Herbaspirillum seropedicae as a degrading bacterium of monofluoroacetate: effects of its inoculation in goats by ingesting Amorimia septentrionalis and the concentrations of this compound in plants sprayed with the bacterium / Herbaspirillum seropedicae como bactéria degradadora de monofluoroacetato de sódio: efeitos de sua inoculação em caprinos ingerindo Amorimia septentrionalis e nas concentrações deste composto em plantas pulverizadas com a bactéria

Herbaspirillum seropedicae is a nitrogen-fixing bacterium capable of using toxic compounds as a source of carbon. Bacteria with this capacity can be used to make animals resistant to plant poisoning containing monofluoroacetate (MFA), such as Amorimia septentrionalis. The aim of this study was to evaluate if H. seropedicae is efficient in the degradation of MFA present in A. septentrionalis and if the inoculation of this bacterium in goats confers protection to A. septentrionalis intoxication. Two experiments were performed: in the first experiment 12 goats were divided into 2 groups. Goats in Group 1 were orally administered a solution containing the H. seropedicae bacterium for 10 days. From day 10 onwards, they received a daily dose of 5g/kg of A. septentrionalis with the bacteriauntil clinical signs of intoxication were observed. Group 2 goats received only the plant at the same dose, also until the observation of clinical signs of intoxication. The amount of MFA found in A. septentrionalis used in the experiment with goats was 1.6±0.058μg/mg. The total plant dose ingested by all goats in Group 1 was 80.83±12.81g/kg (129.33±20.50mg/kg MFA), which were significantly greater ​​(p<0.05) than those of Group 2 goats (39.16±19.08g/kg plant and 62.66±30.53mg/kg MFA). Group 1 goats took an average of 16.16±2.56 days to develop clinical signs of intoxication, significantly longer (p=0.0012) than Group 2 goats (7.83±3.81 days). Two Group 2 goats died on the same day that they developed clinical signs of intoxication. At necropsy of these two animals, no significant changes were observed. In the second experiment, samples of A. septentrionalis were sprayed with a solution containing H. seropedicae. Before and eight days after spraying, the samples were pressed and dried for quantitation of MFA. The amount of MFA present in samples of A. septentrionalis 8 days after spraying with H. seropedicae was significantly lower (p=0.017) than that found prior to spraying. It can be concluded that administration of H. seropedicae in goats is capable of causing greater resistance to A. septentrionalis intoxication, and spraying the plant with this bacterium significantly reduces the amount of MFA in the plant.(AU)

Herbaspirillum seropedicae é uma bactéria fixadora de nitrogênio, capaz de utilizar compostos tóxicos como fonte de carbono. Bactérias com essa capacidade podem ser utilizadas para tornar os animais resistentes à intoxicação por plantas que contém monofluoroacetato (MFA), como Amorimia septentrionalis. O objetivo do presente estudo é avaliar se H. seropedicae é eficiente na degradação do MFA presente em A. septentrionalis e se a inoculação dessa bactéria, em caprinos, confere proteção à intoxicação por A. septentrionalis. Foram realizados dois experimentos: no primeiro experimento foram utilizados 12 caprinos, divididos em dois grupos. Os caprinos do Grupo 1 receberam diariamente, oralmente, uma solução contendo a bactéria H. seropedicae durante 10 dias. A partir do décimo dia passaram a receber, diariamente, além da solução com a bactéria 5g/kg de A. septentrionalis até a observação de sinal clínico de intoxicação. Os caprinos do Grupo 2 receberam apenas a planta na mesma dose, também até que a observação de sinais clínicos de intoxicação. A quantidade de MFA encontrada em A. septentrionalis utilizada no experimento com caprinos foi de 1,6± 0,058µg/mg de planta em média. A dose total de planta ingerida por todos os caprinos do Grupo 1 foi de 80,83±12,81g/kg (129,33±20,50mg/kg de MFA), valores significativamente maiores (p<0,05) do que os dos caprinos do Grupo 2 (39,16±19,08g/kg de planta e 62,66± 30,53mg/Kg de MFA). Os caprinos do Grupo 1 demoraram em média 16,16 ±2,56 dias para desenvolver sinais clínicos da intoxicação, período significativamente maior (p=0,0012) que os caprinos do Grupo 2 (7,83±3,81dias). Dois caprinos do Grupo 2 morreram no mesmo dia que desenvolveram sinais clínicos da intoxicação. Na necropsia desses dois animais não foram observadas alterações significativas. No segundo experimento, amostras de A. septentrionalis foram pulverizadas com uma solução contendo a bactéria H. seropedicae. Antes e oito dias após a pulverização, as amostras foram prensadas e secas para posterior quantificação do MFA. A quantidade de MFA presente nas amostras de A. septentrionalis oito dias após a pulverização com H. seropedicae foi significativamente menor (p=0,017) do que a encontrada antes da pulverização. Pode-se concluir que a administração de H. seropedicae em caprinos é capaz de causar uma maior resistência à intoxicação por A. septentrionalis, e a pulverização da planta com esta bactéria reduz significativamente a quantidade de MFA na planta.(AU)

