Hydrocarbonoclastic Alcanivorax Isolates Exhibit Different Physiological and Expression Responses to n-dodecane

Abstract

Autochthonous microorganisms inhabiting hydrocarbon polluted marine environments play a fundamental role in natural attenuation and constitute promising resources for bioremediation approaches. Alcanivorax spp. members are ubiquitous in contaminated surface waters and are the first to flourish on a wide range of alkanes after an oil-spill. Following oil contamination, a transient community of different Alcanivorax spp. develop, but whether they use a similar physiological, cellular and transcriptomic response to hydrocarbon substrates is unknown. In order to identify which cellular mechanisms are implicated in alkane degradation, we investigated the response of two isolates belonging to different Alcanivorax species, A. dieselolei KS 293 and A. borkumensis SK2 growing on n-dodecane (C12) or on pyruvate. Both strains were equally able to grow on C12 but they activated different strategies to exploit it as carbon and energy source. The membrane morphology and hydrophobicity of SK2 changed remarkably, from neat and hydrophilic on pyruvate to indented and hydrophobic on C12, while no changes were observed in KS 293. In addition, SK2 accumulated a massive amount of intracellular grains when growing on pyruvate, which might constitute a carbon reservoir. Furthermore, SK2 significantly decreased medium surface tension with respect to KS 293 when growing on C12, as a putative result of higher production of biosurfactants. The transcriptomic responses of the two isolates were also highly different. KS 293 changes were relatively balanced when growing on C12 with respect to pyruvate, giving almost the same amount of upregulated (28%), downregulated (37%) and equally regulated (36%) genes, while SK2 transcription was upregulated for most of the genes (81%) when growing on pyruvate when compared to C12. While both strains, having similar genomic background in genes related to hydrocarbon metabolism, retained the same capability to grow on C12, they nevertheless presented very different physiological, cellular and transcriptomic landscapes.

Keywords: Alcanivorax; alkanes; bioremediation; functional redundancy; transcriptomics.

Figure 1

Growth evaluation and physiological features of A. borkumensis SK2 and A. dieselolei KS 293 strains after 7 days incubation on pyruvate (PYR) or n-dodecane (C12). Bars indicate the 95% confidence interval (CI). Stars indicate significantly different values (p < 0.05). (A) Total cell number evaluation with flow cytometry. (B) Measurement of pH variation of the culture media after incubation. (C) MATH test, percentage of cells bound to n-exadecane (C16). (D) Surface tension of the culture media.

Figure 2

(A) Relative abundance of PLFA in cell membranes after 7 days incubation on pyruvate (PYR) or n-dodecane (C12) in A. dieselolei KS 293 and A. borkumensis SK2. (B–E) Transmission electron microscopy of cells of A. dieselolei KS 293 (B,C) and A. borkumensis SK2 (D,E) after 7 days of growth on pyruvate (PYR) (B,D) or n-dodecane (C12) (C,E) as the sole carbon source. Stars indicate electron transparent intracellular bodies inside the cells.

Figure 3

General transcriptomic features. (A) Percentage of gene clusters up-, down- or equally regulated in A. dieselolei KS 293 and A. borkumensis SK2 growing with pyruvate compared to n-dodecane. (B) Comparison of gene clusters transcription (%) in A. dieselolei KS 293 and A. borkumensis SK2 when growing with n-dodecane.

Figure 4

Heat map of the transcription level of genes involved in hydrocarbon degradation and metabolism. Yellow indicates the same level of transcription, light green indicates upregulation, dark green indicates strong upregulation, red indicates downregulation and dark red indicates strong downregulation.

Figure 5

Heat map of the transcription level of genes involved in biosurfactant production and secretion. Yellow indicates the same level of transcription, light green indicates upregulation, dark green indicates strong upregulation, red indicates downregulation and dark red indicates strong downregulation.

Figure 6

Heat map of the transcription level of genes involved in intracellular reservoirs of carbon synthesis. Yellow indicates the same level of transcription, light green indicates upregulation, dark green indicates strong upregulation, red indicates downregulation and dark red indicates strong downregulation.