Quadri L E, Sailer M, Roy K L, Vederas J C, Stiles M E
Department of Chemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1994 Apr 22;269(16):12204-11.
Carnobacteriocins BM1 and B2 are thermostable class II bacteriocins produced by Carnobacterium piscicola LV17B. These bacteriocins were purified by a three-step procedure that included hydrophobic interaction, size exclusion, and reversed-phase high performance liquid chromatography. The purified peptides and fragments derived by enzymatic digestion were analyzed by Edman degradation, amino acid analysis, and mass spectrometry. An oxidized form of carnobacteriocin BM1 (carnobacteriocin B1) was also purified and characterized. Probes synthesized using information from the N-terminal amino acid sequences for the purified bacteriocins were used to locate structural genes for the carnobacteriocins. A 1.9-kilobase (kb) HindIII fragment from a 61-kb plasmid (pCP40) containing the carnobacteriocin B2 structural gene and a 4.0-kb EcoRI-PstI genomic fragment containing the carnobacteriocin BM1 structural gene were cloned and fully or partially sequenced, respectively. Expression of the chromosomal bacteriocin and its immunity function requires the presence of the 61-kb plasmid. The results indicate that both bacteriocins are synthesized as prebacteriocins. Post-translational cleavage of an 18-amino acid N-terminal extension at a Gly-Gly (positions -2 and -1) site takes place in each prepeptide to yield the mature 43-amino acid carnobacteriocin BM1 (molecular mass 4524.6) and the mature 48-amino acid carnobacteriocin B2 (molecular mass 4969.9). These two peptides showed significant amino acid homology to each other and with those class II bacteriocins which contain the YGNGV amino acid motif near the N terminus.
肉杆菌素BM1和B2是由嗜鱼肉杆菌LV17B产生的热稳定II类细菌素。这些细菌素通过三步程序进行纯化，该程序包括疏水相互作用、尺寸排阻和反相高效液相色谱。通过埃德曼降解、氨基酸分析和质谱对纯化的肽段以及酶切产生的片段进行分析。还纯化并表征了肉杆菌素BM1的氧化形式（肉杆菌素B1）。利用纯化细菌素N端氨基酸序列信息合成的探针用于定位肉杆菌素的结构基因。分别克隆了来自含有肉杆菌素B2结构基因的61 kb质粒（pCP40）的1.9 kb HindIII片段和含有肉杆菌素BM1结构基因的4.0 kb EcoRI - PstI基因组片段，并进行了全序列或部分序列测定。染色体细菌素及其免疫功能的表达需要61 kb质粒的存在。结果表明，这两种细菌素均以前细菌素的形式合成。每个前肽在Gly - Gly（位置 -2和 -1）位点处进行18个氨基酸的N端延伸的翻译后切割，产生成熟的43个氨基酸的肉杆菌素BM1（分子量4524.6）和成熟的48个氨基酸的肉杆菌素B2（分子量4969.9）。这两种肽彼此之间以及与那些在N端附近含有YGNGV氨基酸基序的II类细菌素显示出显著的氨基酸同源性。
