Two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8

Abstract

Alkane monooxygenases in Nocardioides sp. strain CF8 were examined at the physiological and genetic levels. Strain CF8 can utilize alkanes ranging in chain length from C(2) to C(16). Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea. Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane. A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne. These results suggest the presence of two monooxygenases in strain CF8. Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase from Pseudomonas oleovorans were used to clone a related gene from strain CF8. Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C(6). These results indicate the presence of two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8.

FIG. 1

Incorporation of ¹⁴C from [¹⁴C]acetylene into cellular proteins of strain CF8 grown on C₄, C₆, C₈, and C₁₀ alkanes (lanes 1, 2, 3, and 5, respectively) and cells grown with C₈ or C₁₀ in the presence of 1-hexyne (lanes 4 and 6, respectively). Incorporation of ¹⁴C into polypeptides was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized with a phosphorimager as described in Materials and Methods. The sizes of molecular mass markers and the apparent molecular mass of the labeled polypeptide (arrow) are shown on the left side of the gel. Each gel lane contains approximately 50 μg of cell extract protein.

FIG. 2

(A) Part of a multiple sequence alignment of AlkB from strain CF8 with other known integral-membrane, binuclear-iron hydrocarbon oxygenases. Eight conserved His residues are indicated by letters in boldface type and marked by asterisks. The positions of the PCR primers used for RT-PCR are shown by arrows. Peptide numbering refers to the position in the full-length enzyme (XXX is shown for the sequences whose full length sequences are not available). Abbreviations: Nc, Nocardioides sp. strain CF8; Ar, A. rugosa; Re, R. erythropolis; Mt, M. tuberculosis; Ac, Acinetobacter sp. strain ADP1; Po, P. oleovorans; Pp, P. putida; Sm, Stenotrophomonas maltophilia. (B) Gene organization and restriction map of the cloned regions including complete AlkB gene. Location of the PCR fragment used as a probe for alkB is shown. Each box indicates an ORF, and the arrows indicate the orientation of each gene.

FIG. 3

RT-PCR (A) and Northern blot analysis (B and C) of alkB expression in strain CF8 grown on alkanes of various chain length. Total RNA was prepared from cells grown on C₄ to C₈ alkanes. RT-PCR was performed using primers shown in Fig. 2A, and the expected product size (525 bp) is indicated (A). Northern blot analysis was performed with the alkB probe shown in Fig. 2B, and the hybridizing band (about 2 kb) is indicated (B). The same blot was stripped and hybridized to the 16S rRNA probe (C).