RESUMO
Bacteria belonging to the genus Herbaspirillum are found in many different ecological niches. Some species are typically endophytic, while others were reported as free-living organisms that occupy various environments. Also, opportunistic herbaspirilli have been found infecting humans affected by several diseases. We have analyzed the production of exopolysaccharides (EPS) by Herbaspirillum strains isolated from different sources and with distinct ecological characteristics. The monosaccharide composition was determined for the EPS obtained for selected strains including free-living, plant-associated and clinical isolates, and the relationship with the ecological niches occupied by Herbaspirillum spp. is proposed.
Assuntos
Bactérias , Meio Ambiente , Herbaspirillum , Polissacarídeos Bacterianos , Bactérias/metabolismo , Herbaspirillum/química , Herbaspirillum/genética , Herbaspirillum/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/químicaRESUMO
The effect of an external electric field on the formation of protein GlnB-Hs films and on its buffer solution on siliconized glass slides has been analyzed by current versus electric field curves and atomic force microscopy (AFM). The Herbaspirillum seropedicae GlnB protein (GlnB-Hs) is a globular, soluble homotrimer (36 kDa) with its 3-D structure previously determined. Concentrations of 10 nM native denatured GlnB-Hs protein were deposited on siliconized glass slides under ambient conditions. Immediately after solution deposition a maximum electric field of 30 kV/m was applied with rates of 3 V/s. The measured currents were surface currents and were analyzed as transport current. Electric current started to flow only after a minimum electric field (critical value) for the systems analyzed. The AFM images showed films with a high degree of directional organization only when the proteins were present in the solution. These results showed that the applied electric field favored directional organization of the protein GlnB-Hs films and may contribute to understand the formation of protein films under applied electric fields.
Assuntos
Proteínas de Bactérias/química , Eletricidade , Herbaspirillum/química , Proteínas PII Reguladoras de Nitrogênio/química , Microscopia de Força Atômica , Silicones/químicaRESUMO
Herbaspirillum bacteria are best known as plant growth-promoting rhizobacteria but have also been recovered from clinical samples. Here, biochemical tests, matrix-assisted laser deionization-time of flight (MALDI-TOF) mass spectrometry, adherence, and cytotoxicity to eukaryotic cells were used to compare clinical and environmental isolates of Herbaspirillum spp. Discrete biochemical differences were observed between human and environmental strains. All strains adhered to HeLa cells at low densities, and cytotoxic effects were discrete, supporting the view that Herbaspirillum bacteria are opportunists with low virulence potential.
Assuntos
Aderência Bacteriana/fisiologia , Microbiologia Ambiental , Infecções por Bactérias Gram-Negativas/microbiologia , Herbaspirillum/fisiologia , Herbaspirillum/patogenicidade , Sobrevivência Celular , Células HeLa , Herbaspirillum/química , Herbaspirillum/classificação , Humanos , Filogenia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Herbaspirillum seropedicae Z67 is a diazotrophic endophyte able to colonize the interior of many economically relevant crops such as rice, wheat, corn and sorghum. Structures of siderophores produced by bacterial endophytes have not yet been elucidated. The aim of this work was to identify and characterize the siderophores produced by this bacterium. In a screening for mutants unable to produce siderophores we found a mutant that had a transposon insertion in a non-ribosomal peptide synthase (NRPS) gene coding for a putative siderophore biosynthetic enzyme. The chemical structure of the siderophore was predicted using computational genomic tools. The predicted structure was confirmed by chemical analysis. We found that siderophores produced by H. seropedicae Z67 are a suite of amphiphilic lipopeptides, named serobactin A, B and C, which vary by the length of the fatty acid chain. We also demonstrated the biological activity of serobactins as nutritional iron sources for H. seropedicae. These are the first structurally described siderophores produced by endophytic bacteria.
