Proof that Burkholderia Strains Form Effective Symbioses with Legumes: a Study of Novel Mimosa-Nodulating Strains from South America

Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other β-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known β-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N₂-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N₂-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.     

Genetic variability and interpopulational differentiation of Artemia strains from South America

Seven Artemia samples from three South American countries (Chile, Brazil, Peru) were studied by starch electrophoresis with the aim of comparing levels of genetic variation and genetic similarity to representative populations of A. franciscana (San Francisco Bay, California, USA) and A. persimilis (Buenos Aires, Argentina), which are species endemic to the New World. Based on the analysis of 22 loci, parameters measuring genetic variability were, for some populations, found to be among the highest reported for Artemia so far. The percentage of polymorphic loci varied from 31.8% (Piura, Peru; Buenos Aires) to 50% (Los Vilos and Salar de Atacama, Chile), while the observed heterozygosity varied from 0.025 (Piura) to 0.165 (Los Vilos, Chile). A dendrogram based on Nei's genetic distance (D) produced four major groups. The Argentinian form, A. persimilis; the San Francisco Bay strain together with samples from Brazil (Macau and Rio Grande do Norte) and Chile (Pichilemu and Salar de Atacama); two coastal populations from Chile (Los Vilos and Iquique) and the sample from Peru (Piura). These four groups have inter-group D values that are, in some cases, far above those normally associated with conspecific populations.     

Genetic characterization of naturally occurring Rhizobium meliloti populations and their potential to form effective symbioses with lucerne

Genetic characterization by Randomly Amplified Polymorphic DNA (RAPD) fingerprinting was employed to study the status of Rhizobium meliloti populations inhabiting nodules of lucerne. Rhizobium strains were isolated from nodules harvested from plants growing in inoculated or uninoculated experimental plots, uninoculated commercial fields and from lucerne grown in pots containing soils of different origin. Dry matter analyses were recorded and rhizobia were assessed for relative genetic diversity between treatments. Inoculated and uninoculated soils did not differ in terms of dry matter production, and lucerne grew, and was adequately nodulated, in soils with no history of lucerne cultivation. These findings, and the demonstration that there is a rich genetic diversity of Rh. meliloti in these soils, show that it is not always necessary to apply a standard commercial inoculant.     

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America

AIMS: The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. METHODS AND RESULTS: Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. CONCLUSIONS: Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.     

The structure of a polysaccharide from infectious strains of Burkholderia cepacia.

The structure of an acidic exopolysaccharide (EPS) from eight strains of Burkholderia cepacia has been investigated by methylation and sugar analysis, periodate oxidation-Smith degradation, and partial acid-hydrolysis. An enzyme preparation obtained from the same organisms producing the EPS was also used to depolymerize the polysaccharide. Detailed NMR studies of the chemical and enzymatic degradation products showed that this EPS consists of a highly branched heptasaccharide-repeating unit with the following structure: [abstract: see text]. About three O-acetyl groups per repeating unit are present at undetermined positions.     

Burkavidin: a novel secreted biotin-binding protein from the human pathogen Burkholderia pseudomallei

The avidin-biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.     

Characterization of Trypanosoma rangeli strains isolated in Central and South America: an overview

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.     

Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia

AIMS: To screen and clone a novel enzyme with specific activity for the resolution of (R)-beta-acetylmercaptoisobutyrate (RAM) from (R,S)-beta-acetylmercaptoisobutyrate [(R,S)-ester]. METHODS AND RESULTS: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis. A novel esterase gene was cloned from the chromosomal DNA of B. cepacia and was designated as cpoA. The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases. In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment. CONCLUSION: The cpoA cloned from B. cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes the isolation and cloning of a serine esterase gene from B. cepacia, which is useful in the chiral resolution of (R,S)-ester. The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.     

Multiple effects of a novel compound from Burkholderia cepacia against Candida albicans

A novel compound (named CF66I) produced by Burkholeria cepacia CF-66 strain was investigated for its antifungal activity against Candida albicans. This compound exhibited excellent antifungal activity in a dose- and time-dependent manner. Uptake analysis revealed that the compound preferentially acted against the fungal cell wall, and was also able to enter the cells. Transmission electron microscopy indicated that this compound caused loosening of the cell wall and a significant increase in the cell wall thickness was noted; however, no alterations were observed in the contents of the cell wall components. CF66I probably affected the normal assembly and integration of fungal cell wall components by interrupting the weak interactions between them, such as hydrogen and hydrophobic bonds. Propidium iodide (PI) staining indicated that on exposure to CF66I C. albicans cells became permeable to PI. Marked alterations in lipid and sterol contents were observed, and the major changes were a depletion of total lipids and ergosterol, concomitant with an increase in lanosterol content. These observations suggested that the novel compound CF66I may have considerable potential for development of a new class of antifungal agents.     

