The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall

Abstract

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Keywords: 3-hydroxy fatty acid; Alcanivorax borkumensis; HPLC; biosurfactants; glucolipid; glucose; glycine; mass spectrometry; oil spill.

FIG 1

Analysis of authentic and derivatized A. borkumensis glycine-glucolipid by mass spectrometry. The glycine-glucolipid was purified by SPE and TLC and analyzed by direct infusion Q-TOF mass spectrometry. (A) Total ion count spectrum of the purified authentic glycine-glucolipid. (B) MS/MS spectrum after fragmentation of the main glycine-glucolipid species Glc(O-10:0)₄Gly. Each neutral loss of an acyl group is represented by two fragment peaks differing by one H₂O group (m/z = 18). (C) MS/MS spectrum after fragmentation of the glycine-glucolipid species Glc(O-8:0)(O-10:0)₃Gly. The m/z for 3-HO-10:0 and 3-HO-8:0 differ by 28. Note that only the fragments representing the losses of the acyl moieties including H₂O are depicted. (D) Calculated masses of ammonium adducts of different molecular species and fragments generated from Glc(O-10:0)₄Gly of the authentic glycine-glucolipid. After the loss of the sugar head group with ammonia, the lipid backbone fragments exist as H⁺ adducts. Fragmentation within the lipid backbone results in radical ions with m/z differences originating from the loss of one H₂O molecule. The glycine ion with m/z = 76.0393 represents the H⁺ adduct. (E) Total ion count spectrum of the purified glycine-glucolipid after conversion into phenacyl esters. (F) MS/MS spectrum after fragmentation of the main species Glc(O-10:0)₄Gly-phenacyl. (G) Calculated masses of ammonium adducts of molecular species and fragments generated from Glc(O-10:0)₄Gly-phenacyl. Glc, glucose; Gly, glycine; O-8:0 or “8,” 3-hydroxy-octanoic acid; O-10:0 or “10,” 3-hydroxy-decanoic acid.

FIG 2

Detection of the A. borkumensis glycine-glucolipid by HPLC-DAD after conversion into phenacyl esters. (A) The glycine-glucolipid was converted into its phenacyl ester after incubation with 2-bromoacetophenone in the presence of triethylamine. Undecanoic acid (11:0) was employed as an internal standard. (B) HPLC-DAD chromatogram of a crude lipid extract from A. borkumensis, after derivatization. (C) HPLC-DAD chromatogram of isolated A. borkumensis glycine-glucolipid (purified by SPE and TLC) after derivatization. Glc, glucose; Gly, glycine; O-8:0, 3-hydroxy-octanoic acid; O-10:0, 3-hydroxy-decanoic acid.

FIG 3

Analysis of 3-hydroxy-fatty acids derived from the A. borkumensis glycine-glucolipid by GC-MS. (A) The four 3-hydroxy-fatty acids in the A. borkumensis glycine-glucolipid were transmethylated, and the free hydroxy groups were converted into trimethylsilyl (TMS) ethers. (B) GC-MS chromatogram (total ion count) of the A. borkumensis glycine-glucolipid, which was purified by SPE and TLC, and the 3-hydroxy fatty acids were converted into methyl esters/TMS ethers, in the presence of the internal standard 3-hydroxy dodecanoic acid (HO-12:0).

FIG 4

Growth of A. borkumensis cultures under different conditions. (A) O₂ (OTR), CO₂ (CTR), and (B) integrals of OTR, CTR of cultures grown in hexadecane or pyruvate medium. The cells were cultivated and monitored in a TOM shaker until the stationary phase was reached. Mean values of two individual cultivations are shown. The error bands indicate the values obtained from the individual cultures. (C) Growth curves (OD₆₀₀) of A. borkumensis cells grown with pyruvate and phosphate, with hexadecane and phosphate, with pyruvate without phosphate, or with hexadecane without phosphate. The plot shows individual, representative growth curves repeated at least two times with similar results.

FIG 5

Quantification of the glycine-glucolipid after growth in different media and in cell pellets and supernatants of A. borkumensis cultures. (A) The lipid extracts were prepared from A. borkumensis cells grown on pyruvate or hexadecane in the presence (+P) or absence (-P) of phosphate and analyzed by direct infusion Q-TOF mass spectrometry. The peak sizes were used for relative quantification. (B) The lipid extracts were derivatized with 2-bromoacetophenone and the glycine-glucolipid quantified by HPLC-DAD. (C) Lipids were extracted from the total culture broth of A. borkumensis cells grown in pyruvate or hexadecane. After centrifugation, lipids were extracted from the cell pellets or the supernatants (Super). The lipid extracts were analyzed by direct infusion Q-TOF mass spectrometry, and the peak sizes were used for relative quantification. (D) The lipid extracts were derivatized with 2-bromoacetophenone and the glycine-glucolipid quantified by HPLC-DAD. All glycine-glucolipid-containing peaks (Glc(O-10:0)₄Gly, Glc(O-8:0)(O-10:0)₃Gly, and Glc(O-8:0)₂(O-10:0)₂Gly) were summarized. The amounts of glycine-glucolipids are expressed in total ion counts per 10¹⁰ cells (Q-TOF) or in nmol per 10¹⁰ cells and in nmol per mg protein (HPLC) Data show means and SD (n = 3). Student T-test, *, P < 0.05; **, P < 0.01; n.s., not significant.