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.

FIG. 1.

Alignments of the DNA sequences at the right end of the variable region. Top panel, the sequence at the end of the A gene. The stop codon of the A gene is indicated by shading. Nucleotides identical to those in P2 are indicated by a dots. Bottom panel, the sequence at the right end, i.e., near cos. The point of sequence divergence at the right end is indicated by shading. Nucleotides identical to those in P2 are indicated by dots. For a map of this part of P2, see top line in Fig. 2.

FIG. 2.

Schematic drawing of the size and possible gene content of the TO region, equivalent to the P2 orf91-tin-old locus (from kb 30.2 to 33.6 in the P2 genome) in P2-like prophages in the ECOR collection. The locations, orientations, and lengths of the open reading frames are indicated by thick arrows. The accession numbers for the TO regions are as follows: P2-EC5, AM157364; P2-EC30, AM157365; P2-EC31, AM157367; P2-EC46, AM157366; P2-EC58, AM157368; and P2-EC67, AM157369.

FIG. 3.

Reverse transcriptase activity as measured by the Quan-T-RT assay system. Fifty microliters of in vitro-coupled transcription and translation (ITT) reaction mixture, containing 3 μg pOTEC30 or 3 μg pCR2.1-TOPO, was used per 200 μl of Quan-T-RT assay mixture. Negative controls were water only and ITT mix with pCR2.1-TOPO. Positive controls were 75 units of recombinant avian myeloblastosis virus (AMV) RT in ITT mix or water. The RT assay mixtures were incubated for 1 hour at 37°C. Results are means ± standard deviations (n = 3).