Su Hsun-Cheng, Hutchison Clyde A, Giddings Morgan C
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
BMC Microbiol. 2007 Jul 2;7:63. doi: 10.1186/1471-2180-7-63.
Little is known regarding the extent or targets of phosphorylation in mycoplasmas, yet in many other bacterial species phosphorylation is known to play an important role in signaling and regulation of cellular processes. To determine the prevalence of phosphorylation in mycoplasmas, we examined the CHAPS-soluble protein fractions of Mycoplasma genitalium and Mycoplasma pneumoniae by two-dimensional gel electrophoresis (2-DE), using a combination of Pro-Q Diamond phosphoprotein stain and 33P labeling. Protein spots that were positive for phosphorylation were identified by peptide mass fingerprinting using MALDI-TOF-TOF mass spectrometry.
We identified a total of 24 distinct phosphoproteins, about 3% and 5% of the total protein complement in M. pneumoniae and M. genitalium, respectively, indicating that phosphorylation occurs with prevalence similar to many other bacterial species. Identified phosphoproteins include pyruvate dehydrogenase E1 alpha and beta subunits, enolase, heat shock proteins DnaK and GroEL, elongation factor Tu, cytadherence accessory protein HMW3, P65, and several hypothetical proteins. These proteins are involved in energy metabolism, carbohydrate metabolism, translation/transcription and cytadherence. Interestingly, fourteen of the 24 phosphoproteins we identified (58%) were previously reported as putatively associated with a cytoskeleton-like structure that is present in the mycoplasmas, indicating a potential regulatory role for phosphorylation in this structure.
This study has shown that phosphorylation in mycoplasmas is comparable to that of other bacterial species. Our evidence supports a link between phosphorylation and cytadherence and/or a cytoskeleton-like structure, since over half of the proteins identified as phosphorylated have been previously associated with these functions. This opens the door to further research into the purposes and mechanisms of phosphorylation for mycoplasmas.
关于支原体磷酸化的程度或靶点，人们了解甚少，然而在许多其他细菌物种中，磷酸化在细胞过程的信号传导和调节中起着重要作用。为了确定支原体中磷酸化的普遍性，我们使用Pro-Q Diamond磷蛋白染色和33P标记相结合的方法，通过二维凝胶电泳（2-DE）检测了生殖支原体和解脲脲原体的CHAPS可溶性蛋白组分。使用基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-TOF MS）通过肽质量指纹图谱鉴定磷酸化呈阳性的蛋白斑点。
我们总共鉴定出24种不同的磷蛋白，分别约占肺炎支原体和解脲脲原体总蛋白补体的3%和5%，这表明磷酸化的发生率与许多其他细菌物种相似。鉴定出的磷蛋白包括丙酮酸脱氢酶E1α和β亚基、烯醇化酶、热休克蛋白DnaK和GroEL、延伸因子Tu、细胞粘附辅助蛋白HMW3、P65以及几种假定蛋白。这些蛋白参与能量代谢、碳水化合物代谢、翻译/转录和细胞粘附。有趣的是，我们鉴定出的24种磷蛋白中有14种（58%）先前被报道可能与支原体中存在的细胞骨架样结构相关，这表明磷酸化在该结构中可能具有潜在的调节作用。
本研究表明支原体中的磷酸化与其他细菌物种相当。我们的证据支持磷酸化与细胞粘附和/或细胞骨架样结构之间的联系，因为超过一半被鉴定为磷酸化的蛋白先前已与这些功能相关。这为进一步研究支原体磷酸化的目的和机制打开了大门。
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