Bulavaitė Aistė, Dalgediene Indre, Michailoviene Vilma, Pleckaityte Milda
Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio al. 7, LT-10257 Vilnius, Lithuania.
Pathogens. 2020 Aug 27;9(9):703. doi: 10.3390/pathogens9090703.
Intensive horizontal gene transfer may generate diversity and heterogeneity within the genus . Restriction-modification (R-M) systems and CRISPR-Cas are the principal defense tools against foreign DNA in bacteria. Nearly half of the tested spp. isolates harbored the CRISPR-Cas system. Several putative R-M systems of spp. strains were identified in the REBASE database. However, there was no experimental evidence for restriction endonuclease (REase) activity in the isolates. We showed that strain ATCC 14018 contains the REase R.Gva14018I, which recognizes GGCC and most probably generates blunt ends on cleavage. Bioinformatics evidence and the activity of recombinant methyltransferase M.Gva14018I in vivo indicate that ATCC 14018 possesses a HaeIII-like R-M system. The truncated R.Gva14018I-4 lacking the C-terminal region was expressed in and displayed wild-type REase specificity. Polyclonal antibodies against R.Gva14018I-4 detected the wild-type REase in the cell lysate of ATCC 14018. The cofactor requirements for activity and bioinformatics analysis indicated that R.Gva14018I belongs to the PD-(D/E)XK family of REases. The REase-like activity was observed in 5 of 31 tested spp. strains, although none of these matched the DNA digestion pattern of R.Gva14018I.
密集的水平基因转移可能在该属内产生多样性和异质性。限制修饰（R-M）系统和CRISPR-Cas是细菌抵御外来DNA的主要防御工具。近一半的受试 属菌株分离株含有CRISPR-Cas系统。在REBASE数据库中鉴定出了几种 属菌株的假定R-M系统。然而，在这些分离株中没有限制内切酶（REase）活性的实验证据。我们发现 菌株ATCC 14018含有REase R.Gva14018I，它识别GGCC，切割时很可能产生平端。生物信息学证据以及重组甲基转移酶M.Gva14018I在体内的活性表明ATCC 14018拥有一个类似HaeIII的R-M系统。缺少C末端区域的截短型R.Gva14018I-4在 中表达，并表现出野生型REase特异性。针对R.Gva14018I-4的多克隆抗体在ATCC 14018的细胞裂解物中检测到了野生型REase。活性的辅因子需求和生物信息学分析表明R.Gva14018I属于REase的PD-(D/E)XK家族。在31个受试 属菌株中的5个中观察到了类似REase的活性，尽管这些都与R.Gva14018I的DNA消化模式不匹配。
