Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora.
Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C(2)-C(9) alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of beta-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O(2) when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH(4)Cl-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 O(2) (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) O(2), CuSO(4) (0.5 microM) repressed 1-butanol-dependent induction of beta-galactosidase activity. Under oxic conditions (20% O(2) [vol/vol]), significantly higher concentrations of CuSO(4) (2 microM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu(2+) reducing agent Na ascorbate (100 microM) and CuSO(4) (0.5 microM) repressed the induction of beta-galactosidase activity under oxic conditions to the same extent that 0.5 microM CuSO(4) alone repressed it under anoxic conditions. Under oxic conditions, 2 microM CuSO(4) 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, CuSO(4) repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.
Doughty
D M
DM
Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA.
Kurth
E G
EG
Sayavedra-Soto
L A
LA
Arp
D J
DJ
Bottomley
P J
PJ
D019677:Artificial Gene Fusion; D002073:Butanes / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D042926:Cytochrome P-450 CYP4A / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D019344:Lactic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011422:Propionates / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000502:physiology*; D001616:beta-Galactosidase / Q000096:biosynthesis
D002073:Butanes; D003470:Culture Media; D011422:Propionates; D019344:Lactic Acid; C046888:butane; D003300:Copper; D042926:Cytochrome P-450 CYP4A; D001616:beta-Galactosidase; C029658:propionic acid; D010100:Oxygen