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S-layers are bacterial structures present on the surface of several Gram-positive and Gram-negative bacteria that play a role in bacterial protection. In Lactobacillus acidophilus (L. acidophilus ATCC 4356), the S-layer is mainly composed of the protein SlpA. A tandem of two copies of the protein domain SLP-A (pfam: 03217) was identified at the C-terminal of SlpA, being this double SLP-A protein domain (in short dSLP-A) necessary and sufficient for the association of the protein to the L. acidophilus cell wall. A variety of proteins fused to the dSLP-A domain were able to spontaneously associate with high affinity to the cell wall of L. acidophilus and Bacillus subtilis var. natto, in a process that we termed decoration. Binding of dSLP-A-containing-proteins to L. acidophilus was stable at conditions that mimic the gastrointestinal transit in terms of pH, proteases, and bile salts. To evaluate if protein decoration of L. acidophilus can be adapted to generate an oral vaccine platform, a chimeric antigen derived from the bacterial pathogen Shiga-toxin-producing Escherichia coli (STEC) was constructed by fusing the sequences encoding the polypeptides EspA36–192, Intimin653–953, Tir240–378, and H7 flagellin352–374 (EITH7) to the dSLP-A domain (EITH7-dSLP-A). Recombinantly expressed EITH7-dSLP-A protein was affinity purified and combined with L. acidophilus cultures to allow the association of the chimeric antigen to the bacterial surface. EITH7-decorated L. acidophilus was orally administered to BALB/c mice and the induction of anti-EITH7 specific antibodies in sera and feces determined by ELISA. Mice presenting significantly higher anti-EITH7 antibodies titers were able to control more efficiently an experimental STEC infection than mice that received the non-decorated L. acidophilus carrier, indicating that antigen-decorated L. acidophilus can be adapted as a mucosal immunization delivery platform to elicit a protective immune response for vaccine purposes. Fil: Uriza, Paula Jimena. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Trautman, Cynthia Veronica. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Palomino, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fina Martin, Joaquina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Ruzal, Sandra Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Roset, Mara Sabrina. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Briones, Carlos Gabriel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina

Mesorhizobium loti MAFF303099 is a rhizobial strain that nodulates Lotus spp. A M. loti MAFF303099 mutant strain affected in the tatC gene was generated. This strain presented an altered protein secretion level to the culture supernatant and also a higher sensitivity to SDS. Its nodulation phenotype on Lotus showed the induction of small and colorless nodules, and in a larger number than those induced by the wild-type strain. In addition, these nodules presented defects in the degree of occupation by rhizobia. Two Rieske Fe/S proteins, encoded by the mll2707 and mlr0970 genes, were predicted as potential Tat substrates in M. loti MAFF303099. The transcriptional expression of mll2707 and mlr0970 genes was analyzed under different oxygen growth conditions. The mll2707 gene was expressed constitutively, while the expression of the mlr0970 gene was only detected under anaerobic and microaerophilic in vitro conditions. Both genes were down-regulated in the tatC mutant strain. mll2707 and mlr0970 mRNAs from the wild-type strain were detected in nodules. Using a translational reporter peptide fusion, we found that the Mll2707 protein was only detectable in the wild-type strain. On the other hand, although Mlr0970 protein was detected in wild-type and tatC mutant strains, its association with the membrane was favored in the wild-type strain. The tatC and the mll2707 mutant strains were affected in the cytochrome c oxidase activity. These results confirm that Mll2707 is required for cytochrome c-dependent respiration and that Tat functionality is required for the correct activity of Mll2707. The mll2707 mutant strain showed a nodulation phenotype similar to the tatC mutant strain, although it presented only a slight difference in comparison with wild-type strain in terms of nodule occupation. No defective phenotype was observed in the nodulation with the mlr0970 mutant strain. These results indicate that, of the two Rieske Fe/S proteins coded by M. loti MAFF303099, only Mll2707 expression is required for the induction of effective nodules, and that the functionality of the Tat system is necessary not only for the correct function of this protein, but also for some other protein required in an earlier stage of the nodulation process. Fil: Basile, Laura Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Zalguizuri, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Lepek, Viviana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH7) from STEC, was constructed and expressed in B. abortus δpgm vaccine strain (Babδpgm). Mice orally immunized with Babδpgm(EITH7) elicited an immune response with the induction of anti-EITH7 antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t=4 days) than mice immunized with Babδpgm carrier strain (t=12 days). As expected, mice immunized with Babδpgm(EITH7) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles-the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC. Fil: Iannino, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Herrmann, Claudia Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Roset, Mara Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Brucella ssp. is a facultative intracellular pathogen that causes brucellosis, a worldwide zoonosis that affects a wide range of mammals including humans. A critical step for the establishment of a successful Brucella infection is its ability to survive within macrophages. To further understand the mechanisms that Brucella utilizes to adapt to an intracellular lifestyle, a differential proteomic study was performed for the identification of intracellular modulated proteins. Our results demonstrated that at 48 hours post-infection Brucella adjusts its metabolism in order to survive intracellularly by modulating central carbon metabolism. Remarkably, low iron concentration is likely the dominant trigger for reprogramming the protein expression profile. Up-regulation of proteins dedicated to reduce the concentration of reactive oxygen species, protein chaperones that prevent misfolding of proteins, and proteases that degrade toxic protein aggregates, suggest that Brucella protects itself from damage likely due to oxidative burst. This proteomic analysis of B. abortus provides novel insights into the mechanisms utilized by Brucella to establish an intracellular persistent infection and will aid in the development of new control strategies and novel targets for antimicrobial therapy. Fil: Roset, Mara Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Alefantis, T. G.. Vital Probes; Estados Unidos Fil: Delvecchio, V. G.. Vital Probes; Estados Unidos Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Type III protein secretion systems, which are organelles with the capacity to deliver bacterial proteins into host cells, have been adapted to deliver heterologous antigens for vaccine development. A limitation of these antigen delivery systems is that some proteins are not amenable to secretion through this pathway. We show here that proteins from the simian and human immunodeficiency viruses that are not permissive for secretion through aSalmonella entericaserovar Typhimurium type III secretion system can be modified to travel this secretion pathway by introduction of discrete mutations. Proteins optimized for secretion were presented more efficiently via the major histocompatibility complex class I pathway and were able to induce a better immune response.

