Sluijter Marcel, Hoogenboezem Theo, Hartwig Nico G, Vink Cornelis
Erasmus MC, Laboratory of Pediatrics, Pediatric Infectious Diseases, PO Box 2040, 3000 CA Rotterdam, the Netherlands.
BMC Microbiol. 2008 Oct 2;8:167. doi: 10.1186/1471-2180-8-167.
Mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms.
The MPN229 ORF has the capacity to encode a 166-amino acid protein with a calculated molecular mass of 18.4 kDa. The amino acid sequence of this protein (Mpn SSB) is most closely related to that of the protein predicted to be encoded by the MG091 gene from Mycoplasma genitalium (61% identity). The MPN229 ORF was cloned, and different versions of Mpn SSB were expressed in E. coli and purified to > 95% homogeneity. The purified protein was found to exist primarily as a homo-tetramer in solution, and to strongly and selectively bind single-stranded DNA (ssDNA) in a divalent cation- and DNA substrate sequence-independent manner. Mpn SSB was found to bind with a higher affinity to ssDNA substrates larger than 20 nucleotides than to smaller substrates. In addition, the protein strongly stimulated E. coli Recombinase A (RecA)-promoted DNA strand exchange, which indicated that Mpn SSB may play an important role in DNA recombination processes in M. pneumoniae.
The M. pneumoniae MPN229 gene encodes a protein, Mpn SSB, which selectively and efficiently binds ssDNA, and stimulates E. coli RecA-promoted homologous DNA recombination. Consequently, the Mpn SSB protein may play a crucial role in DNA recombinatorial pathways in M. pneumoniae. The results from this study will pave the way for unraveling these pathways and assess their role in antigenic variation of M. pneumoniae.
肺炎支原体以前被认为是一种基因高度稳定的微生物。尽管具有这种遗传稳定性，但有人推测同源DNA重组是肺炎支原体主要表面蛋白P1抗原变异的基础。为了鉴定可能参与肺炎支原体同源DNA重组的蛋白质，我们着手对MPN229开放阅读框（ORF）进行表征，该阅读框与其他微生物中编码单链DNA结合（SSB）蛋白的基因具有序列相似性。
MPN229 ORF能够编码一种166个氨基酸的蛋白质，计算分子量为18.4 kDa。该蛋白质（Mpn SSB）的氨基酸序列与生殖支原体MG091基因预测编码的蛋白质序列关系最为密切（同一性为61%）。克隆了MPN229 ORF，并在大肠杆菌中表达了不同版本的Mpn SSB，并将其纯化至>95%的纯度。发现纯化后的蛋白质在溶液中主要以同四聚体形式存在，并以二价阳离子和DNA底物序列无关的方式强烈且选择性地结合单链DNA（ssDNA）。发现Mpn SSB对大于20个核苷酸的ssDNA底物的结合亲和力高于较小的底物。此外，该蛋白质强烈刺激大肠杆菌重组酶A（RecA）促进的DNA链交换，这表明Mpn SSB可能在肺炎支原体的DNA重组过程中起重要作用。
肺炎支原体MPN229基因编码一种蛋白质Mpn SSB，它选择性且有效地结合ssDNA，并刺激大肠杆菌RecA促进的同源DNA重组。因此，Mpn SSB蛋白可能在肺炎支原体的DNA重组途径中起关键作用。本研究结果将为揭示这些途径并评估它们在肺炎支原体抗原变异中的作用铺平道路。
