Effect of nano-micrometric topographies on early steps of biofilm formation

Biofilms are defined as communities of microorganisms that live attached to a surface. They can include a single bacterial specie or multiple species and are formed on both abiotic and biotic surfaces. Thiswell-known phenomenon has undesirable effects for industrial or medical surfaces. Surface properties impact on the first steps of biofilm formation. Nature offers multiple solutions to biofilm formation. Animportant number of biological surfaces prevent microbial colonization due to their surface topographies, e.g.: the shells of mollusks and crabs and the skin of marine mammals and sharks. These facts have encouraged research of bioinspired surface designs. The main objectives of this work were to produce micro-nanometric hierarchical topographies and to analyze the influence of the topography on the bacterial adhesion. The hierarchical surface was designed using surface plasma oxidation of uni-axial stretch of polydimethylsiloxane (PDMS) films. This method has the advantage to allow designing sub-micrometric wrinkle topographic surfaces changing the plasma time exposition and the uniaxial stretch. Different topography surfaces were obtained, surface has wrinkles with different wavelength (from 500 to 3000 nm) and amplitude (from 80 to 700 nm) parameters. The bacterial adhesion on these novel hierarchical surfaces was evaluated through exposing them to a culture of Pseudomonas protegens Pf-5 for different times. The bacterial attachment was evaluated taking images of the wrinkled and smooth surfaces using an Atomic Force Microscopy (AFM). The initial results of this study suggests thatwrinkled surface with a wavelength of 1000 nm (aprox. bacteria size) delay the bacterial adhesion and, on the other hand, wrinkled surface with a wavelength of 3000 nm enhance and encourage bacterial adhesion. These results demonstrate the importance of the topographic surface to inhibit or stimulatethe biofilm development.Fil: Colonnella, Maria Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Paris, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Lizarraga, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaXII Congreso Argentino de Microbiología GeneralTucumánArgentinaSociedad Argentina de Microbiología Genera

Blue-light-dependent inhibition of twitching motility in Acinetobacter baylyi ADP1: additive involvement of three BLUF-domain-containing proteins

Twitching motility in Acinetobacter baylyi ADP1 is inhibited by moderate intensities of blue light in a temperature-dependent manner (maximally at 20 °C). We analysed the involvement of four predicted blue-light sensing using flavin (BLUF)-domain-containing proteins encoded in the genome of this strain in the twitching motility phenotype. All four genes were expressed both in light and in darkness. A phylogenetic tree showed that one BLUF domain, ACIAD2110, grouped separately from the other three (ACIAD1499, ACIAD2125 and ACIAD2129). Individual knockout mutants of the latter three, but not of ACIAD2110, fully abolished the light dependency of the twitching motility response. Quantitative analysis of transcript level of the three genes showed a decreased expression in the light, with dark/light ratios of 1.65±0.28, 1.79±0.21 and 2.69±0.39, for ACIAD2125, ACIAD2129 and ACIAD1499, respectively. Double and triple knockouts of ACIAD1499, ACIAD2125 and ACIAD2129 confirmed the same phenotype as the corresponding single knockouts. Complementation of all the single knockouts and the triple knockout mutants with any of the three BLUF-domain-encoding genes fully restored the inhibition of twitching motility by blue light that is observed in the wild-type strain. A. baylyi ADP1 therefore shows a high degree of redundancy in the genes that encode BLUF-containing photoreceptors. Moreover, all plasmid-complemented strains, expressing any of the BLUF proteins irrespective of the specific set of deleted photoreceptors, displayed increased light-dependent inhibition of twitching motility, as compared to the wild-type (P<0.001). We conclude that the three genes ACIAD1499, ACIAD2125 and ACIAD2129 are jointly required to inhibit twitching motility under moderate blue-light illumination.Fil: Bitrian, Mariana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gonzalez, Rodrigo Horacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Paris, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Hellingwerf, Klaas. University Of Amsterdam; Países BajosFil: Nudel, Berta Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.Fil: Rinaldi, Jimena Julieta. 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: Arrar, Mehrnoosh. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Sycz, Gabriela. 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: Cerutti, Maria Laura. 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; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; ArgentinaFil: Berguer, Paula Mercedes. 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: Paris, Gastón. 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: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Marti, Marcelo Adrian. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Klinke, Sebastian. 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; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; ArgentinaFil: Goldbaum, Fernando Alberto. 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; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; Argentin

