Regular article
Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages1

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Abstract

HK620 is a temperate lambdoid bacteriophage that adsorbs to the O-antigen of its host, Escherichia coli H. The genome of a temperature-sensitive clear-plaque mutant consists of 38,297 nucleotides in which we recognize 60 open reading frames (orfs). Eighteen of these lie in a region of the genome that we call the virion structure domain. The other 42 orfs lie in what we call the metabolic domain.

Virions of HK620 resemble those of phage P22. The virion structural orfs encode three kinds of putative proteins relative to the virion proteins of P22: (1) those that are nearly (about 90 %) identical; (2) those that are weakly (about 30 %) identical; and (3) those composed of nearly and weakly identical segments. We hypothesize that these composite proteins form bridges between the virion proteins of the other two kinds.

Three of the putative virion proteins that are only weakly identical to P22 proteins are 71, 60 and 79 % identical to proteins encoded by the phage APSE-1, whose virions also resemble those of P22. Because the hosts of APSE-1 and HK620 have been separated from each other by an estimated 200 My, we propose using the amino acid differences that have accumulated in these proteins to estimate a biological clock for temperate lambdoid phages.

The putative transcriptional regulatory gene circuitry of HK620 seems to resemble that of phage lambda. Integration, on the other hand, resembles that of satellite phage P4 in that the attP sequence lies between the leftward promoter and int rather than downstream of int.

Comparing the metabolic domains of several lambdoid phage genomes reveals seven short conserved sequences roughly defining boundaries of functional modules. We propose that these boundary sequences are foci of genetic recombination that serve to assort the modules and make the metabolic domain highly mosaic genetically.

Introduction

Dhillon et al.1 described HK620, a temperate general transducing phage for the smooth (O-antigen-producing) Escherichia coli strain H. Virions of HK620 resemble the virions of phage P22 whose hosts are derivatives of Salmonella enterica var. typhimurium, a strain that also produces O-antigen. We have completed the genome sequence of HK620 and here we compare it to the genome sequence of P22 (GenBank accession AF217253), the published sequence of APSE-12 and the sequences of several other phages.

HK620, P22 and APSE-1 belong to the formal tailed-phage category Podoviridae because their virions have very short tails.3 They also belong to the informal category known as lambdoid phages. Lambdoid phages do not necessarily produce virions that resemble the virions of phage lambda. Lambda virions have long non-contractile tails; consequently lambda is classified among the Siphoviridae. Originally lambdoid phages were classified as such because they formed recombinant hybrids with phage lambda DNA.4, 5 Thus even prophages that do not produce infective virions, so-called cryptic prophage, can be classified as lambdoid because they form hybrids in which segments of the lambda genome have been replaced by segments of the prophage genome. Examples include the Rac prophage6 and the DLP12 prophage.7 Both P22 and HK620 merit classification as lambdoid phages because they form genetically verified hybrids with lambda.1, 8

Currently the power of genome sequencing requires that we extend the classification “lambdoid” to three other kinds of phages. First, we include phages whose genomes contain DNA sequences that are similar enough to sequences of phage lambda that the sequences might act as foci for genetic exchange. Phages 933W, N15, HK022 and HK97, VT2-Sa and prophage VT2-Sakai fall into this category.9, 10, 11, 12, 13 Second, we include phages whose genes encode amino acid sequences close enough to the sequences of proteins from phage lambda to indicate that the phages share or have shared a gene pool. The five phages and one prophage mentioned earlier would also qualify as lambdoid by this criterion. Thirdly, we include phages with DNA or amino acid sequences close enough to genome or protein sequences from lambdoid phages other than lambda to indicate that they share or have shared a gene pool. Consequently we consider APSE-1 to be a lambdoid phage because its genome contains sequences similar to those of phage P22, which is classified as a lambdoid phage. Shigella flexneri phage Sf614 would also qualify by this criterion15 (A.J.C., W.I., S. Casjens & R. Morona, unpublished results).

In our analysis of the genome of HK620 we focus on two main points which may illuminate the evolution of lambdoid phages. First, we propose a method for estimating a biological clock for lambdoid phage evolution, i.e. the rate at which amino acid substitutions are fixed in a lambdoid phage population. We also make such an estimate for three virion structural proteins. Second, we discern short conserved sequences that form rough boundaries of functional modules of genes in the metabolic domain of lambdoid phages and propose that recombination at these sequences acts to make phage mosaics.5, 16

Section snippets

The HK620 genome

Electrophoretic examination of the products of restriction nuclease digests of HK620 DNA reveals that the size is about 38.1 kb. (data not shown) This is about 3.6 kb smaller than the genome of P22 phage. Another feature of the HK620 genome by which it differs from that of P22 is that there are no under-represented bands in the electropherograms of restriction fragments obtained by singly digesting HK620 DNA with eight different restriction nucleases (data not shown). Such bands in digested P22

Comparison of the HK620, P22 and APSE-1 genomes

The virions of HK620 resemble those of P22 and APSE-1.1 With two exceptions the putative virion structural proteins of HK620 and APSE-1 are similar in number and size to those of P22. The exceptions are the putative gp9 and gp26 homologs of APSE-1. Both of the gene 9 and gene 26 homologs sport deletions relative to the homologs in the other two phage genomes. Gene 26 of APSE-1 seems to have an internal deletion of 113 codons (Table 1) while gene 9 seems to have a C-terminal deletion of about 1

Library construction and sequencing methods

Wild-type phage HK620 was mutagenized as described by Dhillon et al.1 to produce temperature- sensitive phage HK620cts3. Phage lysates were prepared by inducing an E. coli H lysogen of the cts3 mutant (strain 2951) at non-permissive temperature (37°C). After the cell debris was removed by centrifugation, the phage was titered on non-lysogenic E. coli H (strain 2158). HK620 virions and phage DNA were prepared from the supernatant by the procedure and reagents described in the Invitrogen Easy DNA

Acknowledgements

Public Health Services grant AI05371 from the National Institutes of Health supported this work. The authors are especially grateful to A. Poteete for sharing the sequence of the P22 genome prior to its deposition in GenBank and to R. Hendrix and S. Casjens for sharing the sequences of phages HK022, HK97 and N15 prior to publication. They also acknowledge helpful discussions with R. Hendrix and S. Casjens and comments on the manuscript from S. Casjens.

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    Edited by M. Gottesman

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    Present addresses: A. J. Clark, Department of Molecular and Cellular Biology, Life Sciences South, P.O. Box 210106, Tucson, AZ 85721-0106, USA;T. Cloutier, 1939 Oregon St., Berkeley, CA 94703, USA;T. S. Dhillon, 6133 Ascot Drive, Oakland, CA 94611, USA

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