Septicemia Caused by Herbaspirillum huttiense Secondary to Pneumonia

No abstract available.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

Effect of point mutations on Herbaspirillum seropedicae NifA activity

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

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.

Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.

Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets 'Vitória'

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets 'Vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae.

Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. in sugar cane (Saccharum spp) using differente polyclonal antibodies

The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5) cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.

Associative diazotrophic bacteria in grass roots and soils from heavy metal contaminated sites

This work aimed to evaluate density of associative diazotrophic bacteria populations in soil and grass root samples from heavy metal contaminated sites, and to characterize isolates from these populations, both, phenotypically (Zinc, Cadmium and NaCl tolerance in vitro, and protein profiles) and genotypically (16S rDNA sequencing), as compared to type strains of known diazotrophic species. Densities were evaluated by using NFb, Fam and JNFb media, commonly used for enrichment cultures of diazotrophic bacteria. Bacterial densities found in soil and grass root samples from contaminated sites were similar to those reported for agricultural soils. Azospirillum spp. isolates from contaminated sites and type strains from non-contaminated sites varied substantially in their in vitro tolerance to Zn+2 and Cd+2, being Cd+2 more toxic than Zn+2. Among the most tolerant isolates (UFLA 1S, 1R, S181, S34 and S22), some (1R, S34 and S22) were more tolerant to heavy metals than rhizobia from tropical and temperate soils. The majority of the isolates tolerant to heavy metals were also tolerant to salt stress as indicated by their ability to grow in solid medium supplemented with 30 g L-1 NaCl. Five isolates exhibited high dissimilarity in protein profiles, and the 16S rDNA sequence analysis of two of them revealed new sequences for Azospirillum.

Objetivou-se avaliar a densidade de populações de bactérias diazotróficas associativas em amostras de solos e de raízes de gramíneas oriundas de sítios contaminados com metais pesados, e caracterizar isolados destas populações através da análise fenotípica (tolerância aos metais pesados zinco e cádmio e à NaCl in vitro, perfis protéicos), e genotípica (seqüenciamento de 16S rDNA), comparados às estirpes tipo das mesmas espécies. As densidades foram avaliadas nos meios NFb, Fam e LGI, comumente utilizados para culturas de enriquecimento de populações de bactérias diazotróficas associativas. As densidades encontradas em amostras de solo e raiz de sítios contaminados foram semelhantes àquelas relatadas na literatura para solos agrícolas. Isolados de Azospirillum spp. de solos contaminados e estirpes tipo oriundas de solos não contaminados variaram substancialmente com relação à tolerância a Zn+2 e Cd+2, sendo que Cd+2 mais tóxico que Zn+2. Dentre os isolados mais tolerantes (UFLA 1S, 1R, S181, S34, e S22), alguns(1R, S34 e S22) foram mais tolerantes a metais pesados que rizóbios isolados de solos de áreas tropicais e temperadas. A maioria dos isolados mais tolerantes a metais pesados também foi tolerante ao estresse salino, o que foi indicado por seu crescimento em meio sólido suplementado com 30 g L-1 de NaCl in vitro. Cinco isolados apresentaram alta dissimilaridade em perfis protéicos e o seqüenciamento do gene 16S rDNA em dois deles revelou que apresentam novas seqüências de Azospirillum.

Early colonization pattern of maize (Zea mays L. Poales, Poaceae) roots by Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

The bacterium Herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. However, the mechanisms involved in the interaction between H. seropedicae and these plants are not completely characterized. We investigated the attachment of Herbaspirillum to maize roots and the invasion of the roots by this bacterium using H. seropedicae strain SMR1 transformed with the suicide plasmid pUTKandsRed, which carries a mini-Tn5 transposon containing the gene for the Discosoma red fluorescent protein (Dsred) constitutively expressed together with the kanamycin resistance gene. Integration of the mini-Tn5 into the bacterial chromosome yielded the mutant H. seropedicae strain RAM4 which was capable of expressing Dsred and could be observed on and inside fresh maize root samples. Confocal microscopy of maize roots inoculated with H. seropedicae three days after germination showed that H. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days.