Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. The presence of isomeric FA complicates lipid analysis, because several fatty acids are bound in the intact phospholipids. Furthermore, commercially available reference standards do not fully cover potential lipid isomers.
Therefore, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation.
In addition, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols and cardiolipins, was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was elucidated. Furthermore, the developed LC-MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.
Several bacteria could be differentiated by the developed in-depth phospholipid profiling. The deduced retention order could now pave the way for comprehensive analysis of bacterial phospholipids containing BCFA in further studies, and thus, may provide broader insights into the biological background of bacterial membrane stability and susceptibility.