A novel gene encoding a 54 kDa polypeptide is essential for butane utilization by Pseudomonas sp IMT37

Twenty-three propane- and butane-utilizing bacteria were isolated from soil samples collected from oilfields. Three of them have been identified as Rh odococcus sp. IMT35, Pseudomonas sp. IMT37 and Pseudomonas sp. IMT40. SDS-P AGE analysis of the membrane of Rhodococcus sp. IMT35 revealed the presence of at least four polypeptides induced by propane. Polyclonal antibody rais ed against a 58 kDa polypeptide from Rhodococcus sp. IMT35 specifically det ected bacteria which were actively utilizing propane or butane. Immunoscree ning of a genomic library in lambda gt11 with this antibody resulted in iso lation 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 whic h 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 libr ary of Pseudomonas sp. IMT37 with this 4(.)9 kb EcoRI fragment. The sequenc e 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 28 3 and it had sequences upstream similar to that of a Pseudomonas promoter ( -12, -24 type). Disruption of the ORF by a kanamycin ('kan') cassette preve nted the organism from growing on any alkane but did not affect its ability to utilize the respective alkanols and acids, indicating that alcohol dehy drogenase and subsequent steps in the pathway remained unaltered. The mutan ts 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 th is bacterium either employs a common genetic and metabolic route or at leas t shares the product of this gene for utilization of many alkanes.