A Rhizobium leguminosarum CHDL- (Cadherin-Like-) Lectin Participates in Assembly and Remodeling of the Biofilm Matrix

In natural environments most bacteria live in multicellular structures called biofilms. These cell aggregates are enclosed in a self-produced polymeric extracellular matrix, which protects the cells, provides mechanical stability and mediates cellular cohesion and adhesion to surfaces. Although important advances were made in the identification of the genetic and extracellular factors required for biofilm formation, the mechanisms leading to biofilm matrix assembly, and the roles of extracellular proteins in these processes are still poorly understood. The symbiont Rhizobium leguminosarum requires the synthesis of the acidic exopolysaccharide and the PrsDE secretion system to develop a mature biofilm. PrsDE is responsible for the secretion of the Rap family of proteins that share one or two Ra/CHDL (cadherin-like-) domains. RapA2 is a calcium-dependent lectin with a cadherin-like β sheet structure that specifically recognizes the exopolysaccharide, either as a capsular polysaccharide (CPS) or in its released form [extracellular polysaccharide (EPS)]. In this study, using gain and loss of function approaches combined with phenotypic and microscopic studies we demonstrated that RapA lectins are involved in biofilm matrix development and cellular cohesion. While the absence of any RapA protein increased the compactness of bacterial aggregates, high levels of RapA1 expanded distances between cells and favored the production of a dense matrix network. Whereas endogenous RapA(s) are predominantly located at one bacterial pole, we found that under overproduction conditions, RapA1 surrounded the cell in a way that was reminiscent of the capsule. Accordingly, polysaccharide analyses showed that the RapA lectins promote CPS formation at the expense of lower EPS production. Besides, polysaccharide analysis suggests that RapA modulates the EPS size profile. Collectively, these results show that the interaction of RapA lectins with the polysaccharide is involved in rhizobial biofilm matrix assembly and remodeling.Fil: Vozza, Nicolas Federico. 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: Abdian, Patricia Lorena. 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: Russo, Daniela Marta. 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: Mongiardini, Elias Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Lodeiro, Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Molin, Soren. Technical University of Denmark; DinamarcaFil: Zorreguieta, Ángeles. 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

Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c1 and CycM and a small increase in the amount of cytochrome aa3. In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene

Bridged nucleic acids reloaded

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (20 -O,40 -aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.Fil: Soler Bistue, Alfonso J. C.. 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; ArgentinaFil: Zorreguieta, Ángeles. 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: Tolmasky, Marcelo E.. California State University; Estados Unido

RapA2 is a calcium-binding lectin composed of two highly conserved cadherin-like domains that specifically recognize Rhizobium leguminosarum acidic exopolysaccharides

In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins.Fil: Abdian, Patricia Lorena. 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: Caramelo, Julio Javier. 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. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Ausmees, Nora. Lund University; SueciaFil: Zorreguieta, Angeles. 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. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Identification of the acinetobacter baumannii ribonuclease p catalytic subunit: Cleavage of a target mRNA in the presence of an external guide sequence

The bacterial ribonuclease P or RNase P holoenzyme is usually composed of a catalytic RNA subunit, M1, and a cofactor protein, C5. This enzyme was first identified for its role in maturation of tRNAs by endonucleolytic cleavage of the pre-tRNA. The RNase P endonucleolytic activity is characterized by having structural but not sequence substrate requirements. This property led to development of EGS technology, which consists of utilizing a short antisense oligonucleotide that when forming a duplex with a target RNA induces its cleavage by RNase P. This technology is being explored for designing therapies that interfere with expression of genes, in the case of bacterial infections EGS technology could be applied to target essential, virulence, or antibiotic resistant genes. Acinetobacter baumannii is a problematic pathogen that is commonly resistant to multiple antibiotics, and EGS technology could be utilized to design alternative therapies. To better understand the A. baumannii RNase P we first identified and characterized the catalytic subunit. We identified a gene coding for an RNA species, M1Ab, with the expected features of the RNase P M1 subunit. A recombinant clone coding for M1Ab complemented the M1 thermosensitive mutant Escherichia coli BL21(DE3) T7A49, which upon transformation was able to grow at the non-permissive temperature. M1Ab showed in vitro catalytic activity in combination with the C5 protein cofactor from E. coli as well as with that from A. baumannii, which was identified, cloned and partially purified. M1Ab was also able to cleave a target mRNA in the presence of an EGS with efficiency comparable to that of the E. coli M1, suggesting that EGS technology could be a viable option for designing therapeutic alternatives to treat multiresistant A. baumannii infections.Fil: Davies Sala, Carol Giselle. California State University; Estados Unidos. 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: Jani, Saumya. California State University; Estados UnidosFil: Zorreguieta, Ángeles. 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: Tolmasky, Marcelo E.. California State University; Estados Unido

