ccfA, the Genetic Determinant for the cCF10 Peptide Pheromone in Enterococcus faecalis OG1RF

The nosocomial pathogen Enterococcus faecalis has a unique pheromone-inducible conjugative mating system. Conjugative transfer of the E. faecalis plasmid pCF10 is specifically induced by the cCF10 peptide pheromone (LVTLVFV). Genomic sequence information has recently allowed the identification of putative structural genes coding for the various enterococcal pheromones (D. B. Clewell et al., Mol. Microbiol. 35:246-247, 2000). The cCF10 pheromone sequence LVTLVFV was found within an open reading frame designated ccfA, encoding a putative lipoprotein precursor. Several other pheromone sequences were found in similar locations within other predicted lipoproteins. CcfA shows significant sequence relatedness to the Escherichia coli protein YidC, an inner membrane protein translocase, as well as to a large number of homologs identified in gram-positive and in gram-negative bacteria. Analysis of the deduced CcfA amino acid sequence suggested that mature cCF10 peptide could be formed from the proteolytic degradation of its signal peptide. Expression of the cloned ccfA gene with an inducible expression vector dramatically increased cCF10 production by E. faecalis and also resulted in cCF10 production by Lactococcus lactis, a non-pheromone producer. Site-directed mutagenesis of the ccfA sequence encoding the cCF10 peptide confirmed that ccfA was a functional genetic determinant for cCF10.

医院致病菌粪肠球菌（Enterococcus faecalis）拥有独特的信息素诱导型接合交配系统。粪肠球菌质粒pCF10的接合转移可由cCF10肽信息素（LVTLVFV）特异性诱导。近年来，基因组序列信息已助力鉴定出编码各类肠球菌信息素的推定结构基因（D. B. Clewell et al., Mol. Microbiol. 35:246-247, 2000）。cCF10信息素序列LVTLVFV存在于命名为ccfA的开放阅读框（open reading frame）内，该阅读框编码推定的脂蛋白前体。在其他预测脂蛋白的类似区域中，还发现了数种其他信息素序列。CcfA与大肠杆菌（Escherichia coli）的内膜蛋白转位酶YidC，以及大量在革兰氏阳性菌和革兰氏阴性菌中鉴定出的同源蛋白均存在显著的序列相关性。对推导得到的CcfA氨基酸序列进行分析后表明，成熟的cCF10肽可通过其信号肽的蛋白水解降解产生。将克隆得到的ccfA基因与诱导型表达载体结合表达，可显著提升粪肠球菌的cCF10产量，同时也能使非信息素产生菌乳酸乳球菌（Lactococcus lactis）产生cCF10。对编码cCF10肽的ccfA序列进行定点诱变实验证实，ccfA是cCF10的功能遗传决定因子。