The role of NtrC in the adaptation of Herbaspirillum seropedicae SmR1 to nitrogen limitation and to nitrate.

The RNA-Seq profiling of Herbaspirillum seropedicae SmR1 wild-type and ntrC mutant was performed under aerobic and three nitrogen conditions (ammonium limitation, ammonium shock, and nitrate shock) to identify the major metabolic pathways modulated by these nitrogen sources and those dependent on NtrC. Under ammonium limitation, H. seropedicae scavenges nitrogen compounds by activating transporter systems and metabolic pathways to utilize different nitrogen sources and by increasing proteolysis, along with genes involved in carbon storage, cell protection, and redox balance, while downregulating those involved in energy metabolism and protein synthesis. Growth on nitrate depends on the narKnirBDHsero_2899nasA operon responding to nitrate and NtrC. Ammonium shock resulted in a higher number of genes differently expressed when compared to nitrate. Our results showed that NtrC activates a network of transcriptional regulators to prepare the cell for nitrogen starvation, and also synchronizes nitrogen metabolism with carbon and redox balance pathways.

Keguizhuia sedimenti gen. nov., sp. nov., isolated from river sediment represents a novel genus of the family Oxalobacteraceae.

A Gram-stain-negative bacterium, designated as R-40T, was isolated from sediment of the Mulong river in Mianyang city, Sichuan province, PR China. The cells of strain R-40T were aerobic non-motile and formed translucent white colonies on R2A agar. Growth occurred at 15-37 °C (optimum 30 °C), pH 5.0-9.0 (optimum 7.0) and salinities of 0-3.0 % (w/v, optimum 0 %). R-40T showed 95.2-96.6 % 16S rRNA gene sequence similarities with the type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum in the family Oxalobacteraceae. The results of phylogenetic analysis based on genome sequences indicated that the strain was clustered with type strains of species of the genera Oxalicibacterium and Herminiimonas in the family Oxalobacteraceae but formed a distinct lineage. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and average amino acid identity (AAI) values between R-40T and type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum ranged from 69.3 to 74.1 %, from 18.2 to 21.4 % and from 60.1 to 67.4 %, respectively. The major cellular fatty acids were C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major quinone was ubiquinone-8 (Q-8). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipid and small amounts of glycophospholipids. The genome size of R-40T was 5.1 Mbp with 54.0 % DNA G+C content. On the basis of the evidence presented in this study, strain R-40T represents a novel species of a novel genus in the family Oxalobacteraceae, for which the name Keguizhuia sedimenti gen. nov., sp. nov. (type strain R-40T=MCCC 1K08818T=KCTC 8137T) is proposed.

Nitrogen fertilization regulates crosstalk between marandu palisadegrass and Herbaspirillum seropedicae: An investigation based on 15N isotopic analysis and root morphology.

Strategies seeking to increase the use efficiency of nitrogen (N) fertilizers and that benefit plant growth through multiple mechanisms can reduce production costs and contribute to more sustainable agriculture free of polluting residues. Under controlled conditions, we investigated the compatibility between foliar inoculation with an endophytic diazotrophic bacterium (Herbaspirillum seropedicae HRC54) at control and low, medium and high N fertilization levels (0, 25, 50 and 100 mg of N kg-1 as urea, respectively) in Marandu palisadegrass. Common procedures in our research field (biometric and nutritional assessments) were combined with isotopic techniques (natural abundance - δ15N‰ and 15N isotope dilution) and root scanning to determine the contribution of fixed N and recovery of N fertilizer by the grass. Overall, the combined use of 15N isotopic techniques revealed that inoculation not only improved the recovery of applied N-urea from the soil but also provided fixed nitrogen to Marandu palisade grass, resulting in an increase in the total accumulated N. When inoculated plants grew at control and low levels of N, a positive cascade effect encompassing root growth stimulation (nodes of smaller diameter roots), better soil and fertilizer resource exploitation and increased forage production was observed. In contrast, increasing N reduced the contributions of N fixed by H. seropedicae from 21.5% at the control level to 8.6% at the high N level. Given the minimal to no observed growth promotion, this condition was deemed inhibitory to the positive effects of H. seropedicae. We discuss how to make better use of H. seropedicae inoculation in Marandu palisadegrass, albeit on a small scale, thus contributing to a more rational and efficient use of N fertilizers. Finally, we pose questions for future investigations based on 15N isotopic techniques under field conditions, which have great applicability potential.

