Adaptation of the hydrocarbonoclastic bacterium Alcanivorax borkumensis SK2 to alkanes and toxic organic compounds: a physiological and transcriptomic approach

Abstract

The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is able to degrade mixtures of n-alkanes as they occur in marine oil spills. However, investigations of growth behavior and physiology of these bacteria when cultivated with n-alkanes of different chain lengths (C6 to C30) as the substrates are still lacking. Growth rates increased with increasing alkane chain length up to a maximum between C12 and C19, with no evident difference between even- and odd-numbered chain lengths, before decreasing with chain lengths greater than C19. Surface hydrophobicity of alkane-grown cells, assessed by determination of the water contact angles, showed a similar pattern, with maximum values associated with growth rates on alkanes with chain lengths between C11 and C19 and significantly lower values for cells grown on pyruvate. A. borkumensis was found to incorporate and modify the fatty acid intermediates generated by the corresponding n-alkane degradation pathway. Cells grown on distinct n-alkanes proved that A. borkumensis is able to not only incorporate but also modify fatty acid intermediates derived from the alkane degradation pathway. Comparing cells grown on pyruvate with those cultivated on hexadecane in terms of their tolerance toward two groups of toxic organic compounds, chlorophenols and alkanols, representing intensely studied organic compounds, revealed similar tolerances toward chlorophenols, whereas the toxicities of different n-alkanols were significantly reduced when hexadecane was used as a carbon source. As one adaptive mechanism of A. borkumensis to these toxic organic solvents, the activity of cis-trans isomerization of unsaturated fatty acids was proven. These findings could be verified by a detailed transcriptomic comparison between cultures grown on hexadecane and pyruvate and including solvent stress caused by the addition of 1-octanol as the most toxic intermediate of n-alkane degradation.

Fig 1

Growth rates (bars) and water contact angles (filled circles) of A. borkumensis grown with pyruvate and n-alkanes with different chain lengths as the sole carbon and energy sources.

Fig 2

Fatty acid patterns of A. borkumensis grown with pyruvate (white bars) or hexadecane (black bars) as the sole carbon and energy source.

Fig 3

Gas chromatography patterns of fatty acids obtained from cultures of A. borkumensis grown on pyruvate, n-tridecane, n-tetradecane, n-pentadecane, or n-hexadecane as the sole carbon and energy source. Approximate retention times for C_13:0, C_15:0, and C_17:0 fatty acids are shown in yellow. Approximate retention times for C_15:1 and C_17:1 fatty acids are shown in red. The peak for myristic acid in n-tetradecane-grown cultures is shown in green. Regular even-numbered fatty acid peaks are labeled only in pyruvate- or n-hexadecane-grown samples. Detailed quantification of the data is presented in Table 1.

Fig 4

Effect of 1-decanol on growth rate (●) and trans/cis ratio of unsaturated fatty acids (☐) of A. borkumensis grown with pyruvate (A) or hexadecane (B) as the sole carbon and energy source.

Fig 5

Correlation between hydrophobicity, given as the log(P) value, and growth inhibition caused by chlorophenols (triangles) and n-alkanols (circles) for A. borkumensis grown with pyruvate (open symbols) or hexadecane (filled symbols) as the sole carbon and energy source. Growth inhibition is presented as the EC₅₀s.

Fig 6

Classification of differentially expressed genes of A. borkumensis in response to 1-octanol (15 min after perturbation) in cells grown on pyruvate (A) or hexadecane (B) as the carbon source. Gray bars show upregulated and black bars show downregulated genes. Values represent the percentages of open reading frames that were assigned to each COG category.

Fig 7

Hierarchical cluster of A. borkumensis genes involved in n-alkane degradation. The RNA samples were taken at the given time points after the addition of 1-octanol. Expression ratios of the pyruvate samples are shown in the left-hand columns, and those of the hexadecane samples are shown in the right-hand columns; up- and downregulation as indicated are shown in the color legend at the top of the figure.