complete nucleotide sequence and likely recombinatorial ... · complete nucleotide sequence and...

Complete Nucleotide Sequence and LikelyRecombinatorial Origin of Bacteriophage T3

Maria I. Pajunen1*, Michael R. Elizondo2, Mikael Skurnik1

Jan Kieleczawa3 and Ian J. Molineux2

1Department of MedicalBiochemistry and MolecularBiology, Institute ofBiomedicine, University ofTurku, Kiinamyllynkatu 10FIN-20520 Turku, Finland

2Molecular Genetics andMicrobiology, Institute for Celland Molecular BiologyUniversity of Texas, AustinTX 78712-1095, USA

3Department of BiologyBrookhaven NationalLaboratory, Upton, NY11973-5000, USA

We report the complete genome sequence (38,208 bp) of bacteriophage T3and provide a bioinformatic comparative analysis with other completelysequenced members of the T7 group of phages. This comparison suggeststhat T3 has evolved from a recombinant between a T7-like coliphage and ayersiniophage. To assess this, recombination between T7 and the Yersiniaenterocolitica serotype O:3 phage fYeO3-12 was accomplished in vivo;coliphage progeny from this cross were selected that had many biologicalproperties of T3. This represents the first experimentally observed recom-bination between lytic phages whose normal hosts are different bacterialgenera.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: bacteriophage T3; nucleotide sequence; palindromic sequences;recombination; evolution*Corresponding author

Introduction

The T7 group consists of numerous relatedphages that were originally defined by growth onB strains of Escherichia coli. Many similar phagesare known that grow on different strains andspecies of the family Enterobacteriaceae. Membersof the T7 group of coliphages have been dividedinto three subgroups (T7-likes, BA phages, andT3) on the basis of the efficiency of recombinationwith each other.1 Phages within a group recombinereadily but recombination frequencies betweenphages belonging to different groups are low.2

The complete genome sequence of T7 wasreported in 19833 but a second member of the T7group of phages has been similarly described onlyrecently.4 fYeO3-12, a Yersinia enterocolitica sero-type O:3 (YeO3)-specific bacteriophage, appears tobe related to the coliphage T3 much more closelythan to T7. T3 is one of the original type phagesdefined by Demerec & Fano.5

Base sequence homologies between genomes ofthe T7 group were originally inferred from electronmicroscopy of heteroduplex DNAs.6 9 DNAsequencing of parts of the T3 genome10 15 con-firmed and extended these observations, and itwas noted that many regions with very high levelsof similarity to the corresponding parts of T7 wereflanked by regions of almost no similarity.15 Thisobservation suggested that different genomes ofthe T7 group independently acquired geneticmaterial by recombination. The presence ofS-adenosylmethionine hydrolase (SAMase) activityassociated with both T3 gene 0.3 and its counter-parts in Serratia marcescens phage IV and Klebsiellaphage K11,16 at the time a feature unknown inother T7-like coliphages, led to the hypothesisthat T3 is a recombinant between a T7-likecoliphage and a phage whose normal host was notE. coli.15 When this suggestion was made, the

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

Present addresses: M. I. Pajunen, Institute ofBiotechnology, University of Helsinki, P.O. Box 56(Viikinkaari 9), FIN-00014, Helsinki, Finland;J. Kieleczawa, Wyeth/Genetics Institute, Cambridge, MA02140, USA.

E-mail address of the corresponding author:[email protected]

Abbreviations used: ds, double-stranded; gp, geneproduct; LPS, lipopolysaccharide; ORF, open readingframe; RBS, ribosome-binding sequence; RNAP, RNApolymerase; RNase III, ribonuclease III; SAMase,S-adenosylmethionine hydrolase; SRL, left short repeats;SRR, right short repeats; TR, terminal repeat/repetition;YeO3, Yersinia enterocolitica serotype O:3.

doi:10.1016/S0022-2836(02)00384-4 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 319, 11151132

Table 1. T3 Genes and protein identities with fYeO3-12, T7 and other bacteriophages

Gene Froma ToaLength

(aa)Mass(kDa) RBS and initiation codon

Identity (%)bFunction

fYeO3-12 T7 Other

0.3 901 1359 152 17.0 GAGGTaacaccaaAUG 98.7 16.1 S-Adenosyl-L-methioninehydrolase

0.3B 982 1359 125 13.9 GAGGTGaatAUG 99.20.6A 1430 1627 65 7.7 GAGGTattgaaAUG 79.1 23.10.6B 1430 1781 116 13.6 GAGGTattgaaAUG 86.6 18.70.65 1614 1781 55 6.4 AGGAGtattctacaUUG 94.50.7 1796 2905 369 42.3 AGGAcactgaacgAUG 87.3 48.0 Protein kinase1 2976 5630 884 98.8 GAGGTaagcaAUG 99.1 82.1 72.2

(K11);31.7

(SP6)

RNA polymerase

1.05 5717 5989 90 10.4 GAGattaaatttAUG 97.81.1 6082 6222 46 5.9 GAGGTaagatactAUG 97.8 37.51.2 6222 6500 92 10.6 GGAGtggaactgAUG 93.5 37.0 Deoxyguanosine triphospho-

hydrolase inhibitor; growth incells containing the F plasmid

1.3 6595 7635 346 39.4 GAGGaacaaccgtAUG 93.4 67.1 DNA ligase1.5 7714 7791 25 2.8 AGGAGacacacaccAUG 96.0 33.31.6 7804 8061 85 9.8 TAAGGAGacaacatcAUG 98.8 58.11.7 8061 8552 163 18.3 TAAGGAGGTGctgtaAUG 78.5 41.61.8 8539 8670 43 5.1 GGGGGctgtgttAUG 88.9 24.62 8672 8836 54 6.3 TAAGGAGGcttaacGUG 61.5 40.6 Inhibitor of bacterial RNA

polymerase2.5 8888 9586 232 25.9 AAGGAGaaacaatAUG 98.3 82.6 Single-stranded DNA-binding

protein3 9586 10044 152 17.4 GAGGacttctaAUG 92.8 78.3 39.5

(SIO1)Endonuclease

3.5 10037 10492 151 16.9 AAGGAGtaaagaaaaAUG 96.7 94.7 Amidase (lysozyme; inhibitsT3 RNAP)

3.7 10497 10604 35 4.2 GAGGgtagacctAUG 97.1 Derived from group I intron?4A 10670 12370 566 62.7 AAGGAatgtacaAUG 95.6 80.8 32.5

(SIO1);23.2

(SP6)

DNA primase/helicase

4B 10856 12370 504 55.7 AGGAGGcagcaagcctAUG 99.2 84.5 Primase4.2 12090 12419 109 11.9 AAGGGGaaagcgcAUG 83.5 37.54.3 12466 12678 70 7.7 AGGAGacacatcAUG 97.1 42.94.5 12691 12975 94 10.7 AGGAGcacaacAUG 96.8 53.25 13043 15147 704 80.0 AAGGAGGgcattAUG 87.4 96.7 27.3

