Partial characterization and photolabeling of a Rhizobium meliloti polysaccharide methyltransferase with S-adenosylmethionine

S-Adenosylmethionine (SAM) has been used to directly cross-link a polysaccharide specific methyltransferase isolated from Rhizobium meliloti HA. This peculiar enzyme transfers a methyl group to the 2-O-galacturonosyl residue of a teichuronic type polysaccharide and was very unstable. The apparent Km for SAM was 0.46 mM. The Hill coefficient, n, was 1. The enzyme had an optimum pH of 8.2 and requires Mn2+ at concentration of 2 mM. The enzyme was inactivated by saline concentrations of 120 mM or greater and was eluted from Superose columns with an apparent molecular weight of 28 kDa. The isoelectric point was close to 7.0. To elucidate the relationship between chemical structure and catalytic function, (3H)SAM was cross-linked to the enzyme and the enzymatic activity was assayed in presence and in absence of commercial substrate analogs. Cross-linking was performed by direct irradiation of enzyme and (3H)SAM. The uptake of radioactivity was linear up to about 20 min and then reached a plateau. This irreversible junction is specific, as shown by a number of different criteria. Several competitive inhibitors were able to affect this photoactivated cross-linkage. As the concentration of inhibitors increased, both, the level of photolabeling and enzyme activity always decreased. The SAM-enzyme adduct was shown to be a single protein band by SDS polyacrylamide gel electrophoresis

An Atypical Riboflavin Pathway Is Essential for Brucella abortus Virulence

Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related Brucella spp., which are adapted to intracellular life within cells of a large variety of mammals. Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. In these cells Brucella survives in a replicative niche, which is characterized for having a very low oxygen tension and being deprived from nutrients such as amino acids and vitamins. Among these vitamins, we have focused on riboflavin (vitamin B2). Flavin metabolism has been barely implicated in bacterial virulence. We have recently described that Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway. In the present work we analyze the role of the flavin metabolism on Brucella virulence. Mutants on the two lumazine synthases (LS) isoenzymes RibH1 and RibH2 and a double RibH mutant were generated. These mutants and different complemented strains were tested for viability and virulence in cells and in mice. In this fashion we have established that at least one LS must be present for B. abortus survival and that RibH2 and not RibH1 is essential for intracellular survival due to its LS activity in vivo. In summary, we show that riboflavin biosynthesis is essential for Brucella survival inside cells or in mice. These results highlight the potential use of flavin biosynthetic pathway enzymes as targets for the chemotherapy of brucellosis

Action of glycosidases on the saccharide moiety of the glucose—containing dolichyl diphosphate oligosaccharide

originalFil: Ugalde, Rodolfo Augusto. Instituto de Investigaciones Bioquímicas Fundación Campomar; ArgentinaFil: Staneloni, Roberto Julio. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentinablanco y negro1 ejemplarLFL-PI-O-ART. Artículos científicosUnidad documental simpleAR-HYL-201

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

Background: Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes. Results: Eighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp. Conclusion: The comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus

Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity

In chloroplasts, stromal and thylakoid-bound ascorbate peroxidases (tAPX) play a major role in the removal of H2O2 produced during photosynthesis. Here, we report that hexaploid wheat (Triticum aestivum) expresses three homeologous tAPX genes (TaAPX-6A, TaAPX-6B, and TaAPX-6D) mapping on group-6 chromosomes. The tAPX activity of a mutant line lacking TaAPX-6B was 40% lower than that of the wild type. When grown at high-light intensity photosystem II electron transfer, photosynthetic activity and biomass accumulation were significantly reduced in this mutant, suggesting that tAPX activity is essential for photosynthesis. Despite the reduced tAPX activity, mutant plants did not exhibit oxidative damage probably due to the reduced photochemical activity. This might be the result of a compensating mechanism to prevent oxidative damage having as a consequence a decrease in growth of the tAPX mutant plants.Instituto de Fisiología VegetalFacultad de Ciencias Agrarias y Forestale

Brucella Control of Dendritic Cell Maturation Is Dependent on the TIR-Containing Protein Btp1

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.Instituto de Biotecnologia y Biologia Molecula

An intermediate in cyclic beta 1-2 glucan biosynthesis

Incubation of UDP-[14C]Glc with the inner membranes of Agrobacterium tumefaciens leads to the formation of cyclic beta 1-2 glucan and trichloroacetic acid-insoluble compounds. The proteolysis products of the latter show a positive charge in acid and a negative charge in alkaline buffers. The cyclic beta 1-2 glucan and the trichloroacetic acid insoluble compounds yield the same products on partial acid hydrolysis. Addition of excess non-radioactive UDP-Glc to the reaction mixture nearly stops the formation of radioactive beta 1-2 glucan and leads to a rapid fall of radioactivity in the trichloroacetic acid precipitate. Alkaline treatment of the insoluble compounds under conditions of beta-elimination leads to the partial release of free saccharides (about 30%). It is concluded that beta 1-2 glucan chains are built up joined to a protein and then released as free cyclic beta 1-2 glucan.originalFil: Zorreguieta, Ángeles. Instituto de Investigaciones Bioquímicas Fundación Campomar ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Ugalde, Rodolfo A.. Instituto de Investigaciones Bioquímicas Fundación Campomar ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaEscala de grises6 páginas en pdfLFL-PI-O-ART. Artículos científicosUnidad documental simpleAR-HYL-201