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Microbiology 155 (2009), 761-771; DOI  10.1099/mic.0.021816-0
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Microbiology 155 (2009), 761-771; DOI  10.1099/mic.0.021816-0
© 2009 Society for General Microbiology

Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath

Hanif Ali{dagger} and J. Colin Murrell

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Correspondence
J. Colin Murrell
J.C.Murrell{at}warwick.ac.uk

A series of integrative and versatile broad-host-range promoter-probe vectors carrying reporter genes encoding green fluorescent protein (GFP), catechol 2,3-dioxygenase (XylE) or β-galactosidase (LacZ) were constructed for use in methanotrophs. These vectors facilitated the measurement of in vivo promoter activity in methanotrophs under defined growth conditions. They were tested by constructing transcriptional fusions between the soluble methane monooxygenase (sMMO) {sigma}54 promoter or particulate methane monooxygenase (pMMO) {sigma}70 promoter from Methylococcus capsulatus and the reporter genes. Reporter gene activity was measured under high- and low-copper growth conditions and the data obtained closely reflected transcriptional regulation of the sMMO or pMMO operon, thus demonstrating the suitability of these vectors for assessing promoter activity in methanotrophs. When β-galactosidase expression was coupled with the fluorogenic substrate 4-methylumbelliferyl β-D-glucuronide it yielded a sensitive and powerful screening system for detecting cells expressing this reporter gene. These data were substantiated with independent experiments using RT-PCR and RNA dot-blot analysis.

Abbreviations: BHR, broad host range; DMF, dimethylformamide; MUG, 4-methylumbelliferyl β-D-glucuronide; ONPG, o-nitrophenyl β-D-galactoside; pMMO and sMMO, particulate and soluble methane monooxygenase

{dagger}Present address: Leeds Institute of Molecular Medicine, Wellcome Trust Brenner Building, Section of Experimental Therapeutics, St James's University Hospital, Leeds LS9 7TF, UK.






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