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Journal of Bacteriology, April 2008, p. 2933-2938, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01409-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evidence for Involvement of Copper Ions and Redox State in Regulation of Butane Monooxygenase in Pseudomonas butanovora{triangledown}

D. M. Doughty,1 E. G. Kurth,2 L. A. Sayavedra-Soto,2 D. J. Arp,2 and P. J. Bottomley1,3*

Department of Microbiology,1 Department of Botany and Plant Pathology,2 Department of Crop and Soil Science, Oregon State University, Corvallis, Oregon 97331-38043

Received 30 August 2007/ Accepted 5 February 2008

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C2-C9 alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of β-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O2 when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH4Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O2 (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O2, CuSO4 (0.5 µM) repressed 1-butanol-dependent induction of β-galactosidase activity. Under oxic conditions (20% O2 [vol/vol]), significantly higher concentrations of CuSO4 (2 µM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu2+ reducing agent Na ascorbate (100 µM) and CuSO4 (0.5 µM) repressed the induction of β-galactosidase activity under oxic conditions to the same extent that 0.5 µM CuSO4 alone repressed it under anoxic conditions. Under oxic conditions, 2 µM CuSO4 repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO4 repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

* Corresponding author. Mailing address: Department of Microbiology, Nash Hall Rm. 220, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-4441. Fax: (541) 737-0496. E-mail: Peter.Bottomley{at}oregonstate.edu

{triangledown} Published ahead of print on 15 February 2008.

Journal of Bacteriology, April 2008, p. 2933-2938, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01409-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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