1887

Abstract

Twenty-three propane- and butane-utilizing bacteria were isolated from soil samples collected from oilfields. Three of them have been identified as sp. IMT35, sp. IMT37 and sp. MT40. SDS-PAGE analysis of the membrane of sp. IMT35 revealed the presence of at least four polypeptides induced by propane. Polyclonal antibody raised against a 58 kDa polypeptide from sp. IMT35 specifically detected bacteria which were actively utilizing propane or butane. Immunoscreening of a genomic library in λgt11 with this antibody resulted in isolation of a clone containing a 4·9 kb RI 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 sp. IMT37 with this 4·9 kb RI fragment. The sequence of the ORF (designated ) 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 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.

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2001-09-01
2020-04-06
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