RESUMO
AIMS: The aim of this study was to develop an approach based on a reverse transcriptase (RT)-PCR/denaturing gradient gel electrophoresis (DGGE) for the detection of the functional genes nifH and anfH in Paenibacillus durus. METHODS AND RESULTS: Two sets of primers were employed to study the expression of the nitrogen fixation genes in a pure-culture system of P. durus grown in media with increasing concentrations of ammonium (NH(4)(+)), tungsten (W) or molybdenum (Mo). The results obtained indicate that the expression of nitrogenase genes from P. durus can take place in the presence of relatively high levels of fixed nitrogen. It was also observed that the addition of 20 micromol l(-1) molybdenum and 2 mmol l(-1) tungstate did not interfere in the mRNA levels of nifH and anfH genes. CONCLUSIONS: Our results demonstrate the presence and transcription of nifH and anfH in P. durus under a variety of growth conditions. A specific set of primers was designed for the detection of the alternative system for nitrogen fixation in P. durus. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR/DGGE system enables the rapid gathering of incremental data about the regulation of conventional and alternative nitrogenase genes in P. durus strains.
Assuntos
Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas Formadoras de Endosporo/crescimento & desenvolvimento , Fixação de Nitrogênio , Oxirredutases/metabolismo , Compostos de Amônio Quaternário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas de Bactérias/genética , Meios de Cultura , Bactérias Gram-Positivas Formadoras de Endosporo/classificação , Bactérias Gram-Positivas Formadoras de Endosporo/genética , Bactérias Gram-Positivas Formadoras de Endosporo/metabolismo , Molibdênio/metabolismo , Oxirredutases/genética , Tungstênio/metabolismoRESUMO
Mononuclear molybdenum and tungsten are found in the active site of a diverse group of enzymes that, in general, catalyze oxygen atom transfer reactions. Enzymes of the xanthine oxidase family are the best-characterized mononuclear Mo-containing enzymes. Several 3D structures of diverse members of this family are known. Recently, the structures of substrate-bound and arsenite-inhibited forms of two members of this family have also been reported. In addition, spectroscopic studies have been utilized to elucidate fine details that complement the structural information. Altogether, these studies have provided an important amount of information on the characteristics of the active site and the electron transfer pathways.