Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Bonn, Germany.
Institute of Applied Microbiology, RWTH Aachen, Aachen, Germany.
Appl Environ Microbiol. 2022 Aug 23;88(16):e0112622. doi: 10.1128/aem.01126-22. Epub 2022 Aug 8.
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 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 is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. 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 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 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.
海洋细菌 Alcanivorax borkumensis 在生长过程中会产生一种具有表面活性的甘氨酰-葡糖脂，该物质由长链烷烃诱导生成。我们开发了一种高效液相色谱（HPLC）方法对其进行绝对定量。该方法基于将甘氨酰-葡糖脂转化为苯甲酰酯，然后通过二极管阵列检测高效液相色谱法（HPLC-DAD）进行测量。不同的分子物种通过 HPLC 分离，并通过质谱鉴定为具有不同数量的 3-羟基癸酸或 3-羟基辛酸基团的葡糖基-四（3-羟基酰基）-甘氨酸。与十六烷相比，细胞以丙酮酸为唯一碳源生长时的细胞密度和氧气/二氧化碳转移率均有所提高。与生长在丙酮酸上相比，细胞在十六烷上生长时产生的每细胞甘氨酰-葡糖脂的量更高。与十六烷生长的细胞相比，来自丙酮酸生长的细胞的甘氨酰-葡糖脂中含有相当数量的 3-羟基辛酸，而十六烷生长的细胞几乎仅将 3-羟基癸酸纳入甘氨酰-葡糖脂。甘氨酰-葡糖脂主要存在于细胞沉淀中，而在无细胞上清液中仅存在微量。从细菌细胞培养液、细胞沉淀或无细胞上清液中分离出的甘氨酰-葡糖脂具有相同的结构，其中含有一个甘氨酸残基，这与之前的报道不同，之前的报道表明存在一种分泌到上清液中的无甘氨酸形式的葡糖脂。总之，是 Alcanivorax borkumensis 的甘氨酰-葡糖脂驻留在细胞壁上，使其能够在脂质-水界面结合和溶解烷烃。Alcanivorax borkumensis 是在溢油区发现的最丰富的海洋细菌之一，它能降解烷烃。甘氨酰-葡糖脂的产生被认为是烷烃降解的一个必要因素。我们开发了一种定量方法，并在不同培养基中生长后，确定了培养物不同部分中的 甘氨酰-葡糖脂的结构。我们的结果表明，细胞中甘氨酰-葡糖脂的含量远远超过上清液中测量到的含量，证实了提出的细胞壁定位。这些结果支持这样的假设，即通过产生甘氨酰-葡糖脂，增加了 细胞的表面疏水性，使其能够附着在烷烃-水界面并形成生物膜。我们没有发现无甘氨酸形式的葡糖脂的证据。