(SIO1)DNA polymerase

5.1 13686 13775 29 3.2 GAGGccgttgAUG5B 14039 15157 372 41.6 GGAGcacgtaGUG 87.85.3 15168 15473 101 11.9 GGAGGTattAUG 83.6 17.7 Derived from group I intron?5.5 15487 15783 98 10.9 AAGGAGaaattattAUG 52.5 49.0 Growth on lambda lysogens?5.7 15783 15992 69 7.4 GGAGGTGttctgAUG 88.4 98.65.9 15992 16150 52 6.1 GGAGGTtgtgtctaAUG 30.0 98.1 Inhibits host recBCD nuclease6 16137 17045 302 34.6 AGGAGagaaacttaAUG 79.2 92.1 Exonuclease6.3 17027 17140 37 4.1 AAGGAGatctacttAUG 97.3 25.06.5 17234 17479 81 9.3 GAGGTGAaattAUG 98.8 54.86.7 17484 17735 83 8.8 AGGAGtaattatAUG 98.8 52.1 Adsorption7.3 17762 18082 106 10.8 AGGGGacacattAUG 95.3 57.4 Host range8 18093 19700 535 58.6 AGGAGGactgaAUG 99.1 84.4 Headtail connector9 19802 20734 310 33.7 AGGAGatttaataAUG 94.8 61.9 Scaffolding protein10A 20891 21934 347 36.9 TAAGGAGattcaacAUG 98.8 79.1 Major capsid protein10B 20891 22190 433 45.4 TAAGGAGattcaacAUG 98.2 66.7 Minor capsid protein11 22425 23015 196 22.2 AGGAGGTaacatcAUG 99.0 80.1 Tail tubular protein A12 23031 25436 801 89.9 TAAGGAGGctctAUG 97.4 68.2 Tail tubular protein B13 25521 25931 136 15.8 GGGGGTtaaagcattAUG 92.0 52.5 Internal virion protein A14 25934 26527 197 21.2 AGGAGGTaactAUG 98.5 68.5 Internal virion protein B15 26530 28776 748 84.8 GGAGGTaataAUG 68.8 95.3 Internal virion protein C16 28803 32759 1318 144.0 TAAGGAGGccctaaAUG 66.5 97.4 Internal virion protein D17 32832 34508 558 62.0 TAAGGAGGTcaaAUG 34.8 86.7 Tail fiber protein17.5 34567 34770 67 7.4 TAAGGAGGacacAUG 89.6 97.0 Holin18 34775 35044 89 10.0 TAAGGAGtaactctAUG 71.9 92.1 DNA packaging protein; small

subunit18.5 35138 35581 147 16.9 GGAGGcattAUG 52.6 91.2 Endopeptidase; lambda Rz

homologue18.7 35253 35504 83 9.4 AGGAGGTacacaAUG 46.4 94.0 Lambda Rz1 homologue

(continued)

1116 Bacteriophage T3 Genome

Y. enterocolitica phage fYeO3-12 had not beendescribed. One problem with this idea is that inmixed infections T7 and T3 exhibit mutualexclusion, in part likely reflecting differentpromoter specificities of the phage-encoded RNApolymerase (RNAP).1 Mutual exclusion is thuslikely to extend to phages infecting differentbacterial genera.

In this study, we completed the 38,208 bp nucleo-tide sequence of T3, identified the positions ofindividual genes and genetic signals, and madegenome-wide comparisons with related bacterio-phages, especially T7 and fYeO3-12. We havefocused our discussion on aspects where T3 ismost similar to or most different from the better-characterized phage T7. These comparisons showthat the closest known relative of T3 is theY. enterocolitica phage fYeO3-12. It is likely thatthe specific phage isolated by Demerec & Fano5 asT3 is the product of recombination between a T7-like coliphage and a fYeO3-12-like yersiniophage.We show that T7 indeed recombines with fYeO3-12 in vivo, generating recombinant phages thathave many of the biological properties of T3. Thisis the first experimentally controlled recombinationbetween two obligate lytic phages whose normalhosts come from different bacterial genera. Thedata provide strong experimental support for theidea, arising largely from theoretical bioinformaticanalyses of a large number of phage genomes, thatthose of the T7 group of phages are also mosaicstaken from a natural gene pool.

Results and Discussion

Nucleotide sequence determination of thephage T3 genome

Eight T3 sequences present in GenBank could beassembled into four contigs ranging from 0.4 to19.7 kb. We used the largest contig as an anchorsequence and determined the remainder of thegenome as outlined in Materials and Methods. Anumber of errors in older Genbank entries(especially M14784) have been corrected; mostnotably these corrections change the predictedsequence of gp17.5 (holin) by removing a positive

charge in the middle of a putative transmembranesegment. This change makes T3 gp17.5 conformmuch more closely to the class II holin family.17

Two frame-shift mutations in the published gp17(tail fiber) sequence have been corrected; T3 gp17is now more similar to T7 gp17 than previouslyappreciated (Table 1). Finally, the terminal repeat(TR) of T3 contains an extra C when compared toGenbank M14784. Both copies of TR weresequenced as part of the current study. The DNAsequence in M14784 was obtained using a T3amber mutant that had been isolated aftermutagenesis,18 and the mutational history of thatphage likely accounts for many of the sequencedifferences with T3. Except for the majority ofgene 1, the 38,208 bp of the T3 genome is basedon DNA of a single isolate of T3, the Luria strain(accession no. AJ318471). The T3 Luria strain prob-ably represents the original T3 isolate and wasused by Davis & Hyman in their heteroduplexanalysis.6 Nucleotides 1 to 3621, and 21776 to thegenetic right end of the T3 genome were deter-mined in this study. The size of the genome,38,208 bp, is close to the 38,740 bp estimated byBailey et al.19

Overall organization of the genome

Many T3 genes were identified in previousstudies. Here, we analyzed the sequence data,updated the positions of previously identifiedgenes and made corrections where needed. Weidentified probable protein-encoding sequencesusing a combination of computer analysis andvisual inspection. Altogether, the sequencerevealed 51 genes having good coding potentialand a start codon in appropriate juxtaposition to aplausible ShineDalgarno ribosome-bindingsequence (RBS) (Table 1). Proof that all predictedopen reading frames (ORFs) are expressed requiresmore detailed study. All but five T3 genes initiatewith an AUG codon, the remainder use GUG(four times) or, potentially, UUG (once).

The overall organization of the T3 genome isvery similar to that of T7 and fYeO3-12 (Figures 1and 2). For clarity, genes are presented inthree reading frames corresponding to the sense

Table 1 Continued

Gene Froma ToaLength

(aa)Mass(kDa) RBS and initiation codon

Identity (%)b Function

fYeO3-12 T7 Other

19 35591 37351 586 66.7 TAAGGAGGcaacGUG 97.3 85.7 DNA packaging protein; largesubunit

19.2 36237 36677 146 16.0 AAGGAactcgaagataaccGUG 43.2 28.119.3 36774 36947 57 6.6 GGGTtcccggAUG 70.2 61.419.5 37595 37744 49 5.5 AAGGAGGTGgctcAUG 98.0 63.3

a Nucleotide coordinates corresponding to the first nucleotide of the initiation codon and the last nucleotide of the terminationcodon.

b Pairwise identities calculated using GeneStream (IGH) program.

Bacteriophage T3 Genome 1117

Figure 1. Genomic map of bacteriophage T3. The upper part of the figure shows E. coli and T3 promoters, and the genes in the three forward (left to right) reading frames.Promoters are represented by arrows indicating the orientation of transcription; the major E. coli promoters: A1, A2, and A3 are identified, as are conserved minor promotersA0 (A0-L, A0-R), B and C. Except for fOL and fOR, T3 promoters are identified by a f followed by the number of the gene first transcribed; f17 is in parentheses as it is aconsensus T7 late promoter and is not recognized during infection.96 Genes are represented by open boxes. TR represents the direct terminal repeats of 231 bp, Ori is theprimary origin of replication. TE and Tf are transcription termination sites for E. coli and T3 RNA polymerases, respectively. The lower part of the Figure shows a diagramof T3 RNAs where each transcript is indicated by a horizontal line; thicker lines represent RNAs of known or predicted greater abundance, horizontal broken lines indicateknown transcriptional readthrough of terminators. Vertical broken lines indicate predicted sites of RNase III recognition and are named by the first gene following the siteof cleavage; sites where cleavage may be inefficient are in parentheses.

