Host Takeover by Bacteriophage T4

功能未知的噬菌体基因包含丰富的生物暗物质。 即使在充分研究的噬菌体T4中，约30%的基因组尚未进行功能表征。 T4早期非必需基因motB就是这样一个基因。 尽管有限的先前工作表明motB可能参与T4基因表达，但我们先前对大肠杆菌中T4 motBam感染或T4 wt感染的RNA-seq分析表明，在大肠杆菌中过量表达MotB表明MotB对噬菌体RNA的表达没有显著影响。 然而，MotB的存在使噬菌体爆发增加2倍，并且MotB的产生对E.杆菌 我们已经发现MotB是一种紧密的DNA结合蛋白，没有可检测的序列特异性，并且对未修饰的宿主和修饰的（葡糖基化、羟甲基化胞嘧啶）T4 DNA具有相似的亲和力。 我们的pull-down实验表明，E.已知抑制异种序列的大肠杆菌组蛋白样蛋白H-NS在DNA存在下与MotB共纯化。 此外，还对E.大肠杆菌基因表达的MotB的存在下，揭示了一个子集的H-NS调节宿主基因的差异表达。 为了研究MotB如何在存在或不存在H-NS的情况下与DNA相互作用，我们进行了原子力显微镜（AFM）。 正如预期的那样，H-NS沿着DNA形成细丝或桥沿着。 相比之下，MotB以一种新的方式压缩DNA，形成蛋白质/DNA“意大利面条怪物”。 为了研究MotB的产生对体内DNA的影响，我们在E. coli或E.含有用mcherry标记的H-NS的大肠杆菌。 我们发现，MotB的存在下的结果在显着的细胞延长和DNA压缩，与AFM观察到的DNA压缩一致。 MotB-GFP和H-NS-mcherry都与压缩的DNA结合。 我们推测MotB的存在可能通过隔离H-NS、延长E.通过压缩宿主DNA以更有效地切割或T4 DNA以更有效地包装，和/或简单地通过在细胞内为噬菌体提供更多的空间。

Phage genes of unknown function comprise an abundance of biological dark matter. Even in the well-studied phage T4, approximately 30% of the genome has not been functionally characterized. The T4 early, nonessential gene, motB, is one such gene. Although limited previous work suggested that motB might be involved in T4 gene expression, our previous RNA-seq analyses of a T4 motBam infection or of a T4 wt infection in E. coli with excess production of MotB indicates that MotB does not significantly affect the expression of phage RNA. However, the presence of motB increases phage burst 2-fold and production of MotB is highly toxic for E. coli. We have found that MotB is a tight DNA binding protein with no detectable sequence specificity and with similar affinities for unmodified host and modified (glucosylated, hydroxymethylated cytosines) T4 DNA. Our pull-down assays indicate that the E. coli histone-like protein H-NS, which is known to repress xenogenic sequences, co-purifies with MotB in the presence of DNA. In addition, RNA-seq analyses of E. coli gene expression in the presence of MotB reveals that a subset of H-NS-regulated host genes are differentially expressed. To investigate how MotB interacts with DNA in the presence or absence of H-NS, we have performed atomic force microscopy (AFM). As expected, H-NS forms filaments or bridges along the DNA. In contrast, MotB compacts the DNA in a novel manner, forming protein/DNA 'spaghetti monsters'. To investigate the effect of MotB production on the DNA in vivo, we heterologously produced MotB or MotB-GFP in E. coli or E. coli containing H-NS tagged with mcherry. We find that the presence of MotB results in significant cell lengthening and DNA compaction, consistent with the DNA compaction observed by AFM. Both MotB-GFP and H-NS-mcherry associate with the compacted DNA. We speculate that the presence of MotB may help T4 infection by sequestering H-NS, by extending the exponential phase of E. coli, by compacting the host DNA for more efficient cleavage or T4 DNA for more efficient packaging, and/or simply by providing more room for phage within the cell.