Vandera Elpiniki, Samiotaki Martina, Parapouli Maria, Panayotou George, Koukkou Anna Irini
Sector of Organic Chemistry and Biochemistry, University of Ioannina, Greece.
Biomedical Sciences Research Center "Alexander Fleming", Vari, Athens, Greece.
J Proteomics. 2015 Jan 15;113:73-89. doi: 10.1016/j.jprot.2014.08.018. Epub 2014 Oct 1.
In the present study, by applying comparative quantitative proteomics, we investigated the metabolic adaptation of Arthrobacter phenanthrenivorans Sphe3 when using phenanthrene, phthalate, glucose or glucose plus phenanthrene as sole carbon and energy sources. More than a third of the total Sphe3 proteins, with function prediction within the genome, were identified with confidence. Proteomic analysis data and annotated genomic information coincide, allowing us to clarify the phenanthrene catabolic pathway. We confirmed the implication of several proteins in aromatic substrate degradation by identifying those mediating the initial ring-hydroxylation and ring cleavage of phenanthrene to phthalate, phthalate degradation, as well as ortho- and meta-protocatechuate catabolism. Repression of catabolic genes by glucose was observed by both proteomic and transcriptional analyses. The presence of aromatic substrates resulted in changes in the abundance of proteins involved in substrate and amino acid metabolism, stress response, detoxification and membrane and cell wall metabolism. Uptake and transport associated proteins differ in the substrates used, indicating the use of different uptake mechanisms for transport of each compound in the Sphe3 cells. Our results also suggest the activation of a glyoxylate shunt in the presence of aromatic compounds, based on the up-regulation of the key enzymes of this pathway.
A. phenanthrenivorans Sphe3, isolated from a creosote contaminated soil in Greece, can grow on phenanthrene as the sole source of carbon and energy. To explore the phenanthrene catabolic pathway by determining the key proteins involved in this pathway, as well as the global changes in proteins due to the adaptive response of Sphe3 cells grown on different substrates, we applied a gel-free quantitative proteomic analysis using nanoLC-MS/MS. To our knowledge this is the first study of comparative global proteomic changes occurring in the Sphe3 cells under exposure in different nutritional environments. The extended proteomic changes observed in Sphe3 grown on different substrates provide an insight in the complex interactions occurring in the presence of aromatic compounds and could serve as a basis for further investigations intended to elucidate the general regulatory mechanism by which Sphe3 adapts to such xenobiotic environments. This may light the way for more efficient engineering of bacteria towards more effective bioremediation applications.
在本研究中，我们应用比较定量蛋白质组学方法，研究了食菲节杆菌Sphe3在以菲、邻苯二甲酸酯、葡萄糖或葡萄糖加菲作为唯一碳源和能源时的代谢适应性。在基因组中具有功能预测的Sphe3蛋白总数中，超过三分之一得到了可靠鉴定。蛋白质组学分析数据与注释的基因组信息相吻合，使我们能够阐明菲的分解代谢途径。通过鉴定介导菲的初始环羟基化和环裂解生成邻苯二甲酸酯、邻苯二甲酸酯降解以及邻位和间位原儿茶酸分解代谢的蛋白质，我们证实了几种蛋白质在芳香族底物降解中的作用。蛋白质组学和转录分析均观察到葡萄糖对分解代谢基因的抑制作用。芳香族底物的存在导致参与底物和氨基酸代谢、应激反应、解毒以及膜和细胞壁代谢的蛋白质丰度发生变化。与摄取和转运相关的蛋白质因所使用的底物而异，这表明在Sphe3细胞中，每种化合物的转运使用了不同的摄取机制。我们的结果还表明，基于该途径关键酶的上调，在存在芳香族化合物的情况下乙醛酸循环被激活。
从希腊受杂酚油污染的土壤中分离出的食菲节杆菌Sphe3能够以菲作为唯一碳源和能源生长。为了通过确定参与该途径的关键蛋白质以及Sphe3细胞在不同底物上生长时由于适应性反应而导致的蛋白质全局变化来探索菲的分解代谢途径，我们应用了使用nanoLC-MS/MS的无凝胶定量蛋白质组学分析。据我们所知，这是首次对Sphe3细胞在不同营养环境下暴露时发生的比较全局蛋白质组学变化进行的研究。在以不同底物生长的Sphe3中观察到的广泛蛋白质组学变化，为芳香族化合物存在时发生的复杂相互作用提供了见解，并可作为进一步研究的基础，旨在阐明Sphe3适应此类异源生物环境的一般调节机制。这可能为更有效地改造细菌以实现更有效的生物修复应用指明方向。