(non-transcribed) strand of the genome. No pre-dicted ORFs or regulatory elements were foundon the complementary strand. The nomenclatureused to describe T7 and fYeO3-12 genes and theirregulatory elements has been applied to the T3sequence. Despite some conflicts due to theabsence of genes 0.5, 3.8 and 4.7, the regulatoryelements at the same relative positions in T3 arenamed as their T7 counterparts. The RNase III rec-ognition site upstream of gene 0.6 is thus calledR0.5 (rather than R0.6 ); similarly, the promoter

and possible RNase III processing site upstream ofgene 4 are called f3.8 and R3.8, respectively, andthe RNase III recognition site upstream of gene 5is called R4.7. In these instances, T3 is consideredto be equivalent to a deletion mutant of T7.

Close-packing of T3 genes is comparable to thatof fYeO3-12 and even more extensive than foundin T7. In all but five instances, the terminationcodon overlaps coding sequences or the RBS ofthe downstream gene, or a regulatory geneticelement (e.g. promoter, terminator, RNase III

Figure 2. Pairwise nucleotide similarity between T3, T7 and fYeO3-12. Pairwise nucleotide alignments of T3 versusfYeO3-12 (upper plot), and T3 versus T7 (lower plot) were done using GAP with gap creation penalty of 20 and gapextension penalty of 1. Gapped sequences were then edited manually to exclude regions of non-homology before com-puting levels of similarity. Finally, using a window of 300 bp, the program PLOTSIMILARITY was used to plot thedegree of identity versus nucleotide number. Insertions/deletions (arrows) are indicated by genes in parentheses.


recognition site). There are 19 bp between the gene6.3 termination codon and f6.5, 27 bp separategenes 6.7 and 7.3, 15 bp separate genes 11 and 12,26 bp separate genes 15 and 16, and 58 bp separategenes 17 and 17.5. One notable non-coding gapseparates f13-R13 from gene 13; the 41 bp contains15 consecutive G residues, the last of which mustserve also as the ShineDalgarno sequence forgene 13. No comparable homopolymeric stretchhas been found in T7 or fYeO3-12, or in any othervirus sequence in the databases.

Close-packing is even maintained across regionsof complete non-homology between differentphages e.g. gene 7.7 appears to have been deletedprecisely from T3 (or gene 7.7 was inserted pre-cisely into T7), as the gap between T3 genes 7.3and 8 is similar to that between T7 genes 7.3 and7.7, and there is no spacing between genes 7.7 and8. T7 genes 0.4 and 0.5 appear to be deleted pre-cisely from T3, the TAA termination codon of T30.3 overlaps the 50 end of R0.5, whereas the T7 0.3termination codon overlaps the ATG of 0.4 andthe termination codon of 0.4 is part of R0.5. Thegap between T3 R0.5 and gene 0.6 is similar tothat between T7 R0.5 and gene 0.5, and the latteroverlaps 0.6. Furthermore, in fYeO3-12, gene 13.5has been inserted precisely between genes 13 and14, since there is no spacing between the lattergenes in T7 and T3. A more extensive discussionof those genes not found in all of fYeO3-12, T7,and the previously known parts of T3 was pre-sented recently.4 Completion of the T3 genomeshows that no gene is unique to T3, only fYeO3-12 contains genes 0.45 and 13.5, and only T7 con-tains genes 0.4 and 0.5.

Figure 2 shows most clearly the high degree ofsimilarity between T3 and both T7 and fYeO3-12.The T3 genome is much less similar to thecoliphage T7 than to the yersiniophage fYeO3-12.After removing insertions/deletions, aligning theentire genome sequences gives an overall level ofidentity between T3 and T7 of about 74%, whereasthat between T3 and fYeO3-12 is about 84%. Thelongest stretch of identity between the T3 and T7genomes is 177 nucleotides, and there are onlyfive regions containing greater than 100 identicalnucleotides. In contrast, a 316 identical nucleotidestretch, an additional three greater than 200 nucleo-tides, and a further 32 greater than 100 identicalnucleotides exist between the T3 and fYeO3-12genomes. The only major parts of the T3 genomethat are distinctly more similar to T7 than tofYeO3-12 lie between codon 60 of gene 5 throughthe codon 180 of gene 6 (excepting gene 5.3, seebelow), and from codon 50 of gene 15 throughcodon 75 of gene 19 (positions 1322016680 and2668035820, respectively).

The average G C composition of the T3genome is 49.9%, and that of individual genesvaries within the range of 4456%. As might beexpected, the G C content of genes that differsmost from the whole genome are non-essential forroutine phage growth in the laboratory. However,

genes 10 and 13 are exceptions in T3, in T7 and infYeO3-12. The G C contents of T3 genes 10 and13 are 55.1% and 43.6%, respectively, those offYeO3-12 are 55.8% and 43.5%, and those of T7are 53.1% and 45.8%. Both proteins are part of thephage virion but, whereas gp10, as the major cap-sid protein, is made in very large amounts, gp13 isa minor protein with no defined biochemical func-tion. It is not obvious why these particular genesshould have a G C content so very differentfrom the remainder of the genome. A linear G Ccomposition scan, using a window of 85 nucleo-tides, reveals several other areas in the T3 genomewhere the G C content falls between 30 and40%. These areas are predominantly intergenic,and include promoter and RNase III recognitionsites, where a high A T content might have arole in gene regulation. T7 and fYeO3-12 DNAsalso show a high A T content in the equivalentregions of their genome.

The G C composition of the majority of T3genes is closer to that of fYeO3-12 than to T7genes (data not shown). Two exceptional regionscontain genes 5 through 6 (though not 5.5 ) andgenes 14 through 19.5 (though not 19 ). Theseregions only approximate those where the DNAsequence similarity between T3 and T7 is higherthan T3 and fYeO3-12, suggesting that T3 hasenjoyed more than one recombination event ineach region. The G C composition of T3 genes 2and 3.5 are closer to their T7 than to theirfYeO3-12 counterparts. In general, this trendfollows the percentage identity of the geneproducts (Table 1), although genes 2 and 5.3 areexceptions.

It was noted previously that most of T3 gene 5.3was more similar to part of T7 gene 7.7 than to5.3,15 and both T3 gene 5.3 and T7 gene 7.7 weresuggested to be derived from homing endo-nucleases (group I introns).20,21 It was proposedthat T7 gene 5.3 encodes an endonuclease, becauseits resistance to overexpression in E. coli suggestedthat gp5.3 is toxic;22 the protein has recently beenshown to be related to a cyanobacterial intronendonuclease.23 T3 gene 5.3 is remarkable in that,although its G C content is almost the same asthat of T7 gene 5.3 (42.6% versus 42.7%) and sub-stantially different from that of fYeO3-12 gene 5.3(50.3%), the T3 and fYeO3-12 genes are 72.7%identical, whereas the T3 and T7 genes share only47.4% identity. T3 gp5.3 is also 83.6% identicalwith fYeO3-12 but only 17.7% identical with T7gp5.3. The different G C contents of T3 and T7gene 5.3, with respect to most of their other genes,suggests that it was acquired by each phagerelatively recently; the lack of sequence similaritybetween T7 and T3 gene 5.3 further suggests thatthe donor of the gene to each phage was different.Conversely, the different G C contents but highsequence similarity between T3 and fYeO3-12gene 5.3 suggests that they originated from thesame source but were acquired by the progenitorsof each phage at very different times.


