RESUMO
Twenty-three propane- and butane-utilizing bacteria were isolated from soil samples collected from oilfields. Three of them have been identified as Rhodococcus sp. IMT35, Pseudomonas sp. IMT37 and Pseudomonas sp. MT40. SDS-PAGE analysis of the membrane of Rhodococcus sp. IMT35 revealed the presence of at least four polypeptides induced by propane. Polyclonal antibody raised against a 58 kDa polypeptide from Rhodococcus sp. IMT35 specifically detected bacteria which were actively utilizing propane or butane. Immunoscreening of a genomic library in lambdagt11 with this antibody resulted in isolation of a clone containing a 4.9 kb EcoRI genomic DNA fragment. This 4.9 kb DNA fragment was found to hybridize specifically with organisms which could grow on propane or butane. This fragment could therefore be used as a probe for detection of such bacteria. A 2.3 kb fragment having an ORF encoding a polypeptide of 54 kDa was identified by screening a genomic library of Pseudomonas sp. IMT37 with this 4.9 kb EcoRI fragment. The sequence of the ORF (designated orf54) was found to be novel. Primer extension and S1 nuclease mapping showed that transcription of the ORF starts at base 283 and it had sequences upstream similar to that of a Pseudomonas promoter (-12, -24 type). Disruption of the ORF by a kanamycin ('kan') cassette prevented the organism from growing on any alkane but did not affect its ability to utilize the respective alkanols and acids, indicating that alcohol dehydrogenase and subsequent steps in the pathway remained unaltered. The mutants had no detectable level of butane monooxygenase activity. Therefore, the product of this gene plays a crucial role in the first step of the pathway and is an essential component of monooxygenase. The findings imply that this bacterium either employs a common genetic and metabolic route or at least shares the product of this gene for utilization of many alkanes.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Butanos/metabolismo , Genes Bacterianos , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Bacteriano/genética , Biblioteca Genômica , Dados de Sequência Molecular , Peso Molecular , Mutagênese Insercional , Fases de Leitura Aberta , Plasmídeos/genética , Propano/metabolismo , Pseudomonas/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Microbiologia do SoloRESUMO
Molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. In this research, we used a new approach to determine the frequency spontaneously generated by Clostridium acetobutylicum ATCC 824 cells during the application of a square high-voltage pulse. Once the frequency of 100 kHz was determined we transformed clostridial cells with pSOS84 plasmid DNA using radio-frequency modulated high-voltage square pulses (electric field strength 12 kVcm-1; pulse duration 22.5 ms; frequency of pulse modulation 100 kHz) to reach an efficiency exceeding 106 transformants microg-1 of plasmid DNA. We propose a possible role for cellular membrane structures in affecting the transformation yield.
