Differential protein expression during growth on linear versus branched alkanes in the obligate marine hydrocarbon-degrading bacterium Alcanivorax borkumensis SK2T

Abstract

Alcanivorax borkumensis SK2^T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC-MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n-C₁₄ ) or branched (pristane) alkanes. During growth on n-C₁₄ , A. borkumensis expressed a complete pathway for the terminal oxidation of n-alkanes to their corresponding acyl-CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for β-oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2^T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of β-oxidation proteins to overcome steric hinderance from branched substrates.

Figure 1

A‐C. Volcano plots of normalized LC–MS/MS spectral counts comparing A. borkumensis protein expression during growth on a linear alkane (n‐C₁₄), a branched alkane (Pristane) and a non‐hydrocarbon control (Pyruvate). Data points above horizontal dashed line represent P‐values below 0.05. Vertical dashed lines represent a two‐fold change. D. Principle component analysis of replicate A. borkumensis proteomes (including normalized spectral counts of 1309 proteins) on growth substrates n‐C₁₄, pristane and pyruvate.

Figure 2

Normalized spectral counts (means ± SE; n = 3) of differentially expressed linear alkane oxidation proteins during growth on a linear alkane (n‐C₁₄/C14) a non‐hydrocarbon control (Pyruvate/PYR) and a branched alkane (Pristane/PRI) in A. borkumensis SK2^T; treatments not sharing a letter (a or b) differ at P < 0.05 (Tukey's HSD). Two different alkane oxidation systems were detected. A. 1. Cytochrome P450 introduces oxygen into the alkane at the terminal carbon converting it into a primary alcohol. 2. AlkJ2 further oxidizes the primary alcohol generated into an aldehyde. 3. Ferredoxin reductase oxidizes NADH to NAD⁺ shuttling electrons (e⁻) to the P450 through a ferredoxin. B. 4/5. Monooxygenases introduce oxygen into the alkane at the terminal carbon converting it into a primary alcohol. 6. AlkJ2 further oxidizes the primary alcohol generated into an aldehyde. 7. Aldehyde dehydrogenase converts the aldehyde into a fatty acid, which enters β‐oxidation. 8. Aldehyde reductase also converts the aldehyde into a fatty acid, which enters β‐oxidation. 9. Rubredoxin reductase oxidizes NADH to NAD⁺ shuttling electrons (e⁻) to the monooxygenase through a rubredoxin.

Figure 3

Normalized spectral counts (means ±; n = 3) of differentially expressed branched alkane oxidation proteins during growth on a linear alkane (n‐C₁₄/C14) a non‐hydrocarbon control (Pyruvate/PYR) and a branched alkane (Pristane/PRI) in A. borkumensis SK2^T; treatments not sharing a letter (a or b) differ at P < 0.05 (Tukey's HSD). The alkane monooxygenase (AM) introduces oxygen into the alkane at the terminal carbon converting it into a primary alcohol. This alcohol is further oxidized to an aldehyde by an alcohol oxidase (AO) or alcohol dehydrogenase (AD). An aldehyde dehydrogenase (ALD) converts the aldehyde into a fatty acid, which enters β‐oxidation. Alternatively, a cytochrome P450 (P450) can oxidize the alkane up to it corresponding fatty acid, which enters β‐oxidation.

Figure 4

Normalized spectral counts (means ±; n = 3) of differentially expressed β‐oxidation proteins during growth on a linear alkane (n‐C₁₄/C14) a non‐hydrocarbon control (Pyruvate/PYR) and a branched alkane (Pristane/PRI) in A. borkumensis SK2^T; treatments not sharing a letter (a or b) differ at P < 0.05 (Tukey's HSD). The β‐oxidation proteins have putatively been put on either the left‐ or right‐hand side of the figure to represent involvement in straight‐chain alkane degradation (left) or branched‐chain alkane degradation (right) based on their expression pattern. A and B. Acyl CoA synthetases which activate a fatty acid to form acyl‐CoA constitutively expressed all substrates (A) and differentially expressed in pristane (B). C and D. Acyl‐CoA dehydrogenases, which creates a double bond between the second and third carbons down from the CoA group on acyl‐CoA producing trans‐delta 2‐enoyl CoA differentially expressed in n‐C14 (C) and pristane (D). E and F. Enoyl CoA hydratases, which remove the double bond in trans‐delta 2‐enoyl CoA adding a hydroxyl group to the to the third carbon down from the CoA group and a hydrogen on the second carbon down from the CoA group producing L‐3‐hydroxyacyl CoA differentially expressed in n‐C14 (E) and pristane (F). G and H. The FadAB2 (G) and FadAB (H) operon consisting of β‐oxidation multifunctional enzyme complexes with 3‐hydroxyacyl‐CoA dehydrogenase (removes the hydrogen in the hydroxyl group added to L‐3‐hydroxyacl CoA producing 3‐ketoacyl CoA) and ketoacyl‐CoA thiolase (attaches a CoA group on the third carbon down from the CoA group producing acetyl‐CoA and acyl‐CoA which is two carbons shorter) activities were differentially expressed on n‐C₁₄ and pristane, respectively.