An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which Brucella suis was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98.3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which B. suis was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%.

The animal pathogen Brucella abortus contains a gene, cgs , that complemented a Rhizobium meliloti nodule development ( ndvB ) mutant and an Agrobacterium tumefaciens chromosomal virulence ( chvB ) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens , and nitrogen fixation in R. meliloti ; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn 3 -HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium , are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn 3 -HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in Brucella infection.

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella . To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus Δ cypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus Δ cypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus Δ cypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.

Pathogenic brucella bacteria have developed strategies to persist for prolonged periods of time in host cells, avoiding innate immune responses. Here we show that the cyclic beta-1,2-glucans (CbetaG) synthesized by brucella is important for circumventing host cell defenses. CbetaG acted in lipid rafts found on host cell membranes. CbetaG-deficient mutants failed to prevent phagosome-lysosome fusion and could not replicate. However, when treated with purified CbetaG or synthetic methyl-beta-cyclodextrin, the mutants were able to control vacuole maturation by avoiding lysosome fusion, and this allowed intracellular brucella to survive and reach the endoplasmic reticulum. Fusion between the endoplasmic reticulum and the brucella-containing vacuole depended on the brucella virulence type IV secretion system but not on CbetaG. Brucella CbetaG is thus a virulence factor that interacts with lipid rafts and contributes to pathogen survival.

Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic β-1,2-glucan (CβG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CβG mutants of Brucella, cgs (CβG synthase), cgt (CβG transporter) and cgm (CβG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CβG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CβG in promoting splenomegaly in mice. We showed that CβG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CβG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide. Fil: Roset, Mara Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Ibañez, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Souza Filho, Job Alves de. Universidade Federal do Minas Gerais; Brasil Fil: Spera, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Minatel, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Oliveira, Sergio C.. Universidade Federal do Minas Gerais; Brasil Fil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Cassataro, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina

Biosynthesis of periplasmic cyclic 1,2-β-glucans inBrucella ovisstrain REO198 andB. abortusstrain S19 was found to be carried out by membrane-bound enzymes that use UDP-glucose (UDP-Glc) as donor substrate. Contrary to what happens in species of the generaAgrobacteriumandRhizobium, the accumulation of the reaction products inBrucellaappeared not to be osmoticaliy regulated. Incubation of permeabilized cells with UDP-[14C]Glc led to the formation of soluble neutral cyclic 1,2-β-glucans and [14C]glucose-labelled glucoproteins. PAGE of pulse–chase experiments carried out with permeabilized cells showed that the molecular mass of the labelled protein was indistinguishable fromAgrobacterium tumefaciensA348 andRhizobium frediiUSDA191 glucoproteins known to be intermediates in the synthesis of cyclic glucans.Brucellatotal membrane preparations were less efficient than permeabilized cells in the formation of cyclic glucan; this was attributed to defective cyclization. Accumulation of protein intermediates having oligosaccharides of high molecular mass that were not released from the protein was observed after chase with 2 mM UDP-Glc. This defect was not observed when permeabilized cells were used as enzyme preparation, thus suggesting that inBrucellaa factor(s) that was lost or inactivated upon the preparation of membranes was required for the effective regulation between elongation and cyclization reactions.

We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii. Fil: Ibañez, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Coria, Mirta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Carabajal, Marianela Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Delpino, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Risso, Gabriela Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Gonzalez Cobiello, Paula Lucía. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología E Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Barrionuevo, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Bruno, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Frank, Fernanda Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Medica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones En Microbiología y Parasitología Medica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Pasquevich, Karina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Cassataro, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina

In contrast to what happens in AGROBACTERIUM: tumefaciens and RHIZOBIUM: meliloti, synthesis of periplasmic cyclic 1,2-beta-glucan in BRUCELLA: spp. was not inhibited when bacteria were grown in media of high osmolarity. Studies performed with crude membrane preparations showed that cyclic 1,2-beta-glucan synthetase of BRUCELLA: spp. was not inhibited by 0.5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A. tumefaciens A348 or R. meliloti 102F34, respectively encoded by the chvB or ndvB genes. The BRUCELLA: abortus cyclic 1, 2-beta-glucan synthetase gene (cgs) was introduced into A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants. Synthesis of cyclic 1,2-beta-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0.25 M NaCl or 0.5 M mannitol). On the other hand, when the A. tumefaciens cyclic 1, 2-beta-glucan synthetase gene was introduced into the R. meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-beta-glucan synthesis when grown in high-osmolarity media. However, the same gene introduced into a B. abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity. In vitro studies with crude membranes isolated from recombinant strains revealed that BRUCELLA: cyclic 1, 2-beta-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in AGROBACTERIUM: or RHIZOBIUM: backgrounds. It was concluded that the lack of effect of high osmolarity on 1,2-beta-glucan synthesis in BRUCELLA: is due to two convergent mechanisms: a) the presence of a cyclic 1, 2-beta-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1, 2-beta-glucan synthetase.