T7 group phages contain putative homingendonucleases at several genomic locations

Relative to T3, T7 has acquired genes 0.4, 0.5, 1.4,2.8, 4.7, 7, and 7.7, and lacks only T3 gene 1.05 inequivalent genomic regions. Again, relative to T3,fYeO3-12 has likewise gained genes 0.45, 1.45,and 13.5. Several gene products in these divergentregions are thought to be related to group I intronsor homing endonucleases.20,21,23,24 fYeO3-12 gp1.45and gp13.5 exhibit sequence similarity toendonucleases.4

Homing endonucleases are thought to be mobileelements that often insert into self-splicingelements, making the composite element mobile.25

In this way, a homing endonuclease can insert any-where in the recipient DNA, as the function of thetargeted gene is not affected. However, in the T7group of phages, putative HNH endonucleasesand other inserted genes seem to be confined tointergenic locations and a self-splicing elementwould not be necessary, and there may be a limitednumber of sites on the genome where integrationof the new genetic material can be tolerated. Onthe basis of conserved amino acid sequences,homing endonucleases have been grouped intofour families, LAGLIDADG, GIY-YIG, His-Cysbox and HNH. Structural similarities prompted anewer re-classification of His-Cys box and HNHendonucleases into a single family called the bba-Me family,26 including homing endonucleases andnon-specific colicigenic nucleases, and the Serratiamarcescens extracellular nuclease. Furthermore, byvirtue of a conserved fold comprising its activesite, the DNA junction-resolving phage T4 endo-nuclease VII is considered as a member of this

family.27 Aligning those T7, T3, and fYeO3-12proteins that include the HNH endonucleaseconsensus motif reveals several conserved residueswith the bba-Me family of endonucleases(Figure 3).

Between about 34 kb and 41.5 kb DNA can bepackaged efficiently into a T7 capsid. However,only about 32 kb is accounted for by essential andconditionally essential genes plus all known regu-latory elements. Thus at least 2 kb of DNA mayneed to be added to a basic T7-like genome forefficient packaging. The three T7-like genomessequenced to date all code for proteins that appearto be related to homing endonucleases, some ofthe required extra DNA may thus be derived fromparasitic DNA elements that have found theirecological niche in a larger parasite.

Within the size constraints of a genome that canbe packaged efficiently, acquisition of theseelements by a T7-like phage could occur byhomologous recombination with another memberof the family. However, the question remains ofthe original sources of these genes, which are pre-sumably carried on a bacterial chromosome orplasmid and were first acquired by a phage afterinfection. Transposition of a mobile element ontothe phage genome is one potential mechanismbut, as the T7 group of phages degrade hostchromosomal DNA efficiently, there is little oppor-tunity for this process. However, group II intronmobility may circumvent this difficulty, as RNA isreverse spliced into the recipient DNA duplex.Although T7-like phages shut off host transcriptionthey are not known to enhance RNA degradation,and phage mRNAs appear to be stable throughoutthe latent period. Thus, retrohoming processes

Figure 3. Multiple alignment of the putative homing endonucleases of T3, T7 and fYeO3-12 that contain the HNHendonuclease motif; the consensus sequence is from the Pfam Protein families database. Identical residues are shadedwith black and similar residues are shaded with grey with a threshold value of 0.5. Gaps (indicated by horizontal lines)were introduced into the sequences to maximize the alignments. Numbering is from the N-terminal methionineresidue.


may proceed unhindered by phage infectionprovided that the phage genome contains appro-priate target sites. The observation that newlyacquired elements appear to be inserted preciselybetween existing phage genes may simply be anatural result of selection for sites that confer nodeleterious effect on phage fitness. If one assumesthat all the basic T7 set of genes have somebeneficial function in phage growth, then an inter-genic location for new elements would be the leastdisruptive, although other potential sites of inser-tion cannot be dismissed arbitrarily. The newlyacquired parasitic element itself may not providean advantage to the phage, and the element coulditself then become a target for subsequent inser-tions. Perhaps consistent with this idea is the factthat the N-terminal region of T3 gp5.3 shares simi-larity with T7 gp1.7, whereas the majority of theprotein appears more related to T7 gp7.7.15

RNA metabolism

The three major early promoters (A1, A2 and A3)for E. coli RNAP in the non-coding region near theleft end of T3 DNA were first identified by Brownet al.28 Scanning the complete genome sequencefor E. coli s70 like promoters revealed counterpartsto the minor B and C promoters of T7 and the left-ward promoter A0 (Figure 1). T7 A0 (also calledthe D promoter) lies adjacent to the left terminalrepetition (TR) in a unique sequence of DNA.However, T3 A0 lies in the TR and therefore twocopies are present; they have been named A0-Land A0-R, to indicate the left and right genomeend. fYeO3-12 A0 also lies in the TR.4 No functionof A0 in T7 development is known and it can bedeleted without obvious effect. Deletions of T3 A0have not been sought but such deletions mightreduce the terminal repetition below the lengthnecessary to support efficient phage growth.Several other E. coli promoter-like sequences wereidentified within the T3 genome and similar poten-tial sequences are found in T7 and fYeO3-12. Theirlocations are not conserved among the three phagegenomes. Thus, it is unlikely that they are import-ant in phage development.

The consensus promoter sequence for T3 RNAPhas been well documented.29 There are 15 phagepromoters on the T3 genome, although only 14have T3 RNAP specificity. The complete genomesequence now confirms that the promoter at 64.8map units30 is f13. All phage promoters in T3 andfYeO3-12 lie exactly at the equivalent positionsand the sequences of ten promoters are perfectlyconserved between the two genomes. Only f17,which is T7-specific in T3 but fYeO3-12 (T3)-specific in fYeO3-12, varies at more than a singleposition. There is less promoter conservationbetween T3 and T7; in addition to the difference atf17, several class II promoters are present in onlyone or the other phage genome. No satisfactoryhypothesis has been offered to explain why T7 con-

tains ten promoters in the class II region (T3 has 7),perhaps they are truly redundant for function.

The T3 and fYeO3-12 gene 10 leader RNAs areidentical; with 142 nucleotides they are muchlonger that their 62 nucleotide T7 counterpart. Sec-ondary structure predictions of each leader RNAshow that the 35 to 40 bases 50 to the initiationcodon are relatively unstructured and, apart fromthe ShineDalgarno sequence, are markedlydeficient in G residues. The T3 and fYeO3-12 gene10 leader RNAs also contain two changes in thenine base translational enhancer element 1.31 It isunclear how this element functions32,33 but as theefficiency of gp10 (major capsid protein) synthesisis likely to be comparable in all three phages, 1may not have any independent activity in its natu-ral context of gene 10. In line with this idea, thefYeO3-12 leader RNA was shown to inhibit T3RNAP-driven transcriptiontranslation of a luci-ferase reporter gene in vitro, removing the leadersequence between f10 and the ShineDalgarnosequence restored activity (M.S., unpublishedresults). Translational control sequences mayindeed be present in the leader RNAs but theymay be designed specifically to function with agene 10 message.

The programs Terminator and Stemloop in theGCG suite of programs (Genetics ComputerGroup, Madison, WI) identified the early T3 tran-scriptional terminator TE at positions 7646 to 7667(DG 215:7 kcal=mol 1 cal 4:184 J) for E. coliRNAP, and the late transcriptional terminator Tfat positions 22352 to 22390 DG 223:2 kcal=mol(Figure 1), and a potential terminator at positions32766 to 32804 DG 218:6 kcal=mol that over-laps the T7-specific f17 promoter. This stem-loopis not responsible for the lack of activity of f17during T3 infection because comparable structuresare present at both T7 and fYeO3-12 f17.

