Vibrio furnissii M 1 : Genomic and Biochemical Studies 3 Demonstrating the Absence of an Alkane - Producing Phenotype 4 5

(212 words) 1 2 Vibrio furnissii M1 was recently reported to biosynthesize n-alkanes when grown on 3 biopolymers, sugars or organic acids (Park, M. the present study, V. furnissii M1 was subjected to genomic analysis and studied 5 biochemically. The sequence of the 16S rRNA gene and repetitive (REP)-PCR showed 6 that V. furnissii M1 was not identical to other V. furnissii strains tested, but the level of 7 relatedness was consistent with its assignment as a V. furnissii strain. Pulse field gel 8 electrophoresis showed chromosomal bands at approximately 3.2 and 1.8 Mb, similar to 9 other Vibrio strains. Complete genomic DNA from V. furnissii M1 was sequenced with 10 21-fold coverage. Alkane biosynthetic and degradation genes could not be identified. 11 Moreover, V. furnissii M1 did not produce demonstrable levels of n-alkanes in vivo or in 12 vitro. In vivo experiments were conducted by growing V. furnissii M1 under different 13 conditions, extracting with solvent, and analyzing extracts by gas chromatography-mass 14 spectrometry. A highly sensitive assay was used for in vitro experiments with cell-free 15 extracts and [ 14 C]-hexadecanol. The data are consistent with the present strain being a V. 16 furnissii with similar properties as previously described but lacking the alkane-producing 17 phenotype. V. furnissii ATCC 35016, also reported to biosynthesize alkanes, was found 18 in the present study not to produce alkanes. 3 The need to develop renewable energy sources will require that society develop biofuel 1 options other than ethanol. One excellent fuel option would be bio-alkanes. Alkanes 2 comprise the major component of current petroleum-based fuels. A biological petroleum 3 would be renewable and completely compatible with existing fuel infrastructure. Thus, 4 considerable interest was generated by recent reports of high-level n-alkane formation by 5 the bacterium Vibrio furnissii M1 (22,23,24). 6 7 Vibrio furnissii strains were recognized as a distinct species in 1983 (6). Other Vibrio 8 species, such as V. cholera (8) and V. parahaemolyticus (30), have been more extensively 9 studied because of their significant pathogenicity to humans. Both of these latter species 10 (12), along with V. vulnificus (7) and V. fischeri (25), have been subjected to genomic 11 sequencing that has been completed and published. V. furnissii has been most extensively 12 studied with respect to its physiological and genetic mechanisms of chitin degradation (3, 13 17). Marine Vibrio are prominent chitinolytic organisms (18). 14 15 Thus, …