Host Takeover by Bacteriophage T4

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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 work indicated 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. Using purified MotB, 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 investigation of the phylogeny of motB and its predicted domains has revealed that the gene is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB's ability to bind DNA. While previous work has suggested that MotB is an abundant protein, the level of MotB had not been determined. We constructed T4 containing the His-tagged version of the gene to estimate the level of MotB in an infected cell. We found that MotB level during infection is indeed quite high with 40,000 MotB molecules/cell and 50,000 MotB molecules/cell at 5 and 10 min post-infection, respectively. Our pull-down assays have shown that in the presence of DNA, MotB interacts with the E. coli histone-like protein H-NS, a Nucleoid Associated Protein (NAP) known to help organize the bacterial chromosome within the nucleoid. Besides its role in chromosome organization, H-NS also serves to repress transcription of specific host and xenogeneic genes. To understand the connection between MotB and H-NS, we purified both proteins and performed footprinting and atomic force microscopy (AFM) analyses with each protein separately and together. DNase I footprinting demonstrated that MotB dramatically alters the interaction of H-NS with DNA in vitro. Using AFM, we found that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS, in which H-NS coats the DNA to form filaments and bridges. However, the MotB/DNA complexes do resemble complexes formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo yields a significantly compacted nucleoid containing MotB and H-NS. Importantly, this compaction is observed when the level of MotB is similar to that present in a T4-infected cell. Using RNA-seq, we investigated how overexpression of plasmid-borne motB affects host gene expression in E. coli B and in E. coli K12. In the B strain, motB overexpression up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. In E. coli K12, motB overexpression dysregulates hundreds of host genes, and 70% are within the hns regulon. In infected K12, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up 5-fold. Taken together, our results suggest that MotB represents a phage-encoded NAP that interacts with host and T4 DNA and aids infection in a previously unrecognized way. We speculate that early in infection, MotB-induced compaction of host DNA may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon. Later in infection, when T4 DNA is being replicated and MotB is still abundant, we postulate that MotB binding to phage DNA may aid in replication or packaging.