Institute of Veterinary Bacteriology, University Zurich, Zurich, Switzerland.
BMC Microbiol. 2010 Jul 20;10:194. doi: 10.1186/1471-2180-10-194.
Mycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.Inorganic pyrophosphatases (PPase) are important enzymes that catalyze the hydrolysis of inorganic pyrophosphate PPi to inorganic phosphate Pi. PPases are essential and ubiquitous metal-dependent enzymes providing a thermodynamic pull for many biosynthetic reactions. Here, we describe the identification, recombinant production and characterization of the soluble (s)PPase of Mycoplasma suis.
Screening of genomic M. suis libraries was used to identify a gene encoding the M. suis inorganic pyrophosphatase (sPPase). The M. suis sPPase consists of 164 amino acids with a molecular mass of 20 kDa. The highest identity of 63.7% was found to the M. penetrans sPPase. The typical 13 active site residues as well as the cation binding signature could be also identified in the M. suis sPPase. The activity of the M. suis enzyme was strongly dependent on Mg2+ and significantly lower in the presence of Mn2+ and Zn2+. Addition of Ca2+ and EDTA inhibited the M. suis sPPase activity. These characteristics confirmed the affiliation of the M. suis PPase to family I soluble PPases. The highest activity was determined at pH 9.0. In M. suis the sPPase builds tetramers of 80 kDa which were detected by convalescent sera from experimentally M. suis infected pigs.
The identification and characterization of the sPPase of M. suis is an additional step towards the clarification of the metabolism of hemotrophic mycoplasmas and, thus, important for the establishment of an in vitro cultivation system. As an antigenic and conserved protein the M. suis sPPase could in future be further analyzed as a diagnostic antigen.
猪肺炎支原体属于一组高度专业化的血溶菌，附着在宿主红细胞表面。血溶菌不能培养，其基因组至今尚未测序。因此，需要阐明基本的代谢途径，这可能是建立这些具有重要兽医意义的细菌体外培养系统的关键障碍。无机焦磷酸酶（PPase）是一种重要的酶，能催化无机焦磷酸（PPi）水解为无机磷酸（Pi）。PPases 是必不可少的、普遍存在的、依赖金属的酶，为许多生物合成反应提供热力学驱动力。在这里，我们描述了猪肺炎支原体可溶性（s）PPase 的鉴定、重组生产和特性。
筛选基因组 M. suis 文库用于鉴定编码 M. suis 无机焦磷酸酶（sPPase）的基因。M. suis sPPase 由 164 个氨基酸组成，分子量为 20kDa。与 M. penetrans sPPase 的同源性最高，为 63.7%。在 M. suis sPPase 中也可以发现典型的 13 个活性位点残基和阳离子结合特征。M. suis 酶的活性强烈依赖于 Mg2+，而在 Mn2+和 Zn2+存在时活性显著降低。Ca2+和 EDTA 的加入抑制了 M. suis sPPase 的活性。这些特性证实了 M. suis PPase 属于家族 I 可溶性 PPases。在 pH9.0 时，活性最高。在 M. suis 中，sPPase 构建 80kDa 的四聚体，这可以通过实验感染 M. suis 的猪的恢复期血清检测到。
M. suis sPPase 的鉴定和特性分析是阐明血溶菌代谢的又一步骤，因此对建立体外培养系统很重要。作为一种抗原性和保守性蛋白，M. suis sPPase 可以作为一种诊断抗原在未来进一步分析。