Genome Sequence of Bradyrhizobium japonicum E109, One of the Most Agronomically Used Nitrogen-Fixing Rhizobacteria in Argentina

We present here the complete genome sequence of Bradyrhizobium japonicum strain E109, one of the most used rhizobacteria for soybean inoculation in Argentina since the 1970s. The genome consists of a 9.22-Mbp single chromosome and contains several genes related to nitrogen fixation, phytohormone biosynthesis, and a rhizospheric lifestyle.Fil: Torres, Daniela Soledad. Universidad Nacional de Rio Cuarto; ArgentinaFil: Revale, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Obando, Melissa. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Departamento de Cs.naturales. Laboratorio de Fisiologia Vegetal y de la Interaccion Planta-microorganismo; ArgentinaFil: Maroniche, Guillermo Andres. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Paris, Gastón. Fundación Instituto Leloir; ArgentinaFil: Perticari, Alejandro. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Vazquez, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Wisniewsk Dyé, Florence. Universite Lyon 2; FranciaFil: Martínez Abarca Pastor, Francisco. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidin; EspañaFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto; Argentin

Complete Genome Sequence of the Model Rhizosphere Strain Azospirillum brasilense Az39, Successfully Applied in Agriculture

We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.Fil: Rivera Botia, Diego Mauricio. Universidad Nacional de Rio Cuarto; ArgentinaFil: Revale, Santiago. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Molina, Romina Micaela. Universidad Nacional de Rio Cuarto; ArgentinaFil: Gualpa, Jose. Universidad Nacional de Rio Cuarto; ArgentinaFil: Puente, Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Maroniche, Guillermo Andres. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Paris, Gastón. Fundación Instituto Leloir; ArgentinaFil: Baker, David. The Genome Analysis Centre; Reino UnidoFil: Clavijo, Bernardo. The Genome Analysis Centre; Reino UnidoFil: McLay, Kirsten. The Genome Analysis Centre; Reino UnidoFil: Spaepen, Stijn. Katholieke Universiteit Leuven; Bélgica. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Perticari, Alejandro. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Wisniewski Dyé, Florence. Université Lyon. Ecologie Microbienne; FranciaFil: Whatkins, Christopher. The Genome Analysis Centre; Reino UnidoFil: Martínez Abarca, Francisco. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidin; EspañaFil: Vanderleyden, Jos. Katholieke Universiteit Leuven; BélgicaFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto; Argentin

Relevance of the Diversity among Members of the Trypanosoma Cruzi Trans-Sialidase Family Analyzed with Camelids Single-Domain Antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system

Modular unfolding and dissociation of the human respiratory syncytial virus phosphoprotein p and its interaction with the m(2-1) antiterminator: a singular tetramer-tetramer interface arrangement

