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

Institute of Microbial Technology, Sector 39-A, Chandigarh-160 036, , India¹

Author for correspondence: T. Chakrabarti. Tel: +91 172 690562. Fax: +91 172 690585/690632.e-mail: tapan°imtech.res.in

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 gt11 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.

Keywords: alkane utilization, butane monooxygenase, primer extension, S1 nuclease mapping, insertional inactivation

Abbreviations: BMO, butane monooxygenase; LPG, liquefied petroleum gas; MMO, methane monooxygenase; pMMO, particulate MMO; PMO, propane monooxygenase; sMMO, soluble MMO

The GenBank accession number for the orf54 sequence is L81125.

^a The first four authors contributed equally to this work.

^b Present address: Dept of Internal Medicine, University of Texas-Health Science Center, Houston, TX 77030, USA.

^c Present address: Building 8, Room B2A-15, 8, Center Dr.MSC.0805, LMCB/NIDDK/NIH, Bethesda, MD 20892-0805, USA.

^d Present address: Dept of Neurology, Mount Sinai School of Medicine, New York, NY 10023, USA.

^e Present address: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India.

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