Response characteristics and functional predictions of soil microorganisms to heavy metals, antibiotics, and their resistance genes originating from different animal farms amended with Herbaspirillum huttiense.

Current understanding is limited regarding technologies that use biochar and microorganisms to simultaneously treat soils contaminated with both veterinary antibiotics (VAs) and heavy metals (HMs) from different animal farms. The contributions of the keystone taxa and their similarities from different animal farms under VA and HM stresses before and after soil remediation should be further investigated as well. An innovative treatment of Herbaspirillum huttiense (HHS1) inoculated waste fungus chaff-based (WFCB) biochar was designed for immobilization of copper (Cu) and zinc (Zn), and the removal of oxytetracycline (OTC), enrofloxacin (ENR), and a subsequent reduction in their resistance genes in soils from pig, cow, and chicken farms. Roles of indigenous microorganisms which can treat soils contaminated with VAs and HMs were summarized. Results showed that available Cu and Zn were reduced by 19.5% and 28.1%, respectively, while 49.8% of OTC and 85.1% of ENR were removed by WFCB-HHS1. The decrease in ENR improved overall microbial community diversity, and the increases in genera HHS1, Pedobacter, Flavobacterium and Aequorivita, along with the decreases of genera Bacillus, Methylobacter, and Fermentimonas were indirectly favorable to treat HMs and VAs in soils from different animal farms. Bacterial communities in different animal farm soils were predominantly influenced by stochastic processes. The regulations of functional genes associated with metabolism and environmental information processing, which contribute to HM and VA defense, were altered when using WFCB-HHS1. Furthermore, the spread of their antibiotic resistance genes was restricted.

Response strategies of slash pine (Pinus elliottii) to cadmium stress and the gain effects of inoculation with Herbaspirillum sp. YTG72 in alleviating phytotoxicity and enhancing accumulation of cadmium.

Phytoremediation using fast-growing woody plants assisted by plant growth-promoting bacteria (PGPB) on cadmium (Cd)-contaminated sites is considered a promising technique; however, its remediation efficiency is still affected by multiple factors. In this study, the mining areas' soil conditions were simulated with different Cd addition levels (0, 3, 6, 9 mg kg-1) in order to investigate the response strategy to Cd stress of fast-growing economic tree species, slash pine (Pinus elliottii), and the effects of inoculation with the PGPB strain Herbaspirillum sp. YTG72 on the physiological activity and Cd accumulation of plants. The main results showed that there were significant (p < 0.05) increases in contents of chlorophyll and nutrient elements (P, K, Ca, and Mg) at low Cd addition level (3 mg kg-1) compared to non-Cd addition treatment. When the additive amount of Cd increased, the growth of plants was severely inhibited and the content of proline was increased, as well as Cd in plants. Besides, the ratios of K:P, Ca:P, and Mg:P in plants were negatively correlated with the contents of Cd in plants and soils. Inoculation of P. elliottii with the PGPB strain Herbaspirillum sp. YTG72 improved the physiological functions of the plants under Cd stress and activated the antioxidant system, reduced the accumulation of proline, and decreased the ratios of K:P, Ca:P, and Mg:P in plant. More importantly, planting P. elliottii in Cd-contaminated soil could significantly (p < 0.05) reduce the Cd content in the rhizosphere soil, and furthermore, inoculation treatment could promote the reduction of soil Cd content and increased the accumulation of Cd by root. The results of the present study emphasized the Cd response mechanism of P. elliottii based on multifaceted regulation, as well as the feasibility of strain Herbaspirillum sp. YTG72 assisted P. elliottii for the remediation on Cd-contaminated sites.

Lipopolysaccharides of Herbaspirillum species and their relevance for bacterium-host interactions.

The lipopolysaccharides of Herbaspirillum lusitanum P6-12T (HlP6-12T) and H. frisingense GSF30T (HfGSF30T) was isolated by phenol-water extraction from bacterial cells and was characterized using chemical analysis and SDS-PAGE. It was shown that these bacteria produce LPSs that differ in their physicochemical properties and macromolecular organization. In this paper, the lipid A structure of the HlP6-12T LPS, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To prove the effect of the size of micelles on their bioavailability, we examined the activity of both LPSs toward the morphology of wheat seedlings. Analysis of the HlP6-12T and HfGSF30T genomes showed no significant differences between the operons that encode proteins involved in the biosynthesis of the lipids A and core oligosaccharides. The difference may be due to the composition of the O-antigen operon. HfGSF30T has two copies of the rfb operon, with the main one divided into two fragments. In contrast, the HlP6-12T genome contains only a single rfb-containing operon, and the other O-antigen operons are not comparable at all. The integrity of O-antigen-related genes may also affect LPS variability of. Specifically, we have observed a hairpin structure in the middle of the O-antigen glycosyltransferase gene, which led to the division of the gene into two fragments, resulting in incorrect protein synthesis and potential abnormalities in O-antigen production.