The host enzyme RNase III cleaves T7 primarytranscripts. Processing of T3 early RNAs by RNaseIII has been demonstrated both in vivo and invitro.34 Like T7, T3 plates at normal efficiency butgives somewhat smaller plaques on cells lackingRNase III (I.J.M., unpublished results). The Stem-loop program revealed ten putative RNase III sitesin the T3 genome, all at positions comparable tothose in T7 and fYeO3-12 (Figure 1). T3 R0.3 andR4.7 are identical with, and only R0.5 and R18.5differ substantially in sequence from, their fYeO3-12 counterparts (Figure 4). As the genes followingT3 R0.5 and fYeO3-12 R0.45 are different, sequencedivergence at this site is to be expected. Interest-ingly, fYeO3-12 R0.45 shows greater similarity toT7 R0.5, although there is no obvious similaritybetween fYeO3-12 gene 0.45 and T7 gene 0.5. Theonly example where a T3 RNase III site is moresimilar to its T7 than its fYeO3-12 counterpart isR18.5 but even in this case a number of mismatchesare present. However, the predicted duplex regionsof each equivalent T3, T7, and fYeO3-12 RNase IIIrecognition structure are almost completely main-tained and most sequence differences are confined


Figure 4. Predicted RNase III recognition sites of bacteriophages T7, T3, and fYeO3-12. Underlined bases indicate bulge loops in the otherwise duplex stem structure, bold-face indicates a mismatch with the T3 sequence, and downward arrows indicate known cleavage sites for T7 transcripts.

to the bulge and upper loops (Figure 4). Otherputative stem-loops/terminator structures foundby the computer algorithms are not conservedbetween the T3, T7 and fYeO3-12 genomes andany biological role for them seems unlikely.

Terminal repeats and neighboring conservedputative regulatory signals

The direct TRs of the T7 group of phages aredifferent in both sequence and length. There are231 bp in T3 TR, 232 bp in fYeO3-12, 181 bp inKlebsiella phage K11,35 and 160 bp in T7. Thesequences of the T3 and fYeO3-12 TRs are 85.7%identical, but there is much less conservation withthe T7 and K11 TRs. Alignments of all four TRsreveal high levels of similarity only over the first30 bp and the last 16 bp of the repetitions, whichcorrespond to the extreme left and right ends ofthe genome, respectively. Within the T3 TR liethree direct repeats of the heptamer CCTAAAG,plus a further ten one-base or two-base variants.fYeO3-12 TR contains four exact and eightimperfect copies, T7 contains three exact and fourimperfect copies, and K11 contains five imperfectcopies only. However, most copies of CCTAAAGin T7 lie adjacent to both copies of TR in regionsreferred to as SRL and SRR.3 Both regions containtwo sets of six imperfect but regularly spacedcopies of CCTAAAG; in SRL, the T7 A0 promoterseparates each set. Interestingly, in the left end TRthe only regular spacing of the heptamer repeat inT3 and fYeO3-12 is found to the right of A0 butthe lowest levels of similarity between the T3 TRand those of T7 and K11 (which also lacks A0 inits TR) extend rightward from the A0 transcriptionstart site. Relative to T7, perhaps T3 and fYeO3-12suffered a deletion that fused TR and A0, or per-haps an element containing A0 and the heptamerrepeat inserted at different positions in ancestralphages. The repeats are not as well conserved inT7 SRR as in SRL3 but the reverse is true in T3,where 13 imperfect heptameric repeats, beginningat position 37812, likely correspond to SRR. Anybiological function of these repeats is unknown;T7 SRL has been deleted entirely,36 and a deletionremoving nine of the 12 heptamers in T7 SRR isnon-lethal.37,38 However, no attempt has beenmade to combine the two deletions.

Immediately 50 of T7 SRR lies a potentialimperfect cruciform that may be important induplicating the TR during DNA maturation andencapsidation, and in forming the mature left endof the genome.39 Comparable structures (T3, Nos.3781437774; fYeO3-12, Nos. 3917939139) couldform during T3 and fYeO3-12 infections, althoughboth are predicted to be much less stable thantheir T7 homologue.

The mature right end of T7 is created by cuttingconcatemeric DNA at the right end of the TR.40

Recognition sequences for the packaging enzymescoincide with those required for gp3.5-dependenttranscription termination at the concatamer

junction, which is an important determinant inDNA packaging.41,42 Sequences at the 30 end of TRare well conserved between T7, T3 and fYeO3-12,as are the first seven bases of unique sequenceDNA (Nos. 232238 in T3). The mechanisms ofboth DNA packaging and transcription termin-ation at this site are thus likely conserved, althoughthe 30 ends of the T7, T3, and fYeO3-12 transcriptsare predicted to be different.

Avoidance of palindromic sequences

The paucity of sites for commonly used type IIrestriction enzymes in the T7 and T3 genomes haslong been appreciated,43,44 and fYeO3-12 alsolacks sites for many restriction enzymes.4 gp0.3binds and inactivates type I restriction enzymes45

but does not protect against type II or type IIIenzymes. Unlike T7 gene 0.3, that of T346 andfYeO3-124 encodes SAMase, which should makethese two phages particularly sensitive to restric-tion. It was hypothesized that the T7 group ofphages evolved to avoid sites for restrictionenzymes, at least for enzymes recognizing palin-dromic sequences47 but at the time only the T7 gen-ome sequence was available.

Phage genomes containing about 40 kb ofdouble-stranded DNA are expected to have about20 copies of any hexameric nucleotide sequence.Of the 4096 possible hexamers, T3 lacks 51, T7lacks 57 and fYeO3-12 lacks 103; 26 hexamers arenot present in any of the three genomes. Palin-dromic sequences represent only 1/64 of the totalnumber of hexamers; thus, on statistical grounds,only one or two would be expected to be absentfrom each phage genome. However, 21 palin-dromes are missing from the T3 genome, 19 fromT7, and 29 from fYeO3-12. Of the missing 26hexamers, 14 are palindromic and correspond toknown restriction enzyme specificities. However,nine of the 26 are not expected to be common, asthey contain the sequence GATC, which is stronglyselected against in these genomes (see below).Nevertheless, a strong bias against all palindromichexamers clearly exists in genomes of the T7group of phages.

Sixteen of the 256 possible tetranucleotides arepalindromic, but palindromes represent eight ofthe 15 least common tetranucleotide sites in boththe T3 and T7 genomes, and seven of the 15 infYeO3-12. The sequence GATC is the least frequenttetranucleotide, being found at ten, six, and threesites, respectively, in T3, T7, and fYeO3-12 DNAs.Although several restriction enzymes are knownthat recognize GATC, it is not necessarily avoid-ance of restriction that led to this sequence beingso rare in genomes of the T7 group. The sequenceGATC is also a major regulatory element in theEnterobacteriaceae, Dam methylation of GATCaffects the initiation of DNA replication and theexpression of certain genes, and GATC is the recog-nition element for strand selectivity during post-replication mismatch repair. Nevertheless, the bias


against most palindromic tetranucleotides in thesegenomes suggests strongly that they have beenselected against during phage evolution.

