Exclusion of Glucosyl-Hydroxymethylcytosine DNA Containing Bacteriophages Is Overcome by the Injected Protein Inhibitor IPI*

Abstract

The Escherichia coli isolate CT596 excludes infection by the Myoviridae T4 ip1(-) phage that lacks the encapsidated IPI* protein normally injected into the host with the phage DNA. Screening of a CT596 genomic library identified adjacent genes responsible for this exclusion, gmrS (942 bp) and gmrD (708 bp) that are encoded by a cryptic prophage DNA. The two genes are necessary and sufficient to confer upon a host the ability to exclude infection by T4 ip1(-) phage and other glucosyl-hydroxymethylcytosine (glc-HMC) Tevens lacking the ip1 gene, yet allow infection by phages with non-glucoslyated cytosine (C) DNA that lack the ip1 gene. A plasmid expressing the ip1 gene product, IPI*, allows growth of Tevens lacking ip1 on E. coli strains carrying the cloned gmrS/gmrD genes. Members of the Teven family carry a diverse and, in some cases, expanded set of ip1 locus genes. In vivo analysis suggests a family of gmr genes that specifically target sugar-HMC modified DNA have evolved to exclude Teven phages, and these exclusion genes have in turn been countered by a family of injected exclusion inhibitors that likely help determine the host range of different glc-HMC phages.

Molecular architecture of tailed double-stranded DNA phages

“…13,104 One of these proteins (IPI) helps to protect the phage genome from degradation by the host's endonucleases. 107,108 Capsids of T7-like phages contain internal cores assembled on the portal protein. 81 The cores have a central channel for DNA passage.…”

Restriction Endonuclease Inhibitor IPI* of Bacteriophage T4: A Novel Structure for a Dedicated Target

“…The importance of structural integrity to the function of the protein is demonstrated by the loss of inhibitor function of the single-amino-acid replacement ip1 − mutant KAI − (A40T) protein. 2 A closely related IPI* protein containing the mutations A40T and E53K was found to be completely unfolded in solution (data not shown), and as discussed in previous work, the E53K mutation is strongly suspected to be silent. 2 The current work offers an explanation on why the A40T replacement disrupts the IPI* structure.…”

“…2 A closely related IPI* protein containing the mutations A40T and E53K was found to be completely unfolded in solution (data not shown), and as discussed in previous work, the E53K mutation is strongly suspected to be silent. 2 The current work offers an explanation on why the A40T replacement disrupts the IPI* structure. A40, labeled in red in Fig.…”

“…An E. coli CT596 genomic library identified adjacent glucose-modified restriction genes gmrS (942 bp) and gmrD (708 bp) that are necessary and sufficient to confer upon a host the ability to exclude infection by T4 ip1 − and other glucosyl-hydroxymethylcytosine (g-HMC) T-evens lacking the ip1 gene. 2 However, clones allow infection by phages with nonglucosylated cytosine DNA lacking the ip1 gene (e.g., lambda, T7, and RB49). Thus, g-HMC phages C16 and RB69 containing the ip1 gene grow on the clone, whereas g-HMC phages such as T6, T2, RB15, and RB70 (and T4 ip1 − mutants) lacking the ip1 gene do not.…”

“…Moreover a plasmid expressing the mature encapsidated ip1 gene product, IPI*, allows T4 ip1 − and the other phages lacking ip1 to grow on E. coli strains carrying the cloned gmrS/gmrD (glucose-modified hydroxymethylcytosine restriction endonuclease) genes, whereas an isogenic plasmid expressing the mutant ip1 − product, IPI − * (A40T and E53K), does not. 2 Two proteins, GmrS (36 kDa) and GmrD (27 kDa), encoded by the gmrS/gmrD genes were purified and found to be inactive separately, but together degraded several different g-HMC-modified DNAs (T4, T2, and T6) to low-molecular-mass products (b500 bp), whereas no activity was observed against nonmodified DNA, including unmodified T4 cytosine (C) or nonglucosylated T4 HMC DNA. 3 Enzyme activity could be inhibited by IPI*, which binds to GmrSD proteins.…”