Paramyxoviruses share the essential RNA polymerase complex components, namely, the polymerase (L), phosphoprotein (P), and nucleoprotein (N). Human respiratory syncytial virus (RSV) P is the smallest polypeptide among the family, sharing a coiled coil tetramerization domain, which disruption renders the virus inactive. We show that unfolding of P displays a first transition with low cooperativity but substantial loss of α-helix content and accessibility to hydrophobic sites, indicative of loose chain packing and fluctuating tertiary structure, typical of molten globules. The lack of unfolding baseline indicates a native state in conformational exchange and metastable at 20 °C. The second transition starts from a true intermediate state, with only the tetramerization domain remaining folded. The tetramerization domain undergoes a two-state dissociation/unfolding reaction (37.3 kcal mol(-1)). The M(2-1) transcription antiterminator, unique to RSV and Metapneumovirus, forms a nonglobular P:M(2-1) complex with a 1:1 stoichiometry and a K(D) of 8.1 nM determined by fluorescence anisotropy, far from the strikingly coincident dissociation range of P and M(2-1) tetramers (10(-28) M(3)). The M(2-1) binding region has been previously mapped to the N-terminal module of P, strongly suggesting the latter as the metastable molten globule domain. Folding, oligomerization, and assembly events between proteins and with RNA are coupled in the RNA polymerase complex. Quantitative assessment of the hierarchy of these interactions and their mechanisms contribute to the general understanding of RNA replication and transcription in Paramyxoviruses. In particular, the unique P-M(2-1) interface present in RSV provides a valuable antiviral target for this worldwide spread human pathogen.Fil: Esperante, Sebastian. 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: Paris, Gastón. 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: de Prat Gay, Gonzalo. 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; Argentin

The two-component systems PrrBA and NtrYX co-ordinately regulate the adaptation of Brucella abortus to an oxygen-limited environment

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. The success of Brucella as pathogen relies in its ability to adapt to the harsh environmental conditions found in mammalian hosts. One of its main adaptations is the induction of the expression of different genes involved in respiration at low oxygen tension. In this report we describe a regulatory network involved in this adaptation. We show that Brucella abortus PrrBA is a functional two-component signal transduction system that responds to the redox status and acts as a global regulator controlling the expression of the regulatory proteins NtrY, FnrN and NnrA, which are involved in the adaptation to survive at low oxygen tension. We also show that the two-component systems PrrBA and NtrYX co-ordinately regulate the expression of denitrification and high-affinity cytochrome oxidase genes. Strikingly, a double mutant strain in the prrB and ntrY genes is severely impaired in growth and virulence, while the ntrY and prrB single mutant strains are similar to wild-type B. abortus. The proposed regulatory network may contribute to understand the mechanisms used by Brucella for a successful adaptation to its replicative niche inside mammalian cells.Fil: Carrica, Mariela del Carmen. 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: Fernandez, Ignacio. 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: Sieira, Rodrigo. 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: Paris, Gastón. 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: Goldbaum, Fernando Alberto. 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; Argentin

The NtrY/X two-component system of Brucella spp. acts as a redox sensor and regulates the expression of nitrogen respiration enzymes

Brucella spp. are facultative intracellular bacteria pathogenic for many mammalian species including humans, causing a disease called brucellosis. Learning how Brucella adapts to its intracellular niche is crucial for understanding its pathogenesis mechanism, allowing for the development of new and more effective vaccines and treatments against brucellosis. Brucella pathogenesis resides mostly in its ability to adapt to the harsh environmental conditions encountered during host infection such as the oxygen depletion. The mechanism by which Brucella senses the oxygen tension and triggers its environmental adaptation is unknown. In this work we show that the Brucella abortus NtrY/NtrX two-component system is involved in oxygen sensing through a haem group contained in a Per-ARNT-SIM (PAS) domain of the NtrY histidine kinase. The NtrY haem iron can be reduced to the ferrous form and is rapidly oxidized to the ferric form in presence of oxygen. Importantly, we show that the oxidation state of the haem iron modulates the autokinase activity, being the anoxygenic reduced ferrous form the signalling state of NtrY. Also, we show that ntrY gene expression increases under low oxygen tension and that NtrY transfers its signal to its cognate response regulator NtrX, regulating in this way the expression of nitrogen respiration enzymes. Based on these findings, we postulate that NtrY acts as a redox sensor in Brucella spp.Fil: Carrica, Mariela del Carmen. 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: Fernandez, Ignacio. 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: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Paris, Gastón. 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: Goldbaum, Fernando Alberto. 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; Argentin