Kossykh Valeri G, Lloyd R Stephen
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1071, USA.
J Bacteriol. 2004 Apr;186(7):2061-7. doi: 10.1128/JB.186.7.2061-2067.2004.
DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp. strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT. The yhdJ gene was cloned, and the enzyme was overexpressed and purified. Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT. This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for "cell cycle-regulated methyltransferase"). The CcrM DNA adenine methyltransferase is required for viability in E. coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans. The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off. Overexpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population. Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology. Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.
DNA序列分析显示，大肠杆菌假定的yhdJ DNA甲基转移酶基因与编码DNA甲基转移酶AvaIII的蓝藻菌株PCC7120基因有55%的同一性，AvaIII在识别序列ATGCAT中使腺嘌呤甲基化。克隆了yhdJ基因，并对该酶进行了过表达和纯化。甲基化和限制性分析表明，该DNA甲基转移酶使序列ATGCAT中的第一个腺嘌呤甲基化。发现这种DNA甲基化在细胞周期中受到调控，并且该DNA腺嘌呤甲基转移酶被命名为M.EcoKCcrM（“细胞周期调控甲基转移酶”）。CcrM DNA腺嘌呤甲基转移酶是大肠杆菌生存所必需的，因为只有在反式提供ccrM的额外拷贝时，才能分离出缺乏功能性基因组拷贝的菌株。当诱导性质粒ccrM基因的表达被关闭时，这种敲除菌株的细胞停止生长。M.EcoKCcrM的过表达减缓了细菌生长，并且在整个细胞周期中ATGCAT位点完全甲基化；在这个群体中发现了高比例的细胞，其大小分布和DNA含量异常。因此，这种甲基转移酶的时间控制可能有助于细胞分裂和细胞形态的精确细胞周期控制。M.EcoKCcrM的同源物存在于属于变形菌纲γ亚群的其他细菌中，这表明在ATGCAT位点的甲基化在该类群的其他成员中可能具有类似的功能。
