Madsen A, Josephsen J
Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Appl Environ Microbiol. 1998 Jul;64(7):2424-31. doi: 10.1128/AEM.64.7.2424-2431.1998.
The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GC decreases NGC-3', where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stemloop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.
在乳酸乳球菌亚种cremoris W39的天然存在的8.9 kb质粒pHW393上发现了LlaDII限制/修饰（R/M）系统。插入大肠杆菌-乳酸乳球菌穿梭载体pCI3340的一个2.4 kb PstI-EcoRI片段赋予乳酸乳球菌LM2301和乳酸乳球菌SMQ86对三种最常见的乳球菌噬菌体种类（936、P335和c2）代表株的抗性。LlaDII核酸内切酶被部分纯化，发现其识别并切割序列5'-GC↓NGC-3'，其中箭头指示切割位点。因此，它是市售限制性核酸内切酶Fnu4HI的同裂酶。对2.4 kb PstI-EcoRI片段进行测序，发现有两个串联排列的开放阅读框，中间由一个105 bp的基因间隔区隔开。543 bp的核酸内切酶基因位于954 bp的甲基化酶基因之前。LlaDII R/M系统推导的氨基酸序列与其唯一测序的同裂酶——芽孢杆菌属菌株RFL6的Bsp6I的氨基酸序列具有高度同源性，核酸内切酶之间的同一性为41%，甲基化酶之间的同一性为60%。LlaDII和Bsp6I R/M系统的基因组织相同。两种甲基化酶都有两个识别位点（5'-GCGGC-3'和5'-GCCGC-3'），形成一个推定的茎环结构，跨越推定的-35序列的一部分以及-35和-10序列之间间隔区部分。LlaDII和Bsp6I甲基化酶与其他m5C甲基化酶的比对表明，蛋白质一级结构具有相同的组织形式。
