Identification of a Gene Encoding a Functional Reverse Transcriptase within a Highly Variable Locus in the P2-Like Coliphages

Abstract

The P2-like coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.Temperate coliphage P2 has three nonessential lysogenic conversion genes, Z/fun, tin, and old, conferring resistance to infections by phage T5, T-even phages, and lambda phages, respectively (1,8,11,12,15,21). These genes have been shown to have a higher AT content than the rest of the genome and a codon usage that differs from that of the host, which suggests that they are horizontally transferred genes (3). P2-like prophages are common in Escherichia coli; about 30% of the ECOR collection (20) contain P2-like prophages. At the region equivalent to the P2 Z/fun gene, i.e., the Z region, which is located between the well-conserved tail genes G and F I , these P2-like prophages have been shown to contain different gene cassettes surrounded by a highly similar inverted repeat, indicating a site-specific integration event. Similar inverted repeats, spacing other genes, can be found in genetically unstable regions in pathogenic enterobacteria (19). The tin and old genes are located at the right end of the P2 genetic map close to the cos site. The old gene encodes an exonuclease that blocks multiplication of lambdoid phages (16), and tin encodes a protein that inhibits T4 DNA synthesis by poisoning the T4 single-stranded binding protein (15). These lysogenic conversion genes have an unusual AT content and codon utilization and are believed to have been additions from foreign genomes. This is supported by the fact that genes for hypothetical proteins similar to Old are found in various bacterial genomes in the UniProt GenBank (Table 1), while the Tin protein so far is unique. Furthermore, P2-related phages found in other enterobacteria contain other genes at this locus (4-6, 14, 17, 18). To clarify the nature of this locus in the P2-like coliphages, we have sequenced and characterized the region equivalent to the P2 tin and old genes, i.e., the TO region, in seven of the P2-like prophages in the ECOR library.Sequence variation between the A genes and the cos sites of the prophages. To sequence the region located between the A genes and the cos sites of the prophages, DNA was extracted from seven strains of the ECOR collection, which are known to contain P2-like prophages (P2-ECnb). A primer located at the catalytic site of the A gene and a primer located to the left of the cos sequence were used for the PCR amplifications, and specific amplified DNA fragments of variable length were obtained. The fragments were either sequenced directly or first cloned and then sequenced. To obtain the complete region, plasmid primers and internal primers were used. All strains contained different D...

Evolution of a Self-Inducible Cytolethal Distending Toxin Type V-Encoding Bacteriophage from Escherichia coli O157:H7 to Shigella sonnei

“…The gene content of the TO region varies extensively among P2-like prophages found in E. coli (49,50). A previous sequence of a prophage harboring the cdt operon indicates the same insertion site of the genes (12).…”

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

“…The Abi system blocks phages at different stages in the infection cycle and is generally mediated by a single plasmid-encoded gene. The AbiA, AbiK, and Abi-P2 systems are known to include a RT [16] – [18] . Mutations of the gene encoding the RT of AbiK have been shown to block infection [16] , and this enzyme has recently been shown to have genuine RT activity [19] .…”

An Unexplored Diversity of Reverse Transcriptases in Bacteria

“…doi:10.1128/microbiolspec.MDNA3-0058-2014.f5 share ∼75% identity. When ORF570 of ECOR30 was expressed on a plasmid in E. coli strain BL21(DE3), the strain was rendered resistant at levels of 10 -7 relative to infection by T5 phage, but there was no effect on infection by lambda, T2, T4, or T6 phages (106). The expressed ORF570 was reported to have RT activity in a biochemical assay, but the assays were with crude extracts under conditions where artifactual incorporation can occur (96).…”