Madsen A, Josephsen J
Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark.
Biol Chem. 1998 Apr-May;379(4-5):443-9. doi: 10.1515/bchm.1998.379.4-5.443.
The genes encoding the restriction-modification (R/M) system LlaCI have been found on the naturally occurring 7.0 kb plasmid pAW153 in L. lactis subsp. cremoris W15. The R/M system was isolated on a chloramphenicol resistant derivative of the wild type plasmid (pAW153cat). Plasmid pAW153cat and a 2.4 kb HincII-SphI fragment cloned into a high- and a low-copy vector conferred decreased sensitivity in L. lactis LM2301 and L. lactis SMQ86 against small isometric-headed phages of the 936 or P335 species, respectively. Increased plasmid copy number enhanced the level of phage restriction. Sequencing the 2.4 kb HincII-SphI fragment revealed two open reading frames arranged convergently with a 94 bp separation. IlaCIM showed 66% identity to hindIIIM, and IlaCIR showed 45% identity to hindIIIR. The organization of the LlaCI operon differs from the HindIII operon, where the endonuclease and methylase genes overlap and are transcribed in the same direction. The LlaCI methylase is predicted to be 296 amino acids long, with 63% identity to the HindIII methylase, while the LlaCI endonuclease is predicted to consist of 324 or 332 amino acids, depending on the position of the start codon. It shows 24% identity to the HindIII endonuclease.
编码限制修饰（R/M）系统LlaCI的基因已在乳酸乳球菌亚种cremoris W15天然存在的7.0 kb质粒pAW153上发现。该R/M系统是在野生型质粒（pAW153cat）的氯霉素抗性衍生物上分离得到的。质粒pAW153cat以及克隆到高拷贝和低拷贝载体中的一个2.4 kb HincII-SphI片段，分别使乳酸乳球菌LM2301和乳酸乳球菌SMQ86对936或P335种的小等轴头噬菌体的敏感性降低。质粒拷贝数的增加提高了噬菌体限制水平。对2.4 kb HincII-SphI片段进行测序，发现有两个反向排列的开放阅读框，间隔94 bp。IlaCIM与hindIIIM的同一性为66%，IlaCIR与hindIIIR的同一性为45%。LlaCI操纵子的组织方式与HindIII操纵子不同，后者的内切酶和甲基化酶基因重叠且转录方向相同。LlaCI甲基化酶预计有296个氨基酸长，与HindIII甲基化酶的同一性为63%，而LlaCI内切酶预计由324或332个氨基酸组成，具体取决于起始密码子的位置。它与HindIII内切酶的同一性为24%。