A random pentamer and its complementarysequence would be expected 75 to 80 times in theT3, T7, or fYeO3-12 genomes. Ignoring thesequences NGATC and GATCN, the most infre-quent pentamers have the form CCWGG, which ispresent only three times in T3, twice in T7 and notat all in fYeO3-12. CCWGG is the recognition sitefor Eco RII and its isoschizomers and for E. coliDNA-cytosine methylase. The counterpart palin-dromic sequence CCSGG, the recognition site forNci I and its isoschizomers, is present at 54 sites inT3, 83 sites in fYeO3-12, but at only nine sites inthe T7 genome. CCSGG is thus strongly selectedagainst in T7, but not in T3 or the yersiniophagefYeO3-12. Among other significantly under-represented palindromic pentamers in T3 andfYeO3-12 is GGNCC, which is present, at 46 and29 sites, respectively. However, this sequence ispresent 79 times in the T7 genome and is thus notselected against. Considering all pentamers presentat 50 sites or less in the T3, T7, and fYeO3-12genomes, nine of 98, 96, and 88 pentamers,respectively, all about one-tenth of the total, arepalindromic. As one-sixteenth of all pentamershave a palindromic form, selection against themhas been weaker than for palindromic tetra- andhexanucleotides. Nevertheless, the T7 group ofphages appear to be under selective pressure tominimize the frequency of palindromic restrictionenzyme sites in their genomes. As the phage pro-tein gp0.3 is active only against type I restrictionenzymes, avoidance of the palindromic sites usedby many type II enzymes provides an obviousbenefit to phage growth and spread in theenvironment.

Proteins

The similarities between T3 proteins and those ofother bacteriophages were determined by databasesearches. All known functions and previously pro-posed putative functions, based on similarities orequivalent genome positions, are summarized inTable 1. Proof that all predicted ORFs areexpressed requires more biochemical studies.

T3 gene 0.3 encodes two proteins (gp0.3 andgp0.3B) by virtue of an internal in-frame initiationat amino acid residue 27.12 Any difference in theactivity between the two gene 0.3 products isunknown; both are thought to bind to S-adenosyl-methionine and to type I restriction enzymes.fYeO3-12 gene 0.3 could direct a similar internalin-frame initiation but T7 gene 0.3 has littlesequence similarity to its T3 and fYeO3-12counterparts.

T3 gene 0.6 contains two ORFs, which are foundalso in T7 and fYeO3-12. It was initially proposedthat T7 made two independent proteins, gp0.6 andgp0.65, both initiated at an AUG codon but with apoor ribosome-binding sequence for gene 0.65.48

In order to accommodate experimental data, itwas later suggested that a 1 frameshift duringtranslation of gene 0.6A could provide a gp0.6Bproduct whose C-terminal sequences were thoseof the previously defined gp0.65.3 It is not clearwhat happens in T3 or in fYeO3-12. In both gen-omes, the putative gene 0.65 would be initiated ata UUG codon that is preceded by a good ShineDalgarno sequence (Table 1). The nucleotidesequences thought to contain the 1 frame-shiftingsignal in T7 gene 0.6A are not conserved in T3 orfYeO3-12 and the two possibilities cannot be dis-tinguished without additional information. Table 1shows both gp0.6B and gp0.65 as putativeproducts, although only one may be made in vivo.

The structure and properties of T7 RNAP (gp1)have been studied extensively, and a crystal struc-ture of the enzyme complexed to its inhibitor,gp3.5 lysozyme, has been determined.49 Asexpected, many of the regions of RNAP demon-strated to be important for transcription are wellconserved in all three phage enzymes. All residuesin T7 RNAP known to affect its sensitivity to inhi-bition by lysozyme50 are conserved in both T3 andfYeO3-12 RNAP. Modulation of T3 RNAP activityby T3 lysozyme is thus expected to parallel thatfound in T7.

The structures of T7 DNA ligase (gp1.3), single-stranded DNA-binding protein (gp2.5), and endo-nuclease I (gp3) have been determined.51 53 Thecounterpart T3 proteins retain, 67.1%, 82.6%, and78.3% identity, respectively. Specific residues orregions of the T7 proteins that are known to beimportant for function are largely conserved, andunstructured or flexible regions account for mostof the amino acid differences with the T3 andfYeO3-12 proteins. T3 primase/helicase is 80.8%identical with T7 gp4 and T3 gene 4 complementsT7 mutants.54 A hybrid T3T7 gp4 is active bothin vivo and in vitro; despite several amino acidchanges in the T3-derived primase domain, thehybrid enzyme still recognizes T7 recognitionsites, therefore T3 primase likely has the same rec-ognition sequence as T7.55,56 The C terminus of T3gp4 differs substantially from T7 gp4, but mostacidic residues necessary for interaction with gp557

are conserved.DNA polymerase (gp5) is one of the most highly

conserved proteins between T3 and T7, and allknown functional residues are identical. However,fYeO3-12 gene 5 is somewhat less similar (Figure2). Comparison of fYeO3-12 gp5 to the crystalstructure of T7 DNA polymerase58 shows thatmost important residues are conserved except fora N335K substitution in a region that contacts theprimer-template and a change in the thioredoxin-binding domain. Mutations affecting three lysineresidues in the thioredoxin-binding loop of T7 gp5(K300E, K302E and K304E) all prevent phagegrowth, decrease the affinity of the polymerase forDNA, and lower its processivity.59 A K302A substi-tution also leads to reduced activity. fYeO3-12 gp5contains a K302R substitution, but it is not clear if


this change affects activity. Although fYeO3-12grows in E. coli trxA strains that express YeO3O-antigens, and fails to grow in trxA mutants,60 ithas not been shown that all aspects of fYeO3-12growth in E. coli are normal. The sequence ofY. enterocolitica trxA would help to determine ifTrxA has an altered interaction with fYeO3-12 gp5.

A predicted recombinatorial origin forcoliphage T3

Temperate phages enjoy the luxury of recombi-nation with prophages resident in the host cellsthat they infect. Indeed, genomics has confirmedthe modular theory of phage evolution61 by reveal-ing that an individual phage genome can berepresented as a mosaic of genetic modules takenfrom a large natural gene pool.62 Evolution of tem-perate phages is thus thought to result from recom-binatorial exchange of one module for another.63,64

For example, changes in host range can resultfrom recombination of the immunity regions of aninfecting phage and a resident prophage;65,66 hostrange switches can be obtained by recombinatorialreassortment of tail fiber genes or parts of thosegenes.67 69 A second mode of evolution of temper-ate phages appears to be due to insertion ofmorons.70 Whether morons are purely parasiticgenetic elements that confer no real benefit to thephage during lytic development and/or in thelysogenic state, or whether they provide a selectiveadvantage in certain environments has not beenestablished. It may be significant that moronshave not been recognized in the obligate lytic T7group of phages.

A priori, one might expect the rate of evolutionby recombinatorial exchange of gene modules tobe lower for lytic phages like T7 and T4 than thatfor their temperate counterparts. Inter-phagerecombination requires coinfection, which may beless common than a single infection. T7-like phagesefficiently degrade the host chromosome to mono-nucleotides, which are then used to support phageDNA replication. The host chromosome is thusrapidly rendered incapable of providing geneticcontent to an infecting phage. However, lessopportunity for genetic exchange may be partiallyoffset by highly efficient recombination processes.Indeed, there is evidence that T4 has benefitedfrom recombination of its tail fiber genes with tem-perate phages,67,71 and the RZ and RZ1 homologues

in genomes of the T7 group (genes 18.5 and 18.7 )may have been transferred by recombinationbetween T7 and a lambdoid phage.72 Of course,these examples of recombination may haveoccurred by T4 or T7 superinfection of a cell thatwas actively replicating the temperate phage ratherthan by infection of an uninduced lysogen.

The comparisons presented above show that T3is more similar to fYeO3-12 than it is to T7 and itcan now be hypothesized that recombinationbetween a T7-like coliphage and a fYeO3-12-likeyersiniophage led to the appearance of T3. Theproblem is then to demonstrate that the twophages recombine. fYeO3-12 does not yieldplaques on E. coli, similarly T3 does not yieldplaques on YeO3. Some T7 stocks have beenobserved to plate on YeO3 at very low efficiency(e.g. Table 2) but such behavior is uncommon andthe low frequency of plating does not make theisolation of recombinants from a cross between T7and fYeO3-12 in Y. enterocolitica serotype O:3 aprobable event.

