Baranyi U, Klein R, Lubitz W, Krüger D H, Witte A
Institute of Microbiology and Genetics, University of Vienna, Dr Bohr-Gasse 9, A-1030 Vienna, Austria.
Mol Microbiol. 2000 Mar;35(5):1168-79. doi: 10.1046/j.1365-2958.2000.01786.x.
The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100-800 nucleotides. A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.phiCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of phiCh1. After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the phiCh1 progeny genomes after infection of N. magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3'. Additionally, expression of M.phiCh1-I in dam- E. coli cells led to a complete substitution of the function of M. Dam in DNA mismatch repair.
感染嗜盐碱红菌（原嗜盐碱杆菌）的古菌病毒phiCh1的基因组由58.5千碱基对的线性双链DNA组成。病毒颗粒含有几种大小在100 - 800个核苷酸的RNA种类。通过各种限制性酶切分析确定，phiCh1基因组的一部分在5'-GATC-3'及相关序列内被修饰。高效液相色谱显示，除了四种核苷外，还有第五种碱基，经鉴定为N6-甲基腺嘌呤。遗传分析及后续测序导致鉴定出一个DNA（N6-腺嘌呤）甲基转移酶（甲基转移酶）基因。该蛋白质产物被命名为M.phiCh1-I。通过定位最保守的基序（在FxGxG之前出现的DPPY基序），该酶被归为（N6-腺嘌呤）甲基转移酶类的β亚组。phiCh1的甲基转移酶基因被归类为“晚期”基因，这是通过在phiCh1裂解周期中测量嗜盐碱红菌中mRNA和蛋白质表达的动力学来确定的。细胞感染后，与从溶原细胞诱导病毒的情况相比，能检测到的M.phiCh1-I mRNA和蛋白质的量较少。因此，感染嗜盐碱红菌后，只有约5%的phiCh1子代基因组携带M.phiCh1-I甲基化，而phiCh1溶原性嗜盐碱红菌细胞诱导产生的病毒基因组中有50%携带该甲基化。来自胞质盐浓度为3M的嗜盐菌的甲基转移酶的异源表达显示出一个意外的特征：该蛋白质在大肠杆菌的低环境中具有活性，并且能够在体内使DNA甲基化。有趣的是，它似乎在大肠杆菌中表现出更高的序列特异性，导致仅在5'-GATC-3'序列中发生腺嘌呤甲基化。此外，在dam-大肠杆菌细胞中表达M.phiCh1-I导致DNA错配修复中M. Dam的功能完全被替代。