Assuntos
Herbaspirillum/metabolismo , Sideróforos/química , Endófitos/química , Endófitos/genética , Endófitos/metabolismo , Herbaspirillum/química , Herbaspirillum/genética , Ferro/metabolismo , Modelos Químicos , Mutação , Peptídeo Sintases/genética , Poaceae/microbiologia , Estrutura Terciária de Proteína , Sideróforos/isolamento & purificaçãoRESUMO
DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum/química , Recombinases Rec A/metabolismo , DNA Bacteriano , Escherichia coli/metabolismo , Ligação ProteicaRESUMO
DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum/química , Recombinases Rec A/metabolismo , DNA Bacteriano , Escherichia coli/metabolismo , Ligação ProteicaRESUMO
Lipid-A was isolated by mild acid hydrolysis from lipopolysaccharides extracted from cells of Herbaspirillum seropedicae, strain SMR1, and from two mutants deficient in the biosynthesis of rhamnose (rmlBâ» and rmlCâ»). Structural analyzes were carried out using MALDI-TOF and derivatization by per-O-trimethylsilylation followed by GC-MS in order to determine monosaccharide and fatty acid composition. De-O-acylation was also performed to determine the presence of N-linked fatty acids. Lipid-A from H. seropedicae SMR1 showed a major structure comprising 2-amino-2-deoxy-glucopyranose-(1â6)-2-amino-2-deoxy-glucopyranose phosphorylated at C4' and C1 positions, each carrying a unit of 4-amino-4-deoxy-arabinose. C2 and C2' positions were substituted by amide-linked 3-hydroxy-dodecanoic acids. Both rhamnose-defective mutants showed similar structure for their lipid-A moieties, except for the lack of 4-amino-4-deoxy-arabinose units attached to phosphoryl groups.
Assuntos
Herbaspirillum/genética , Herbaspirillum/fisiologia , Lipídeo A/química , Mutação , Raízes de Plantas/microbiologia , Zea mays/microbiologia , Herbaspirillum/química , Herbaspirillum/metabolismo , Lipídeo A/isolamento & purificação , Mutagênese , Ramnose/biossínteseRESUMO
The RNA chaperone Hfq is a homohexamer protein identified as an E. coli host factor involved in phage Qß replication and it is an important posttranscriptional regulator of several types of RNA, affecting a plethora of bacterial functions. Although twenty Hfq crystal structures have already been reported in the Protein Data Bank (PDB), new insights into these protein structures can still be discussed. In this work, the structure of Hfq from the ß-proteobacterium Herbaspirillum seropedicae, a diazotroph associated with economically important agricultural crops, was determined by X-ray crystallography and small-angle X-ray scattering (SAXS). Biochemical assays such as exclusion chromatography and RNA-binding by the electrophoretic shift assay (EMSA) confirmed that the purified protein is homogeneous and active. The crystal structure revealed a conserved Sm topology, composed of one N-terminal α-helix followed by five twisted ß-strands, and a novel π-π stacking intra-subunit interaction of two histidine residues, absent in other Hfq proteins. Moreover, the calculated ab initio envelope based on small-angle X-ray scattering (SAXS) data agreed with the Hfq crystal structure, suggesting that the protein has the same folding structure in solution.
Assuntos
Herbaspirillum/química , Fator Proteico 1 do Hospedeiro/química , Chaperonas Moleculares/química , Sequência de Aminoácidos , Cromatografia em Gel , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Histidina/química , Fator Proteico 1 do Hospedeiro/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espalhamento a Baixo ÂnguloRESUMO
This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.
Assuntos
Membrana Celular/química , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Herbaspirillum/química , Proteínas de Membrana Transportadoras/análise , Proteoma/análise , Compostos de Amônio Quaternário/metabolismo , Proteínas de Bactérias/análise , Proteínas de Transporte de Cátions/análise , Eletroforese em Gel Bidimensional , Herbaspirillum/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Bacteria from the genus Herbaspirillum are endophytes responsible for nitrogen fixation in gramineous plants of economic importance such as maize, sugarcane, sorghum, rice, and wheat. Some species are known to produce plant growth substances. In contrast, Herbaspirillum rubrisubalbicans strains are known to be mild plant pathogens. The molecular communication between the plant and the microbes might involve lipopolysaccharides present in the outer membrane of these gram-negative bacteria. Phenol-water extraction was used to obtain lipopolysaccharides from 7 strains of Herbaspirillum seropedicae (SmR1, Z67, Z78, ZA95, and M2) and H. rubrisubalbicans (M1 and M4). The electrophoretic profiles and chemical composition of the lipopolysaccharides obtained in the phenol and aqueous extracts were shown herein.
