Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli.

BACKGROUND: The genus Burkholderia is widespread in diverse ecological niches, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants. Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant. Therefore these bacteria are promising candidates for biotechnological applications. RESULTS: Burkholderia sp. (R 3.25 isolate) was isolated from agricultural soil in Ponta Grossa-PR-Brazil and identified through analysis of the 16S rDNA as a strain classified as Burkholderia gladioli. The expression of membrane-bound acid phosphatase (MBAcP) was strictly regulated with optimal expression at a concentration of phosphorus 5 mM. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate (PNPP) was 6.0. The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. Kinetic data revealed that the hydrolysis of PNPP exhibited cooperative kinetics with n = 1.3, Vm = 113.5 U/mg and K0.5 = 65 µM. The PNPPase activity was inhibited by vanadate, p-hydroxymercuribenzoate, arsenate and phosphate, however the activity was not inhibited by calcium, levamisole, sodium tartrate, EDTA, zinc, magnesium, cobalt, ouabain, oligomycin or pantoprazol. CONCLUSION: The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element, for specific crops.

Molecular characterization of bacterial populations of different soils

Until recently, few studies were carried out in Brazil about diversity of bacterial soil communities. Aiming to characterize the bacterial population in the soil through 16S rRNA analysis, two types of soil have been analyzed: one of them characterized by intensive use where tomato, beans and corn were cultivated (CS); the other analyzed soil was under forest (FS), unchanged by man; both located in Guaíra, São Paulo State, Brazil. Using specific primers, 16S rRNA genes from metagenomic DNA in both soils were amplified by PCR, amplicons were cloned and 139 clones from two libraries were partially sequenced. The use of 16S rRNA analysis allowed identification of several bacterial populations in the soil belonging to the following phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria Verrucomicrobia in addition to the others that were not classified, beyond Archaea domain. Differences between FS and CS libraries were observed in size phyla. A larger number of phyla and, consequently, a greater bacterial diversity were found in the under-forest soil. These data were confirmed by the analyses of genetic diversity that have been carried out. The characterization of bacterial communities of soil has made its contribution by providing facts for further studies on the dynamics of bacterial populations in different soil conditions in Brazil.

Até o momento poucos estudos foram realizados no Brasil a respeito da diversidade de comunidades bacterianas no solo. Com o objetivo de caracterizar as populações bacterianas presentes no solo através da análise do gene 16S rRNA, foram analisados dois solos: um caracterizado pelo uso intensivo, principalmente para a produção de tomate, feijão e milho (CS); e outro sob floresta (FS), não modificado pelo homem, ambos do município de Guaíra, no estado de São Paulo, Brasil. Usando oligonucleotídeos específicos, de genes 16S rRNA do DNA metagenomico de ambos os solos foram amplificados por PCR, amplicons foram clonados e 139 clones de duas bibliotecas foram seqüenciados. O uso da técnica de 16S rRNA, gerou a identificação de diferentes populações de bactérias de solo pertencentes aos filos Acidobacteria Actinobacteria Bacteroidetes Firmicutes Proteobacteria Verrucomicrobia, Archaea, além das não classificadas. Diferenças entre as bibliotecas FS e CS foram observadas no tamanho dos filos. Um grande número de filos e, consequentemente, uma grande diversidade bacteriana foi observada no solo sob floresta. Estes dados foram confirmados pela análise de diversidade genética realizada. A caracterização de comunidades do solo apresentada neste trabalho contribuiu fornecendo dados para estudos posteriores sobre a dinâmica das populações bacterianas em solos de diferentes condições no Brasil.