Enhanced removal of antibiotics and heavy metals in aquatic systems using spent mushroom substrate-derived biochar integrated with Herbaspirillum huttiense.

A novel integrated removal strategy was developed to enhance the concurrent elimination of copper (Cu), zinc (Zn), oxytetracycline (OTC), and enrofloxacin (ENR) from the aqueous environments. The underlying adsorption mechanisms of spent mushroom substrate (SMSB) and the Herbaspirillum huttiense strain (HHS1), and their efficacy in removing Cu, Zn, OTC, and ENR was also examined. Results showed that the SMSB-HHS1 composite stabilized 29.86% of Cu and 49.75% of Zn and achieved removal rates of 97.95% for OTC and 59.35% for ENR through a combination of chemisorption and biodegradation. Zinc did not affect Cu adsorption, and ENR did not impact the adsorption of OTC on SMSB. However, the co-presence of OTC and ENR modified the adsorption behaviors of both Cu and Zn. Copper and Zn enhanced the adsorption of OTC and ENR by serving as bridging agents, facilitating the interaction between the contaminants and SMSB. Conversely, OTC and ENR inhibited the adsorption process of Cu by obstructing its interaction with the SMSB and occupying the oxygen-containing functional groups. The ‒OH (3415 cm-1) and C-O-C (1059 cm-1) functional groups were identified as the principal active sites to form hydrogen bonds and interact with Cu and Zn, leading to the formation of CuP4O11 and Zn4CO3(OH)6H2O. HHS1 also enhanced antibiotic removal through biodegradation, as evidenced by the decrease of ‒C‒O and increase of ‒C = O groups. This study underscores the innovative potential of the SMSB-HHS1 composite, offering a sustainable approach to addressing multifaceted pollution challenges in the aquatic environments.

Catheter-related Infections in Pediatric Patients Due to a Rare Pathogen: Herbaspirillum huttiense.

BACKGROUND: Herbaspirillum species are nonfermenting, aerobic, helical or curved, Gram-negative bacteria belonging to the class Betaproteobacteria, order Burkholderiales. To date, only a few studies have reported on the epidemiology, clinical symptoms, antibiotic susceptibility profiles, treatment and outcomes of Herbaspirillum huttiense -related infections in pediatric patients. METHODS: The aim of this study was to present 3 years of H.huntiense data, antibiotic susceptibility profiles, systemic antibiotics and antibiotic lock therapy (ALT) options and clinical outcomes. RESULTS: Fourteen episodes of infection in 12 patients were included in this retrospective study. The patients had a male/female ratio of 1:1 and a median age of 160.5 months (range, 3-198 months). Catheter-related bloodstream infection (CRBSI) was detected in 11 patients. Only 1 patient developed catheter-related infective endocarditis. The patient's catheter was removed, and she was successfully treated with systemic antibiotics for 4 weeks. Systemic antibiotics were used in all infections related to H. huttiense . In septic, critically ill patients, the catheter was removed, and systemic antibiotics were started. Port catheters were removed in 5 patients. ALT was performed in clinically stable patients. ALT using amikacin was administered to 6 patients through the port catheter. Two patients had a 2nd attack. After the 2nd ALT treatment, 1 patient cured, and the catheter of the other patient was removed due to persistent microbial growth in cultures. Antimicrobial susceptibility testing of the reported isolates showed susceptibility to meropenem (90%), ceftazidime (87%) and piperacillin/tazobactam (65%), with 92% resistance to colistin. CONCLUSION: H. huttiense is an emerging pathogen in CRBSI. Piperacillin/tazobactam, ceftazidime and meropenem appear to be good therapeutic options for the treatment of H. huttiense infections. ALT and systemic antibiotics can be used in H. huttiense -CRBSI to sterilize and preserve the central venous catheter.

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)

Bacteriemia por Herbaspirillum huttiense en un paciente neonato / Bacteremia caused by Herbaspirillum huttiense in a newborn

No disponible

Herbaspirillum huttiense pneumonia in a patient with essential thrombocythaemia / Neumonía por Herbaspirillum huttiense en un paciente con trombocitemia esencial

No disponible

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.

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.

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.