Differential Protein Expression During Growth on Medium Versus Long-Chain Alkanes in the Obligate Marine Hydrocarbon-Degrading Bacterium Thalassolituus oleivorans MIL-1.

Benjamin H Gregson,Metodi V Metodiev,Gergana Metodieva,Boyd A Mckew,Peter N Golyshin

doi:10.3389/fmicb.2018.03130

Benjamin H Gregson, Metodi V Metodiev + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2018.03130

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Journal: Frontiers in microbiology	Publication Date: Dec 17, 2018
Citations: 33	License type: CC BY 4.0

Affiliation: University of Essex, Bangor University

Abstract

The marine obligate hydrocarbonoclastic bacterium Thalassolituus oleivorans MIL-1 metabolizes a broad range of aliphatic hydrocarbons almost exclusively as carbon and energy sources. We used LC-MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on medium- (n-C14) or long-chain (n-C28) alkanes. During growth on n-C14, T. oleivorans expresses an alkane monooxygenase system involved in terminal oxidation including two alkane 1-monooxygenases, a ferredoxin, a ferredoxin reductase and an aldehyde dehydrogenase. In contrast, during growth on long-chain alkanes (n-C28), T. oleivorans may switch to a subterminal alkane oxidation pathway evidenced by significant upregulation of Baeyer-Villiger monooxygenase and an esterase, proteins catalyzing ketone and ester metabolism, respectively. The metabolite (primary alcohol) generated from terminal oxidation of an alkane was detected during growth on n-C14 but not on n-C28 also suggesting alternative metabolic pathways. Expression of both active and passive transport systems involved in uptake of long-chain alkanes was higher when compared to the non-hydrocarbon control, including a TonB-dependent receptor, a FadL homolog and a specialized porin. Also, an inner membrane transport protein involved in the export of an outer membrane protein was expressed. This study has demonstrated the substrate range of T. oleivorans is larger than previously reported with growth from n-C10 up to n-C32. It has also greatly enhanced our understanding of the fundamental physiology of T. oleivorans, a key bacterium that plays a significant role in natural attenuation of marine oil pollution, by identifying key enzymes expressed during the catabolism of n-alkanes.

Highlights

Thalassolituus oleivorans MIL-1 is a motile aerobic bacterium belonging to the Gammaproteobacteria class, which was first isolated from seawater and sediment samples collected in Milazzo Harbor, Sicily, Italy, supplemented with the n-alkane, tetradecane (n-C14) (Yakimov et al, 2004)
12 LC-MS/MS runs were performed consisting of four independent biological replicates of three treatments [medium-chain alkane (n-C14), long-chain alkane, non-hydrocarbon control (Tween 80)] resulting in 191647 spectral counts that were assigned to 1792 proteins, representing 50% of the total protein-coding genes on the T. oleivorans MIL-1 genome (Supplementary Table 1)
This range of utilizable alkane substrates is far greater than the n-C7 to n-C20 range previously documented for T. oleivorans (Yakimov et al, 2004; Wentzel et al, 2007) and similar to Alcanivorax borkumensis SK2 which is capable of growth on alkanes up to n-C32 (Schneiker et al, 2006), T. oleivorans cannot grow on the branched alkane pristane

Summary

Introduction

Thalassolituus oleivorans MIL-1 is a motile aerobic bacterium belonging to the Gammaproteobacteria class, which was first isolated from seawater and sediment samples collected in Milazzo Harbor, Sicily, Italy, supplemented with the n-alkane, tetradecane (n-C14) (Yakimov et al, 2004). OHCB are present in non-polluted marine environments at low numbers, but following oil spills, they typically bloom and become dominant members of the microbial community (Kasai et al, 2002a,b; Cappello et al, 2007; McKew et al, 2007a; Teramoto et al, 2009; Vila et al, 2010). Previous reports have shown that Thalassolituus-related species were among the most dominant members of hydrocarbon/petroleum enriched consortia and can outcompete other OHCB such as Alcanivorax, which is often the most dominant alkane degrader in marine oil spills (Harayama et al, 2004; Kostka et al, 2011). T. oleivorans became abundant in crude oil-amended North Sea microcosms at both 4◦C and 20◦C and was the most dominant bacterium at 20◦C, with total extractable hydrocarbons 15% their original value, confirming the important role this species plays in crude oil degradation in seawater (Coulon et al, 2007). Thalassolituus spp. dominated the microbial communities present in water samples collected from oil production wells in Canada (Kryachko et al, 2012)

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