Comparative Proteomics of Marinobacter sp. TT1 Reveals Corexit Impacts on Hydrocarbon Metabolism, Chemotactic Motility, and Biofilm Formation

Abstract

The application of chemical dispersants during marine oil spills can affect the community composition and activity of marine microorganisms. Several studies have indicated that certain marine hydrocarbon-degrading bacteria, such as Marinobacter spp., can be inhibited by chemical dispersants, resulting in lower abundances and/or reduced biodegradation rates. However, a major knowledge gap exists regarding the mechanisms underlying these physiological effects. Here, we performed comparative proteomics of the Deepwater Horizon isolate Marinobacter sp. TT1 grown under different conditions. Strain TT1 received different carbon sources (pyruvate vs. n-hexadecane) with and without added dispersant (Corexit EC9500A). Additional treatments contained crude oil in the form of a water-accommodated fraction (WAF) or chemically-enhanced WAF (CEWAF; with Corexit). For the first time, we identified the proteins associated with alkane metabolism and alginate biosynthesis in strain TT1, report on its potential for aromatic hydrocarbon biodegradation and present a protein-based proposed metabolism of Corexit components as carbon substrates. Our findings revealed that Corexit exposure affects hydrocarbon metabolism, chemotactic motility, biofilm formation, and induces solvent tolerance mechanisms, like efflux pumps, in strain TT1. This study provides novel insights into dispersant impacts on microbial hydrocarbon degraders that should be taken into consideration for future oil spill response actions.

Keywords: Corexit; Marinobacter; WAF; biofilm formation; chemotactic motility; dispersant; hexadecane; hydrocarbon metabolism; proteomics.

Figure 1

Growth and biodegradation of carbon sources supplied to Marinobacter sp. TT1 cultures, sampled for proteomic analysis after 1 day (pyruvate treatments) or 4 days (all other treatments) of incubation. Treatments contained the following carbon sources: 3 mM pyruvate (Pyruvate), 100 mg L⁻¹ n-hexadecane (Hxdc), 100 mg L⁻¹ n-hexadecane and 10 mg L⁻¹ Corexit (Hxdc+Cxt), 100 mg L⁻¹ Corexit (Corexit), no carbon source (Control), 6 mg L⁻¹ water-accommodated fraction (WAF)-derived dissolved organic carbon (DOC) (WAF) or 6 mg L⁻¹ chemically-enhanced WAF (CEWAF)-derived DOC (CEWAF). Results shown are averages of sacrificial, triplicate cultures (standard deviations are based on triplicates). ab. = abiotic controls without inoculum. (A) Cell numbers determined by fluorescence microscopy are presented using a divided y-axis in order to better visualize the lower cell numbers in WAF and CEWAF treatments. (B) Remaining pyruvate and n-hexadecane concentrations were determined via HPLC or GC-MS measurements, respectively.

Figure 2

Normalized, relative mean abundances (symbolized by circle size and color; sum per protein = 100%) of a selection of significantly (q-value < 0.05) differentially abundant proteins associated with alkane metabolism, alginate synthesis, non-alkane hydrocarbon (HC) metabolism, chemotaxis, motility, and transmembrane transport systems during growth of Marinobacter sp. TT1 cultures on different carbon sources. Treatments received either pyruvate, n-hexadecane (Hxdc), n-hexadecane and Corexit (Hxdc+Cxt), only Corexit (Corexit), crude oil WAF, or chemically enhanced WAF (CEWAF). 1 = proteins belonging to the proposed alk operon (plus AlkB1 homologue); 2 = Cycloaliphatic HC metabolism; 3 = Phenol metabolism; 4 = Aromatic HC metabolism; 5 = Aminobenzoate metabolism; 6 = Type IV pilus assembly; 7 = Twitching motility; 8 = Proteins belonging to the proposed alg operon. See Table S9 for protein names.

Figure 3

Schematic overview of the metabolism of Marinobacter sp. TT1 when (A) utilizing hydrocarbons (n-hexadecane or WAF), or (B) growing on components of Corexit and/or with Corexit exposure. Abbreviations: LPS = lipopolysaccharide, PG = peptidoglycan, HC = hydrocarbon, WAF = water-accommodated fraction, MCP = methyl-accepting chemotaxis protein, TRAP = tripartite ATP-independent periplasmic, ABC = ATP-binding cassette. See Table S9 for protein names.