Diversity in butane monooxygenases among butane-grown bacteria

Abstract

Butane monooxygenases of butane-grown Pseudomonas butanovora, Mycobacterium vaccae JOB5, and an environmental isolate, CF8, were compared at the physiological level. The presence of butane monooxygenases in these bacteria was indicated by the following results. (i) O(2) was required for butane degradation. (ii) 1-Butanol was produced during butane degradation. (iii) Acetylene inhibited both butane oxidation and 1-butanol production. The responses to the known monooxygenase inactivator, ethylene, and inhibitor, allyl thiourea (ATU), discriminated butane degradation among the three bacteria. Ethylene irreversibly inactivated butane oxidation by P. butanovora but not by M. vaccae or CF8. In contrast, butane oxidation by only CF8 was strongly inhibited by ATU. In all three strains of butane-grown bacteria, specific polypeptides were labeled in the presence of [(14)C]acetylene. The [(14)C]acetylene labeling patterns were different among the three bacteria. Exposure of lactate-grown CF8 and P. butanovora and glucose-grown M. vaccae to butane induced butane oxidation activity as well as the specific acetylene-binding polypeptides. Ammonia was oxidized by all three bacteria. P. butanovora oxidized ammonia to hydroxylamine, while CF8 and M. vaccae produced nitrite. All three bacteria oxidized ethylene to ethylene oxide. Methane oxidation was not detected by any of the bacteria. The results indicate the presence of three distinct butane monooxygenases in butane-grown P. butanovora, M. vaccae, and CF8.

FIG. 1

Incorporation of ¹⁴C from [¹⁴C]acetylene into cellular proteins of P. butanovora (lanes 1 and 2), CF8 (lanes 3 and 4), and M. vaccae (lanes 5 and 6). Butane-grown cells were incubated with [¹⁴C]acetylene (lanes 1, 3, and 5) or [¹⁴C]acetylene in the presence of 50% (vol/total vial vol) butane (lanes 2, 4, and 6). Incorporation of ¹⁴C into polypeptides was analyzed by SDS-PAGE and visualized by a phosphorimager as described in Materials and Methods. Each gel lane contains approximately 50 μg of cell extract protein.

FIG. 2

Induction of specific polypeptides during the induction of butane degradation activity in P. butanovora (lanes 1 to 3), CF8 (lanes 4 to 6), and M. vaccae (lanes 7 to 9). (A) Coomassie blue-stained SDS-polyacrylamide gel of butane-grown cells (lanes 1, 4, and 7), butane-induced cells (lanes 2, 5, and 8), and lactate- or glucose-grown cells (lanes 3, 6, and 9). (B) Phosphorimager image of [¹⁴C]acetylene-labeled butane-grown cells (lanes 1, 4, and 7), butane-induced cells (lanes 2, 5, and 8), and lactate- or glucose-grown cells (lanes 3, 6, and 9). The apparent molecular masses of the labeled polypeptides are shown on the left of each panel.