A multimeric matrix-associated lectin (RapD) affects proper exopolysaccharide processing in Rhizobium leguminosarum

Rhizobium leguminosarum synthesizes an acidic polysaccharide formed by the polymerization of octasaccharide repeating units containing glucose (Glc), glucuronic acid (GlcA) and galactose (Gal) in a 5:2:1 ratio with particular substitutions; most of it is secreted to the extracellular medium (EPS) and part of it is retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, substrates of the PrsDE type I secretion system (TISS) share at least one Ra/CDHL (cadherin-like) domain and are involved in biofilm and matrix development either by cleaving the polysaccharide (Ply glycanases) or by altering the bacterial adhesive properties. Previous studies have shown that RapA2 is a monomeric calcium-binding lectin capable of binding specifically the R. leguminosarum CPS through a Ra/CDHL domain. It was shown that the absence or excess of RapA2 in the extracellular medium alters the biofilm matrix's properties. In this work we identified a new Rap protein (RapD), which comprises an N-terminal Ra/CDHL domain and a C-terminal domain of unknown function. By Western blot analysis using specific polyclonal antibodies we showed that in planktonic cultures RapD is co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, under conditions that favor EPS production, a prominent RapD secretion was observed. In addition, colony blot assays indicated that RapD is associated with the biofilm matrix. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2 ΔrapD double mutant showed differences in the EPS profiles compared with those of the single mutants and the wild type strain, thus suggesting a functional interaction between the RapA2 and RapD proteins.Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further RapD conformation changes were observed in the presence of EPS. ELISA and BIA (binding inhibition assay) assays showed that in the presence of calcium, RapD specifically binds the EPS and that galactose residues would be involved in this interaction. In conclusion, RapD is a multimeric calcium-dependent EPS lectin that is co-secreted with the other Rap proteins via TISS PrsDE. Unlike RapA2, RapD is not retained on the bacterial surface but would rather interact with the released EPS. Finally, our results suggest that the interaction of RapA2 and RapD with the CPS or the EPS somehow affects the polysaccharide processing and therefore the biofilm matrix.Fil: Tarsitano, Julián. 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: Russo, Daniela Marta. 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: Alonso, Leonardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Zorreguieta, Ángeles. 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; ArgentinaProceedings of the Biofilms 9 online conferenceKarslruheAlemaniaKarlsruhe Institute of Technolog

Draft genome sequence of <i>Burkholderia cordobensis</i> type strain LMG 27620, isolated from agricultural soils in Argentina

Bacteria of the genus Burkholderia are commonly found in diverse ecological niches in nature. We report here the draft genome sequence of Burkholderia cordobensis type strain LMG 27620, isolated from agricultural soil in Córdoba, Argentina. This strain harbors several genes involved in chitin utilization and phenol degradation, which make it an interesting candidate for biocontrol purposes and xenobiotic degradation in polluted environments.Instituto de Biotecnologia y Biologia Molecula

Draft genome sequence of <i>Burkholderia cordobensis</i> type strain LMG 27620, isolated from agricultural soils in Argentina

Bacteria of the genus Burkholderia are commonly found in diverse ecological niches in nature. We report here the draft genome sequence of Burkholderia cordobensis type strain LMG 27620, isolated from agricultural soil in Córdoba, Argentina. This strain harbors several genes involved in chitin utilization and phenol degradation, which make it an interesting candidate for biocontrol purposes and xenobiotic degradation in polluted environments.Instituto de Biotecnologia y Biologia Molecula

RapD Is a Multimeric Calcium-Binding Protein That Interacts With the Rhizobium leguminosarum Biofilm Exopolysaccharide, Influencing the Polymer Lengths

Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix’s properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.Fil: Tarsitano, Julián. 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: Ramis, Lila Yanina. 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: Alonso, Leonardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Russo, Daniela Marta. 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: Zorreguieta, Ángeles. 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

Draft genome sequence of <i>Burkholderia cordobensis</i> type strain LMG 27620, isolated from agricultural soils in Argentina

Bacteria of the genus Burkholderia are commonly found in diverse ecological niches in nature. We report here the draft genome sequence of Burkholderia cordobensis type strain LMG 27620, isolated from agricultural soil in Córdoba, Argentina. This strain harbors several genes involved in chitin utilization and phenol degradation, which make it an interesting candidate for biocontrol purposes and xenobiotic degradation in polluted environments.Instituto de Biotecnologia y Biologia Molecula