Recombination between coliphage T7 andyersiniophage fYeO3-12

It is appreciated that the O-antigen compositionis not constant over the entire cell surface.73 Alllipopolysaccharide chains on a cell surface are notidentical, failures to make a complete O-antigenchain are not uncommon and, in the case of theYeO3 O-antigen, the number of repeating unitsper LPS molecule is affected by temperature.74 If asmall patch of O-antigen were missing on the sur-face of a YeO3 cell, a coliphage may be able toadsorb. On other parts of this same cell, where theO-antigen structure was complete, a yersiniophagemay also be able to adsorb.

We reasoned that we could increase the fre-quency of T7 and fYeO3-12 coinfection by incor-porating the O-antigen gene cluster from YeO3into E. coli. Because of the unnatural situation, wesurmised that the frequency of forming incompleteO-antigens would be increased. The E. coli K-12strain IJ511, which has been used extensively as ahost for T7, was transformed with the plasmidpAY100. The plasmid expresses the YeO3 O-anti-gen genes and allows the yersiniophage fYeO3-12to adsorb and infect E. coli.75 This same strain sup-ports T7 plaque formation at an efficiency of 0.9,relative to IJ511 (Table 2). We therefore coinfected

Table 2. Plating efficiencies

Host bacteria

Phage IJ511 IJ511/F0lac IJ511(pAY100) YeO3-c

T7 1 ,1028 0.9 1.9 1027LG12 1 ,1028 0.9 ,1028

fYeO3-12 ,1029 ,1029 1.1 1T7 fYeO3-12 1 0.8 0.7 ,1027LG12 fYeO3-12 1 0.7 0.7 ,1027T3 1 0.7 0.6 ,1029


IJ511(pAY100) with fYeO3-12 and T7, both at amultiplicity of 10, and plated the resulting lysateon IJ511/F0lac. The presence of F in the IJ511 back-ground suppresses T7 plaque formationcompletely76 and, although fYeO3-12 is not subjectto exclusion by F,60 the lack of YeO3 O-antigensprevents plaque formation. The strain IJ511/F0lactherefore selects recombinant phages that haveacquired gene 1.2 from fYeO3-12 but have theadsorption characteristics associated with T7 gene17. A second experiment used the ligase deletionmutant LG12 rather than T7. The rationale wasthat it provided an independent selection forligase-proficient recombinants and a stronger selec-tion for fYeO3-12 gene 1.2 activity was imposed.T7 mutants that lack DNA ligase cannot avoid Fexclusion by acquiring gene 10 mutations (I.J.M.,unpublished results).76 Recombinants betweenfYeO3-12 and T7 or LG12 were detected at a fre-quencies of 1.6 1026 and 2.6 1027, respectively.Recombinants plate on IJ511/F0lac as efficiently aswild-type T3 and, like T3, do not give plaques onYeO3 (Table 2). The recombination frequenciesobserved are about an order of magnitude lowerthan found in T3 T7 crosses, where mutual exclu-sion occurs; it is likely that fYeO3-12 and T7exclude each other after coinfection. The greatersequence divergence of fYeO3-12 from T7 likelyaccounts for the additional reduction.

One representative from each of the abovecrosses was purified and its genome analyzed byrestriction enzyme digestion, Figure 5 shows data

for Xmn I. From these and other data, both recom-binants appear to be largely derived from T7 andboth appear to contain a single insertion offYeO3-12 DNA. The T7 fYeO3-12 recombinantappears to have undergone two crossovers, onewithin 1200 bp region between positions 3650 and4870 (T7 coordinates), which lies within gene 1,the second within a 2 kb region between positions7550 and 9574, which includes T7 genes 1.5through the 50 60% of gene 2.5. The fYeO3-12 LG12 recombinant likewise enjoyed one cross-over in the 1200 bp gene 1 region and one withinT7 gene 10.

Aligning the nucleotide sequences of fYeO3-12and T7 DNAs reveals several extended regions ofidentity where recombination could occur. Withingene 1, beginning at positions 4410 (T7) and 4555(fYeO3-12), lie 38 identical base-pairs. This regionis one likely site for a crossover event for both theT7 fYeO3-12 and LG12 fYeO3-12 recombi-nants. T7T3 hybrid RNAP have been obtainedboth in vivo and in vitro.77 81 The C-terminal regionof the protein determines promoter specificity79,81

and the hybrid enzyme from these experimentswould thus have T3 (fYeO3-12) promoterspecificity. RNAP from T3 and fYeO3-12 are99.1% identical (Table 1), it is highly probabletherefore that a T7-fYeO3-12 hybrid gp1 would beviable. The second crossover in the T7 fYeO3-12 recombinant most likely occurred within gene2.5. Two extended regions of identical nucleotides,29 bp and 27 bp long, lie at positions 9434 and9340 (T7), and 9904 and 9810 (fYeO3-12). Gene 2.5is thought to be hybrid in some viable T7 T3recombinants, hybrid gp2.5 proteins have not beencharacterized biochemically but the high levels ofsimilarity between T7, T3 and fYeO3-12 gp2.5(Table 1) suggests strongly that any hybrid proteinwould be functional. Use of either region in gene2.5, together with that in gene 1, for crossoverswould produce a T7 fYeO3-12 recombinantwhose restriction map is consistent with thatobtained. This recombinant therefore likely con-tains a predominantly T7 genome with a fYeO3-12 replacement of the 30 half of gene 1 through the50 40% of gene 2.5. By restriction mapping, thesecond crossover in the LG12 fYeO3-12 recombi-nant occurred within gene 10. Within gene 10, atpositions 23291 (T7) and 21978 (fYeO3-12) beginsa 33 bp stretch of identity. A crossover at thispoint would result in a hybrid capsid protein butsuch a protein may well be functional. Not only isfYeO3-12 gp10 98.8% identical with T3 gp10, butthe latter is known to fully substitute for T7 gp10in T7 particle assembly.14 It is possible that twoother 26 bp regions of T7 and fYeO3-12 gene 10provide the nucleotide identity necessary to initiatehomologous recombination. The first would resultin a hybrid major capsid protein, the secondwould lead to a crossover within unique gene 10Bsequences, which would yield a normal fYeO3-12gp10A and a hybrid gp10B. gp10B results fromprogrammed ribosomal frameshifting near the 30

Figure 5. Characterization of fYeO3-12 T7 recombi-nants. Xmn I restriction digestion patterns of LG12,LG12 fYeO3-12, fYeO3-12, T7 fYeO3-12, and T7DNAs. The GibcoBRL 1 kb DNA ladder was used as amarker.


end of gene 10A.14,82 As the frameshifted gp10B isnon-essential for phage growth, it is improbablethat a crossover within gene 10B would be highlydetrimental. Nevertheless, the longer 33 bp regionof nucleotide identity within the middle of gene10A is the more probable crossover site. Thus theLG12 fYeO3-12 recombinant likely containsfYeO3-12 DNA between the middle of gene 1through the first 5558% of gene 10, but is other-wise derived from T7.

T7 and T3 are known to exclude each other inmixed infections,83 and exclusion likely affectscoinfections by T7 and fYeO3-12. In part, exclusionmay therefore be responsible for the low frequen-cies of recombination between T7 and fYeO3-12.In addition, the proposed sites of crossoverbetween the T7 and fYeO3-12 genomes are quiteshort. In crosses involving deletions, it was foundthat T7 recombination frequencies sharply declinedwhen the length of homology was reduced toabout 40 bp.84 The observed low frequencies ofrecombination between T7 and fYeO3-12 maythus be unsurprising. However, deletions appar-ently resulting from recombination over as little as4 bp have been isolated,84 86 and in evolutionarytime-frames, considerably less than 40 identicalnucleotides should suffice for recombination.

