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.

Figure 1. Exclusion properties of E. coli CT596 clones toward phages with modified and unmodified DNAs

Compared to growth of phages on standard E. coli such as DH10B (with a BAC vector, HS995) (a) and (d), E. coli CT596 excludes growth of most phages except T4 and close relatives with the ip1 gene (b). DH10B BAC transformant B11N containing the gmrS/gmrD genes cloned from CT596 specifically excludes growth of glucosyl HMC DNA phage T4 and close relatives that lack ip1 (c) and (f). However the gmrS/gmrD genes do not affect the growth of phages such as T3, T5 and T7 containing unmodified cytosine DNA (e). Phage growth is assayed by serial hundred fold dilutions from ~10¹⁰/ml.

Figure 2a. A summary diagram of the E. coli CT596 four ORF pCONTIG clone containing the gmrS/gmrD genes

The four ORFs of pCONTIG display homology to ORFs in other organisms, sometimes with fused gmrS/gmrD genes. The pCONTIG Protein 3 and Protein 4 sequences display evidence of a prophage origin. Also shown are putative regulatory elements with the strength of the regulatory elements indicated by symbol size. .

Figure 2b. Nucleotide sequence of the entire 3.6 kb E. coli CT596 clone pCONTIG containing gmrS, gmrD, Protein 3 and Protein 4

ORFs are underlined, with arrows depicting the direction of transcription. The −10 and −35 regions are uncapitalized, the ribosome binding sites are underlined prior to the start of the gmrS and gmrD coding regions. The putative rho termination sequence is in italics. The transposon (Tn10) insertion site resulting in the loss of T4 ip1⁻ exclusion is indicated with the symbol §.

Figure 3. Schematic of E. coli CT596 derived clones, their gene content, and their exclusion properties

Those conferring T4 ip1⁻ exclusion to the cell are underlined.

Figure 4. The amino acid sequences of GmrS and GmrD

Highlighted are regions containing putative functional motifs. In GmrS, the endonuclease-like sequences are in lower case and underlined, and the DUF domain is underlined. In GmrD the putative DNA binding motifs are underlined.