Zameioscirpus, a new genus of Cyperaceae from South America

Systematic studies of scirpoid species in the Andes showed the necessity to exclude one species each from Scirpus and Carex. They are combined in a new genus Zameioscirpus and a third new species is described. The autonomous generic position of Zameioscirpus within the Scirpeae is supported by a phylogenetic analysis based on rbcL and trnL-F sequencing data and by conspicuous morphological similarities.     

Monopera,a new genus of scrophulariaceae from South America

Monopera is described as a new genus in the Scrophulariaceae, subfamily Scrophularioideae, tribe Angeloneae. Two species are transferred fromAngelonia,Monopera micrantha andMonopera perennis. These species are described and illustrated, and a key is provided.     

Isolation and characterization of a novel haloacid permease from Burkholderia cepacia MBA4

Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated deh4p, was found 353 bases downstream of the dehalogenase gene deh4a. Quantitative analysis of the expression of deh4p showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of deh4a. The nucleotide sequence of deh4p was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [(14)C]MCA into the cell was Deh4p dependent. Deh4p has apparent K(m)s of 5.5 and 8.9 muM and V(max)s of 9.1 and 23.1 nmol mg(-1) min(-1) for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when deh4a was provided in trans.     

Antifungal activity of a novel compound from Burkholderia cepacia against plant pathogenic fungi

AIMS: To investigate antifungal activity of a novel compound (named as CF66I provisionally) against plant pathogenic fungi, mainly including Fusarium sp., Colletotrichum lindemuthianum, Rhizoctonia solani, etc. METHODS AND RESULTS: Minimal inhibition concentrations (MIC) and minimal fungicidal concentrations (MFC) of CF66I for each fungi were determined using serial broth dilution method. The data demonstrated MIC ranged from 2.5 to 20.0 microg ml(-1) and MFC were shown at levels of < or =7.5 microg ml(-1) except Fusarium sp. With reverse microscopy, profound morphological alterations of fungal cells were observed after exposure to CF66I. Conidiospores were completely inhibited, and protoplasm aggregated to form chalamydospores because of the changes of cell permeability. Some chalamydospores were broken, suggesting the compound probably possessed strong ability of damaging the cell wall. In addition, CF66I was investigated for its antifungal stability against Curvularia lunata. The results showed CF66I kept strong fungi-static activity over-wide pH range (pH 4-9) and temperature range (from -70 to 120 degrees C). CONCLUSIONS: The compound CF66I exhibited strong and stable broad-spectrum antifungal activity, and had a significant fungicidal effect on fungal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from prebiocontrol evaluations performed to date are probably useful in the search for alternative approaches to controlling serious plant pathogens.     

Molecular characterization of a novel family VIII esterase from Burkholderia multivorans UWC10

An esterase producing Burkholderia multivorans UWC10 strain was isolated by culture enrichment. A shotgun library of B. multivorans UWC10 genomic DNA was screened for esterase activity and a recombinant clone conferring an esterolytic phenotype was identified. Full-length sequencing of the DNA insert showed that it consisted of a single open reading frame (ORF1) encoding a predicted protein of 398 amino acids. ORF1 (termed EstBL) had a high protein sequence identity to family VIII esterases. The EstBL primary structure showed two putative serine motifs, G-V-S(149)-D-G and S(74)-V-T-K. The estBL gene was successfully over-expressed in E. coli and the encoded protein purified by a combination of ammonium sulphate fractionation, hydrophobic interaction, ion exchange and size exclusion chromatographies. Biochemical assays confirmed EstBL esterase activity and revealed a preference for short-chain p-nitrophenyl and beta-naphthyl esters (C2-C4) with no activity against beta-lactam substrates. Secondary structure predictions indicated that EstBL adopts the alpha/beta fold, which is common to all esterases.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity

A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa. Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.     

A modern pollen rain study from the central Andes region of South America

Aim To provide a greater understanding of the modern pollen rain for the central Andes region of South America, thus laying the groundwork for future pollen studies and the derivation of pollen‐climate transfer functions and response surfaces. Location Peru, Bolivia and Chile. Methods Standard palynological techniques for surface soil sampling and discriminant analysis. Results Statistical analysis of the 40 surface samples revealed four palynologically distinct assemblages: (1) the Yungas on the eastern slopes of the Andes; (2) the puna grasslands of the Altiplano; (3) the Coastal Deserts to the west of the Andes; and (4) the Southern Bolivian Highlands. Main conclusions The Yungas group is characterized by high percentages of Urticaceae/Moraceae pollen and fern spores along with trace amounts of pollen from common zoophilous lowland taxa such as Piperaceae, Bignoniaceae and Malpighiaceae. The puna assemblage of the Altiplano is characterized by the dominance of grass (Poaceae) in combination with moderate frequencies of Asteraceae pollen, which tend to increase slightly towards the southern (drier) locations of the Altiplano. Lower percentages of grass pollen and the greater prevalence of Asteraceae, Solanaceae, and the Chenopodiaceae/Amaranthaceae families define the coastal deserts to the west of the Andes. Samples taken from the Southern Bolivian Highlands are characterized by the dominance of Solanaceae in the area, in combination with the pollen of other xerophytic taxa.