Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp. strain Q15

Abstract

We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.

FIG. 1

Growth of Rhodococcus sp. strain Q15 at 5°C with glucose-acetate (A) or diesel fuel (B) as the carbon and energy source and changes in the surface tensions in the cultures, the supernatants, and the pellet fractions. The surface tension values are means based on duplicate determinations. ST, surface tension; A600, absorbance at 600 nm.

FIG. 2

TEM micrographs of Rhodococcus sp. strain Q15 cells obtained during growth on glucose-acetate or diesel fuel at 5°C. (A) Strain Q15 grown on glucose-acetate and fixed in the presence of ruthenium red. The organism occurred as single cells which were surrounded by EPS consisting of a delicate web of fibers consistent with bacterial capsular material. (B) Floc phase cells of strain Q15 grown on diesel fuel and fixed in the presence of ruthenium red. In thin sections, finely contrasted clusters of EPS material surrounded cells within the flocs. The arrow indicates an intracellular inclusion. Bars = 500 nm.

FIG. 3

SEM micrograph of Rhodococcus sp. strain Q15, showing groups of cells colonizing the surface of an octacosane crystal during growth at 5°C. Strands and fibers (fingerlike projections) of EPS were observed on the cell surface between cells and between cells and the surface of the octacosane crystal.

FIG. 4

CSLM of Rhodococcus sp. strain Q15 cells grown on diesel fuel at 5°C. (A) Three-dimensional projections presented as a stereo pair showing a z series through an SYTO 9-stained strain Q15 biofilm grown on diesel fuel as the sole C source. Note the basal layer of attached cells on the slide surface and the microdroplets of diesel fuel surrounded by cells of strain Q15. (B) Series of xz optical sections through the diesel fuel microdroplets shown in panel A. The upper surface was a glass coverslip overlying a well slide. The cells grown on diesel fuel were transferred to the well slide and covered with a glass coverslip, and microdroplets were observed after they rose to contact the upper glass surface. The cells were maintained at 5°C, and no fixation or immobilization of the droplets was performed.

FIG. 5

CSLM xy images showing binding of the C. ensiformis (A) and T. purpureas (B) lectins to cells of strain Q15 grown at 5°C with diesel fuel as the sole carbon source. C. ensiformis lectin appeared to bind to the exterior surfaces of the diesel fuel microdroplets in the biofilm, creating a bright boundary region. There is also some evidence that there were binding sites within the diesel fuel droplets. The T. purpureas lectin binding was extensive at the cell surface and occurred between cells and within the microdroplets. In addition, there is evidence that an emulsion was formed within the microdroplets and in the vicinity of Q15 cells.

FIG. 6

Comparison of fatty acid profiles of Rhodococcus sp. strain Q15 cells grown on glucose-acetate, hexadecane, or diesel fuel at 5 or 24°C. Unk, unknown, putative fatty acid; c, cis, t, trans; cyc, cylcopropane; Me, methyl. The values are means based on triplicate samples (standard deviation, <10% of mean). Unknown fatty acids 3 and 4, which were present in relatively large quantities in cells grown on hexadecane at 5°C, were tentatively identified by gas chromatography-mass spectrometry as C_16:1 and methyl-C_15:0, respectively.