Twine Susan M, Reid Christopher W, Aubry Annie, McMullin David R, Fulton Kelly M, Austin John, Logan Susan M
Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
J Bacteriol. 2009 Nov;191(22):7050-62. doi: 10.1128/JB.00861-09. Epub 2009 Sep 11.
In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein.
在本研究中，从艰难梭菌菌株中纯化出完整的鞭毛蛋白，并使用四极杆飞行时间质谱仪和线性离子阱质谱仪进行分析。自上而下的研究表明，鞭毛蛋白的质量大于根据相应基因序列预测的质量。这些自上而下的研究揭示了聚糖修饰的特征性标记离子。此外，在不同菌株之间观察到聚糖修饰质量的多样性。电子转移解离质谱法用于证明，在所检测的所有菌株中，聚糖通过一个己糖胺残基以O-连接的方式连接到鞭毛蛋白主链上。对艰难梭菌基因组的生物信息学分析显示，鞭毛生物合成位点内聚糖生物合成基因含量存在多样性，这可能反映在观察到的鞭毛聚糖多样性上。在艰难梭菌630菌株中，所有已测序基因组中都存在的一个糖基转移酶基因（CD0240）的插入失活导致无法在细胞表面产生鞭毛丝，并且仅产生少量未修饰的鞭毛蛋白。
