Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1

Abstract

Vibrio furnissii M1 was recently reported to biosynthesize n-alkanes when grown on biopolymers, sugars, or organic acids (M. O. Park, J. Bacteriol. 187:1426-1429, 2005). In the present study, V. furnissii M1 was subjected to genomic analysis and studied biochemically. The sequence of the 16S rRNA gene and repetitive PCR showed that V. furnissii M1 was not identical to other V. furnissii strains tested, but the level of relatedness was consistent with its assignment as a V. furnissii strain. Pulsed-field gel electrophoresis showed chromosomal bands at approximately 3.2 and 1.8 Mb, similar to other Vibrio strains. Complete genomic DNA from V. furnissii M1 was sequenced with 21-fold coverage. Alkane biosynthetic and degradation genes could not be identified. Moreover, V. furnissii M1 did not produce demonstrable levels of n-alkanes in vivo or in vitro. In vivo experiments were conducted by growing V. furnissii M1 under different conditions, extracting with solvent, and analyzing extracts by gas chromatography-mass spectrometry. A highly sensitive assay was used for in vitro experiments with cell extracts and [(14)C]hexadecanol. The data are consistent with the present strain being a V. furnissii with properties similar to those previously described but lacking the alkane-producing phenotype. V. furnissii ATCC 35016, also reported to biosynthesize alkanes, was found in the present study not to produce alkanes.

FIG. 1.

REP-PCR of genomic DNA from V. furnissii M1 and control strains. Percent relatedness was determined as described in Materials and Methods. The lanes contained DNA from the following strains: 1, V. furnissii ATCC 33841; 2, V. furnissii ATCC 35627; 3, V. furnissii ATCC 35016; 4, V. furnissii M1; 5, V. furnissii ATCC 35628; 6, V. parahaemolyticus LM5312; 7, V. harveyi B392; 8, E. coli K-12.

FIG. 2.

Comparative PFGE of genomic DNA from V. furnissii M1 (lane 2), V. furnissii 35016 (lane 3), V. furnissii 35627 (lane 4), V. furnissii 35628 (lane 5), V. parahaemolyticus LM5312 (lane 6), V. harveyi B392 (lane 7), and E. coli DH5α (lane 8). Hansenula wingei (lane 1) and Schizosaccharomyces pombe (lane 9) chromosomes were used as size markers.

FIG. 3.

Genome region from V. furnissii M1 showing similarities to a gene region in Salmonella. Identified protein types are highlighted: carboxysome shell proteins (black), aldehyde dehydrogenases (vertical lines), and alcohol dehydrogenases (diagonal lines). The ORF numbers (275, 277, 278, and 279) are given for proteins discussed in the text.

FIG. 4.

Initial gas chromatograms of V. furnissii M1 (A) and a chloroform blank showing the extent of contamination (B). The prominent peaks in both were identified by MS as methyl palmitate (12.5 min), dibutyl phthalate (13.0), octadecenoic acid methyl ester (14.2 min), diethylhexyl phthalate (17.9 min), and octacosane (19.7 min).

FIG. 5.

Gas chromatograms of extracts of V. furnissii M1 (A) and E. coli K-12 (B) using cleaner solvents and methods. Cultures were spiked with octacosane prior to extraction and workup. The 19.7-min peak was confirmed by MS to be octacosane.