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

Gergana Metodieva,Metodi V. Metodiev,Benjamin H. Gregson,Boyd A. McKew

doi:10.1111/1462-2920.14620

Gergana Metodieva, Metodi V. Metodiev + Show 2 more

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https://doi.org/10.1111/1462-2920.14620

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Journal: Environmental Microbiology	Publication Date: Apr 21, 2019
Citations: 31	License type: CC BY 4.0

Affiliation: University of Essex

Abstract

Summary Alcanivorax borkumensis SK2T 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 SK2T 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.

Highlights

A. borkumensis SK2T is a model strain of a group of organisms known as obligate hydrocarbonoclastic bacteria (OHCB) which grow on a highly restricted spectrum of substrates, predominantly alkanes and their derivatives, with carbon chain length from n-C9 to at least n-C32 (Schneiker et al, 2006; Yakimov et al, 1998; 2007; Naether et al, 2013)
Analysis revealed proteins required to oxidize n-alkanes up to the corresponding acyl-CoA derivative are coded by the alkSB1GJH operon which has >80% amino acid similarity to the corresponding well-characterized alkane degradation components in Pseudomonas putida Gpo1 (Kok et al, 1989; van Beilen et al, 2001; van Beilen et al, 2003; van Beilen et al, 2004)
Nine LC–MS/MS runs were performed consisting of three independent biological replicates of three treatments (linear alkane (n-C14), branched chain alkane, non-hydrocarbon control) resulting in 112,095 spectral counts that were assigned to 1309 proteins, representing 48% of the total protein-coding genes on the A. borkumensis SK2T genome (Supporting Information Table S1)

Summary

Introduction

A. borkumensis SK2T is a model strain of a group of organisms known as obligate hydrocarbonoclastic bacteria (OHCB) which grow on a highly restricted spectrum of substrates, predominantly alkanes and their derivatives, with carbon chain length from n-C9 to at least n-C32 (Schneiker et al, 2006; Yakimov et al, 1998; 2007; Naether et al, 2013). Alcanivorax borkumensis SK2T (DSM 11573) was the first OHCB to have its genome sequenced revealing a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oildegradation capabilities (Reva et al, 2008; Schneiker et al, 2006). A microarray-based study led to the identification of up-regulated genes predicted to be involved in the terminal oxidation of n-hexadecane (Sabirova et al, 2011). These included genes coding for two alkanes monooxygenases (alkB1 and alkB2), three flavin-binding monooxygenases (ABO_0282, ABO_1097, ABO_2107),

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