2009; 12(3):187-192

Characterization of the gene cluster involved in allantoate catabolism and its transcriptional regulation by the RpiR-type repressor HpxU in Klebsiella pneumoniae

Bacteria, fungi, and plants have metabolic pathways for the utilization of nitrogen present in purine bases. In Klebsiella pneumoniae, the genes responsible for the assimilation of purine ring nitrogen are distributed in three separated clusters. We characterized the gene cluster involved in the metabolism of allantoate (genes KPN_01761 to KPN_01771). The functional assignments of HpxK, as an allantoate amidohydrolase, and of HpxU, as a regulator involved in the control of allantoate metabolism, were assessed experimentally. Gene hpxU encodes a repressor of the RpiR family that mediates the regulation of this system by allantoate. In this study, the binding of HpxU to the hpxF promoter and to the hpxU-hpxW intergenic region containing the divergent promoter for these genes was evidenced by electrophoretic mobility shift assays. Allantoate released the HpxU repressor from its target operators whereas other purine intermediate metabolites, such as allantoin and oxamate, failed to induce complex dissociation. Sequence alignment of the four HpxU identifi ed operators identifi ed TGAA-N8-TTCA as the consensus motif recognized by the HpxU repressor. [Int Microbiol 2013; 16(3):XXX-XXX]Keywords: Klebsiella pneumoniae · allantoate metabolism · allantoate amidohydrolase · purine catabolism · RpiR-type represso

Proteomic profile of extracellular vesicles released by Lactiplantibacillus plantarum BGAN8 and their internalization by non-polarized HT29 cell line

In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhibitors of endocytic pathways: chlorpromazine and dynasore (inhibitors of clathrin-mediated endocytosis-CME) and filipin III and nystatin (disrupting lipid rafts). For the first time, our results revealed that the internalization was specifically inhibited by dynasore and chlorpromazine but not by filipin III and nystatin implying that one of the entries of L. plantarum vesicles was through CME pathway

The Gene yjfQ Encodes the Repressor of the yjfR-X Regulon (ula), Which Is Involved in l-Ascorbate Metabolism in Escherichia coli

Mutations in yjfQ allowed us to identify this gene as the regulator of the operon yjfS-X (ula operon), reported to be involved in l-ascorbate metabolism. Inactivation of this gene renders constitutive the expression of the ula operon, indicating that YjfQ acts as a repressor. We also demonstrate that this repressor regulates the nearby yjfR gene, which in this way constitutes a regulon with the ula operon

Dual Role of LldR in Regulation of the lldPRD Operon, Involved in l-Lactate Metabolism in Escherichia coli▿

The lldPRD operon of Escherichia coli, involved in l-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by l-lactate is lost in an ΔlldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions −105 to −89) and O2 (positions +22 to +38), with O1 being filled at a lower concentration of LldR. l-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by l-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence of l-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of l-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex