Quantitative and Functional Characterization of the Hyper-Conserved Protein of <i>Prochlorococcus</i> and Marine <i>Synechococcus</i>

A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.任何细菌基因组的绝大部分均由编码假想蛋白的开放阅读框（Open Reading Frames, ORFs）构成。尽管这类开放阅读框大多仅存在于单个或少数已测序基因组中，但仍有少数具有保守性，且保守范围往往跨越较大的系统发育距离。这种保守性可为亟待阐明的未表征细胞功能提供线索。属于原绿球藻（Prochlorococcus）/海洋聚球藻（Synechococcus）演化支的海洋蓝细菌，是海洋水域中的优势菌群，同时也是全球初级生产力的重要贡献者。一种功能未知的高度保守蛋白（Hyper Conserved Protein, PSHCP）在原绿球藻/海洋聚球藻的基因组中于氨基酸水平上完全保守，但在该演化支之外未发现其同源蛋白。本研究针对海洋原绿球藻（Prochlorococcus marinus）菌株MED4、MIT 9313以及聚球藻属（Synechococcus）菌株WH 8102，采用逆转录定量PCR（RT-Q-PCR）检测PSHCP基因的转录情况，通过定量免疫印迹法检测其蛋白产物的存在，并利用下拉实验（pull down assay）鉴定该蛋白的结合伴侣。所有菌株中均检测到该基因的显著转录。PSHCP蛋白含量因菌株不同而异，每微克总蛋白的含量介于8±1飞摩尔至26±9飞摩尔之间。在所有菌株中，50S核糖体蛋白L2、光系统I蛋白PsaD以及Ycf48样蛋白均被发现与PSHCP蛋白存在结合互作，而对照实验中未检测到此类结合或仅存在极微量的结合。本研究推测，PSHCP是一种与核糖体结合的蛋白，可能参与光系统的组装过程。