Khan Sharik R, Kuzminov Andrei
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
PLoS One. 2017 Dec 21;12(12):e0190177. doi: 10.1371/journal.pone.0190177. eCollection 2017.
The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF) was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU) caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.
大肠杆菌的类核包含DNA、类核相关蛋白（NAPs）和RNA，但其作用尚不清楚。我们发现，在核糖核酸酶存在的情况下裂解包埋在琼脂糖凝胶块中的细菌细胞会导致染色体DNA大量片段化。这种核糖核酸酶诱导的染色体片段化（RiCF）完全依赖于裂解细胞周围核糖核酸酶的存在，而最大程度的染色体断裂需要快速细胞裂解。在没有核糖核酸酶的凝胶块中进行细胞裂解会使染色体DNA对随后的核糖核酸酶处理产生抗性。RiCF不受DNA超螺旋变化的影响，但受生长温度或培养物年龄的影响。RiCF部分依赖于H-NS，即类组蛋白核结构和全局转录调节蛋白。热不稳定类核蛋白（HU）的hupAB缺失导致自发片段化增加，当与两个非编码RNA（nc1和nc5）的缺失相结合时，自发片段化进一步增加。RiCF完全依赖于核酸内切酶I，这是一种通常被细胞RNA抑制的周质脱氧核糖核酸酶。与RiCF不同，hupAB nc1 nc5四重突变体中的自发片段化对核酸内切酶I的缺失具有抗性。如果在细胞裂解过程中显著降低EDTA，则在不向琼脂糖凝胶块中添加核糖核酸酶的情况下也会观察到类似RiCF的现象。在这种条件下添加核糖核酸酶具有协同作用，会将染色体断裂成太小而无法被脉冲场凝胶保留的片段。不依赖核糖核酸酶的片段化在质量和数量上与RiCF相当，并且部分由核酸内切酶I介导。
