Tasmanian Institute of Agriculture, University of Tasmania, Hobart, Tasmania, Australia.
Central Science Laboratory, University of Tasmania, Hobart, Tasmania, Australia.
PLoS One. 2018 Oct 25;13(10):e0206317. doi: 10.1371/journal.pone.0206317. eCollection 2018.
Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (log2-fold change of 7) at 45°C. We identified a novel SecB homolog as a protein export chaperone putatively involved in posttranslational translocation systems, which was down-regulated as growth temperature increased and where the modelled 3D-structure shared architectural similarities with the Escherichia coli SecB protein. Membrane lipid analyses revealed temporal changes in fatty acid composition, cyclization of oleic acid to cyclopropane and novel cyclopentenyl moieties, and reduced synthesis of vaccenic acid, at higher temperatures. An 18kDa α-crystallin domain, Hsp20 family heat shock protein was more highly up-regulated in response to heat stress compared to other molecular chaperones, suggesting this protein could be a useful biomarker of prolonged heat stress in Lb. casei GCRL163.
在乳制品生产中，干酪乳杆菌作为非 starter 乳酸菌，会遇到持续的高温热应激。为了了解 Lb. casei GCRL163 适应持续高温的生理和分子机制，使用无标记定量蛋白质组学方法分析了细胞游离提取物，以表征该菌株在生物反应器中厌氧培养时的全局响应，培养条件为 30 至 45°C、pH6.5，同时在不同生长阶段进行 GC-MS 分析脂肪酸甲酯。在较高的生长温度下，观察到能量消耗代谢途径（如脂肪酸、核苷酸和氨基酸生物合成）受到抑制，而 PTS 和 ABC 型转运系统与氮源和碳源的摄取相关，被上调。碱性应激蛋白 Asp23_2 仅在 45°C 时检测到，表达丰度高，假定的α-L-岩藻糖苷酶仅在 40 和 45°C 时检测到，在 45°C 时丰度显著增加（对数 2 倍变化为 7）。我们鉴定出一种新型 SecB 同源物作为一种蛋白输出伴侣，可能参与翻译后转运系统，随着生长温度的升高，该蛋白下调，其建模的 3D 结构与大肠杆菌 SecB 蛋白具有结构相似性。膜脂分析显示脂肪酸组成、油酸环化形成环丙烷和新型环戊烯基部分、以及在较高温度下疫苗酸合成减少的时间变化。与其他分子伴侣相比，18kDaα-晶状体蛋白结构域、Hsp20 家族热休克蛋白在应对热应激时更高地被上调，表明该蛋白可能是 Lb. casei GCRL163 中长时间热应激的有用生物标志物。
