Structure, function, and insights into the biosynthesis of a head-to-head hydrocarbon in Shewanella oneidensis strain MR-1

Abstract

A polyolefinic hydrocarbon was found in nonpolar extracts of Shewanella oneidensis MR-1 and identified as 3,6,9,12,15,19,22,25,28-hentriacontanonaene (compound I) by mass spectrometry, chemical modification, and nuclear magnetic resonance spectroscopy. Compound I was shown to be the product of a head-to-head fatty acid condensation biosynthetic pathway dependent on genes denoted as ole (for olefin biosynthesis). Four ole genes were present in S. oneidensis MR-1. Deletion of the entire oleABCD gene cluster led to the complete absence of nonpolar extractable products. Deletion of the oleC gene alone generated a strain that lacked compound I but produced a structurally analogous ketone. Complementation of the oleC gene eliminated formation of the ketone and restored the biosynthesis of compound I. A recombinant S. oneidensis strain containing oleA from Stenotrophomonas maltophilia strain R551-3 produced at least 17 related long-chain compounds in addition to compound I, 13 of which were identified as ketones. A potential role for OleA in head-to-head condensation was proposed. It was further proposed that long-chain polyunsaturated compounds aid in adapting cells to a rapid drop in temperature, based on three observations. In S. oneidensis wild-type cells, the cellular concentration of polyunsaturated compounds increased significantly with decreasing growth temperature. Second, the oleABCD deletion strain showed a significantly longer lag phase than the wild-type strain when shifted to a lower temperature. Lastly, compound I has been identified in a significant number of bacteria isolated from cold environments.

FIG. 1.

Gas chromatograph of the S. oneidensis hydrocarbon compound I (20.2 min) (A) and the product of its hydrogenation (20.8 min) that comigrates with and has an identical mass spectrum to n-hentriacosane (B).

FIG. 2.

NMR spectrum of the hydrocarbon compound I produced by S. oneidensis strain MR-1 in deuterated chloroform (CHCl₃) with tetramethylsilane (TMS) as the reference standard. The fragment representing each resonance and the number of protons on integration are indicated. The structure of the compound represented by the spectrum is shown at the top.

FIG. 3.

The oleABCD genes are required for long-chain olefin production by S. oneidensis. (a) Illustration of the oleABCD and oleC regions deleted and plasmid pOleC containing the oleC gene that complemented the oleC deletion. (b) DNA gel confirming gene deletion and complementation (primers used for analysis were SO1744CompF and SO1744CompR). (c) Gas chromatograph of solvent extracts from S. oneidensis wild-type (i), the oleC deletion mutant (ii), and the oleC mutant complemented with the pOleC plasmid (iii).

FIG. 4.

GC results for a solvent extract from recombinant S. oneidensis expressing the heterologous S. maltophilia OleA protein. Compounds were identified as hydrocarbons or ketones by mass spectrometry as described in the text and are designated by the molecular formula next to each major GC peak. The asterisk indicates compound I, which is endogenously produced by wild-type S. oneidensis MR-1.

FIG. 5.

Product structures and proposed pathways in S. oneidensis MR-1 wild-type and mutant strains for head-to-head hydrocarbon and ketone formation, respectively. (A) Structure of compound I, identified as described in the text. (B) Proposed role of OleA in the head-to-head biosynthetic pathway. (C) A proposed pathway to ketones in the presence of the OleA protein alone.

FIG. 6.

Long-chain polyunsaturated compounds as a function of growth temperature in S. oneidensis MR-1 wild type and an oleABCD deletion mutant. (a) Hydrocarbon (blue) and ketone (red) contents at different temperatures relative to the maximum observed (at 4°C). (b) Wild-type MR-1 (black) and the corresponding oleABCD-deficient mutant (green) were downshifted from 30°C to 4°C, and the cold temperature growth curves are shown. Experimental points are average triplicate samplings from six treatments. Variation is shown as the standard deviation.