Known biological differences between T3 and T7include the ability of T3 to grow in the presence ofthe F plasmid (gene 1.2 ),87 to grow on Shigellasonnei D2 371-48 (gene 1.3 or 10 ),

88 and to code forSAMase as part of its gene 0.3 anti-type I restrictionactivity.89 fYeO3-12 is known to share the first twoproperties60 and was inferred from nucleotide simi-larity to contain a T3-like gene 0.3.4 fYeO3-12 T7recombinants were selected directly for the first ofthese properties; both recombinants plate at highefficiency on the S. sonnei strain but by restrictionmapping neither are likely to contain fYeO3-12gene 0.3. There are, however, stretches of nucleo-tide identity between fYeO3-12 and T7 DNAs,29 bp and 26 bp long that lie, both upstream(between the A1 and A2 promoters) and immedi-ately downstream of gene 0.3, respectively. Thesestretches could be used to recombine fYeO3-12gene 0.3 into a genome derived predominantlyfrom T7 DNA. There are many regions of nucleo-tide identity across the entire T7 and fYeO3-12genomes. A 62 nucleotide segment is common toT7 and fYeO3-12 gene 17.5, a 41 nucleotide seg-ment in gene 4 is common, and 76 other regionscontain a contiguous sequence of 20 or more iden-tical nucleotides. These regions provide ampleopportunity for recombination between thesephages in an environment where they can bothinfect the same host.

There is, of course, no reason to believe that thephage defined by Demerec & Fano5 as T3 is theimmediate product of recombination between thespecific phage isolates defined as T7 and fYeO3-12. It is quite likely that T3 has a more complex his-tory, where more than a single genetic exchangeoccurred between its immediate yersiniophage

and coliphage ancestors. Simply noting whethereach T3 gene is more similar to T7 than fYeO3-12(Table 1) suggests that at least six crossoversbetween T7 and fYeO3-12 would be required toyield a recombinant that most closely resemblesT3. However, about 60 T7-like phages have beenisolated1 and yersiniophages that are clearly T7-related, and thus similar to fYeO3-12, are known.Phage T3 may be a recombinant between twophages that are close relatives of the known coliph-age and yersiniophage collections. Nevertheless,by allowing T7 to recombine with fYeO3-12 invivo, we have provided experimental evidencestrongly supporting the idea that bacteriophage T3is the product of recombination between an E. coliand a Y. enterocolitica phage.

Materials and Methods

Phage and bacterial strains

Bacteriophages T3, T7, LG12 (T7 ligase deletionmutant),90 and fYeO3-1275 were from laboratory collec-tions. Large-scale preparation of T3 was propagated at30 8C in IJ511 (E. coli K-12 DlacX74, supE44, galK2,galT22, mcrA, rfbD1, mcrB1, hsdS3).91 Other strains usedin this study were an F plasmid derivative of IJ511(IJ511/F0lac ),91 a virulence plasmid-negative derivativeof Y. enterocolitica serotype O:3 (YeO3-c),92 S. sonnei D2371-48,93 and a plasmid expressing YeO3 O-antigens(pAY100)75 in IJ511.

Sequence determination

Genomic phage DNA was isolated from CsCl-purifiedphage stock as described for bacteriophage lambda,94

and used as template for sequencing. An aliquot (0.51 mg) of genomic DNA was combined with 1 ml of 5 mMprimer and 4 ml of ABI PRISMe BigDyee TerminatorCycle Sequencing Ready Reaction Kit mix. The volumewas adjusted to 20 ml with 10 mM TrisHCl (pH 8.0),and amplification reactions were carried out on PTC-200cycler (MJ Research, Waltham, MA) for 25 cycles (96 8Cfor ten seconds, 50 8C for 45 seconds, 60 8C for fourminutes). The excess dye was removed by precipitationin ethanol; samples were air-dried and resuspended in 3ml of formamide/50 mM EDTA (5:1 (v/v) in 3% (w/v)dextran blue). Samples were heat-denatured for twominutes at 94 8C and the entire content was loaded ontothe gel and electrophoresed for 1012 hours on an ABI377 DNA analyzer as recommended by the manufacturer(ABI Prisme. (1995). DNA Sequencing. Chemistry andSafety Guide. Part Number 903437. Rev. A. Applied Bio-systems, Foster City, CA). All previously unpublishedparts of the T3 genome were determined completely onboth strands.

Sequence analysis

Sequences were assembled and edited using GelAs-semble and other programs included in the GeneticsComputer Group (Accelrys, San Diego, CA) suite(version 10.0). Sequencher for Mac version 3.1.1 (GeneCodes, Ann Arbor, MI) was also used to edit andassemble the sequencing traces. Open reading frameswere searched for similarities in the databases using


BLAST and FASTA. Pairwise nucleotide alignments weredone using GAP using default parameters (gap creationpenalty of 50 and gap extension penalty of 3). Pairwiseidentities at the protein level were calculated by Alignat the Genestream using default parameters.95 Multiplealignments used CLUSTALW at the EMBL-EuropeanBioinformatics Institute. Prokaryotic promoter predic-tions used an Internet-available program with a cutoffvalue of 0.95. Before acceptance, putative s70-like promo-ters were checked manually for three hits out of six at the235 (TTGACA) and 210 (TATAAT) boxes (including theinvariant T) with a spacer of 16 to 18 bp, and preferably apurine about seven bases downstream of the 210 box.Compositional heterogeneity (G C content) wascalculated{ with a window of 85 nt. The DG-values forsecondary structures were calculated with the GCGprogram Mfold.

Data Bank accession numbers

The complete nucleotide sequence data for T3 havebeen deposited in the GenBank/EMBL/DDBJ databasesunder accession no. AJ318471 (includes X17255).

Acknowledgments

We express our thanks to the personnel of the Sequen-cing Laboratory of Department of Genetics (University ofTurku, Finland) and to Priscilla Kemp (University ofTexas) for sequencing. Teemu Toivanen and TapioSalakoski (Turku Centre for Computer Science, TUCS,Finland) and Jim Bull (University of Texas) are thankedfor helping with the oligonucleotide distributionanalysis. M.I.P. thanks the Turku Graduate School forBiomedical Sciences (TuBS) for financial support. Workat the University of Turku was supported by theAcademy of Finland and the National TechnologyAgency of Finland. J.K. was supported by the Office ofBiological and Environmental Research of the U.S.Department of Energy under Prime Contract no DE-AC02-98CH10886 with Brookhaven National Laboratory.Work at the University of Texas was supported by grantN00014-97-1-0295 from the Office of Naval Research.

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Edited by M. Gottesman

(Received 17 December 2001; received in revised form 27 March 2002; accepted 15 April 2002)


Complete Nucleotide Sequence and Likely Recombinatorial Origin of Bacteriophage T3IntroductionResults and DiscussionNucleotide sequence determination of the phage T3 genomeOverall organization of the genomeT7 group phages contain putative homing endonucleases at several genomic locationsRNA metabolismTerminal repeats and neighboring conserved putative regulatory signalsAvoidance of palindromic sequencesProteinsA predicted recombinatorial origin for coliphage T3Recombination between coliphage T7 and yersiniophage PhiYeO3-12

Materials and MethodsPhage and bacterial strainsSequence determinationSequence analysisData Bank accession numbers

AcknowledgmentsReferences

complete nucleotide sequence and likely recombinatorial ... · complete nucleotide sequence and...

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