Assuntos
Herbaspirillum/química , Lipopolissacarídeos/química , Poaceae/microbiologia , Herbaspirillum/fisiologia , Fixação de NitrogênioRESUMO
The adsorption of proteins and its buffer solution on mica surfaces was investigated by atomic force microscopy (AFM). Different salt concentration of the Herbaspirillum seropedicae GlnB protein (GlnB-Hs) solution deposited on mica was investigated. This protein is a globular, soluble homotrimer (36kDa), member of PII-like proteins family involved in signal transducing in prokaryote. Supramolecular structures were formed when this protein was deposited onto bare mica surface. The topographic AFM images of the GlnB-Hs films showed that at high salt concentration the supramolecular structures are spherical-like, instead of the typical doughnut-like shape for low salt concentration. AFM images of NaCl and Tris from the buffer solution showed structures with the same pattern as those observed for high salt protein solution, misleading the image interpretation. XPS experiments showed that GlnB protein film covers the mica surface without chemical reaction.
Assuntos
Silicatos de Alumínio/metabolismo , Proteínas de Bactérias/metabolismo , Herbaspirillum/química , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Adsorção , Proteínas de Bactérias/ultraestrutura , Biofilmes , Herbaspirillum/ultraestrutura , Microscopia de Força Atômica , Proteínas PII Reguladoras de Nitrogênio/ultraestrutura , Soluções , Análise Espectral , Propriedades de SuperfícieRESUMO
Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Herbaspirillum/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Sequência Conservada , Cisteína/química , Cisteína/genética , Regulação Bacteriana da Expressão Gênica , Herbaspirillum/química , Herbaspirillum/genética , Oxigênio/metabolismo , Fatores de Transcrição/genéticaRESUMO
The Herbaspirillum seropedicae genome sequence encodes a truncated hemoglobin typical of group II (Hs-trHb1) members of this family. We show that His-tagged recombinant Hs-trHb1 is monomeric in solution, and its optical spectrum resembles those of previously reported globins. NMR analysis allowed us to assign heme substituents. All data suggest that Hs-trHb1 undergoes a transition from an aquomet form in the ferric state to a hexacoordinate low-spin form in the ferrous state. The close positions of Ser-E7, Lys-E10, Tyr-B10, and His-CD1 in the distal pocket place them as candidates for heme coordination and ligand regulation. Peroxide degradation kinetics suggests an easy access to the heme pocket, as the protein offered no protection against peroxide degradation when compared with free heme. The high solvent exposure of the heme may be due to the presence of a flexible loop in the access pocket, as suggested by a structural model obtained by using homologous globins as templates. The truncated hemoglobin described here has unique features among truncated hemoglobins and may function in the facilitation of O(2) transfer and scavenging, playing an important role in the nitrogen-fixation mechanism.
Assuntos
Proteínas de Bactérias/química , Herbaspirillum/química , Herbaspirillum/metabolismo , Nitrogênio/metabolismo , Hemoglobinas Truncadas/química , Absorção , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Heme/metabolismo , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peróxidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Espectrofotometria Ultravioleta , Hemoglobinas Truncadas/metabolismoRESUMO
Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.
Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Herbaspirillum/genética , Mutagênese Insercional/métodos , Compostos de Amônio Quaternário/farmacologia , Genes Bacterianos/fisiologia , Herbaspirillum/química , Herbaspirillum/efeitos dos fármacos , Herbaspirillum/metabolismo , Nitrogênio/fisiologiaRESUMO
Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.