Two distinct alcohol dehydrogenases participate in butane metabolism by Pseudomonas butanovora

Abstract

The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD(+)-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora.

FIG. 1.

Induction of BOH and BDH total activity and mRNAs by butane. (a) Development of ethylene oxidation (dashed line, solid squares) and 1-butanol oxidation (solid line, open squares) activities. Lactate-grown cells were washed and then incubated in basal medium with butane for the indicated times. (b) BOH and BDH mRNA levels during the induction of butane oxidation. The same blot was used for the three hybridizations after probe stripping. The three arrows represent the relative position of each mRNA with respect to the other two. The numbers in the first frame are the estimated sizes of the mRNAs and of the 16S rRNA.

FIG. 2.

Induction of the mRNA for BOH and BDH upon incubation with different alcohols and butane. The same RNA preparation was probed for the presence of the BOH and BDH mRNAs. For comparison among treatments, the blots were stripped and hybridized to a probe for the 16S rRNA. Lactate-grown cells were washed and then incubated for 2 h in medium containing the indicated substrate and then tested for the presence of BOH and BDH mRNAs by Northern hybridization. Cells were incubated with lactate (1), ethanol (e), 1-propanol (1-p), 2-propanol (2-p), 1-butanol (1-b), 2-butanol (2-b), 1-pentanol (1-pe), and butane (but). The numbers above the 16S rRNA blot were calculated from two batches of cells and are the ratios of the BOH or BDH mRNA signal to the rRNA signal normalized to the ratios obtained with cells exposed to butane.

FIG. 3.

Maps of the loci of boh (a) and bdh (b) and phosphorimage of the Southern blot of DNA (c) from the wild-type and mutant strains of P. butanovora. The maps show the locations of the adjacent genes and the sites of insertion of the antibiotic-conferring cassettes. The arrows under the genes show the direction of transcription. The nucleotide sequences of the genes adjacent to the alcohol dehydrogenase-encoding genes are incomplete but show similarity to a regulatory element (orf1) and to genes coding for aldehyde dehydrogenase (orf2), to another aldehyde dehydrogenase (orf3), to an orf of unknown function (orf4), and to a regulatory element (orf5). The dashed lines represent undetermined sequences. In the Southern hybridization two restriction digests were used for clarity to show the different loci of boh and bdh and the increase in size as a result of the cassette insertion. The strains, restriction enzymes, and probes used are indicated.

FIG. 4.

Growth of the wild-type, boh::tet mutant, bdh::kan mutant, and boh::tet-bdh::kan mutant strains of P. butanovora. The growth substrates were butane and 1-butanol as indicated. Symbols: ▴, wild-type; ▪, boh::tet; ◊, bdh::kan; and •, boh::tet-bdh::kan P. butanovora strains.