Genome Sequence and Global Gene Expression of Q54, a New PhageSpecies Linking the 936 and c2 Phage Species of Lactococcus lactis
Louis-Charles Fortier, Ali Bransi, and Sylvain Moineau*Departement de biochimie et de microbiologie, Faculte des sciences et de genie, Groupe de recherche en ecologie buccale, Faculte de
medecine dentaire, Felix d’Herelle Reference Centre for Bacterial Viruses, Universite Laval, Quebec, Quebec, Canada G1K 7P4
Received 24 April 2006/Accepted 19 June 2006
The lytic lactococcal phage Q54 was previously isolated from a failed sour cream production. Its completegenomic sequence (26,537 bp) is reported here, and the analysis indicated that it represents a new Lactococcuslactis phage species. A striking feature of phage Q54 is the low level of similarity of its proteome (47 openreading frames) with proteins in databases. A global gene expression study confirmed the presence of two earlygene modules in Q54. The unusual configuration of these modules, combined with results of comparativeanalysis with other lactococcal phage genomes, suggests that one of these modules was acquired throughrecombination events between c2- and 936-like phages. Proteolytic cleavage and cross-linking of the majorcapsid protein were demonstrated through structural protein analyses. A programmed translational frameshiftbetween the major tail protein (MTP) and the receptor-binding protein (RBP) was also discovered. A “shiftystop” signal followed by putative secondary structures is likely involved in frameshifting. To our knowledge,this is only the second report of translational frameshifting (�1) in double-stranded DNA bacteriophages andthe first case of translational coupling between an MTP and an RBP. Thus, phage Q54 represents a fascinatingmember of a new species with unusual characteristics that brings new insights into lactococcal phage evolution.
Lactococcus lactis is a low-G�C gram-positive bacteriumextensively used by the dairy industry for its ability to convertsugars into lactic acid, leading to various fermented milk prod-ucts. However, L. lactis strains are susceptible to attacks bylytic bacteriophages with concomitant low-quality products andeconomic losses (56). Different strategies have been developedover the past 70 years with the aim of better controlling theindigenous phage population within the dairy environment (54,71). Nevertheless, the phage population is constantly evolvingand new phage isolates are still frequently isolated. Some ofthese phages are emerging due to current manufacturing prac-tices (49).
Classification of the phages is still controversial, and differ-ent propositions have been made in recent years (10, 65, 74).Previous classification studies relied on the comparison of virusmorphology and DNA-DNA hybridizations using whole ge-nomes. According to these criteria, 12 lactococcal phage spe-cies representing distinct phage groups were proposed (37, 38).This classification of lactococcal phages was recently revisited,and 10 genetically diverse groups of phages, sharing very lim-ited nucleotide similarities, were proposed (26). Among these,two new species were identified, one of which was the typephage Q54.
Phage genomics has considerably expanded in the last 5years, and as of June 2006, 362 complete genomes are availableat GenBank. Of those, 14 are from lactococcal phages. Amongthe available genomes, only members of the three main groupsof L. lactis phages are represented, namely, the 936, c2, andP335 species. Ten of the genomes are from phages of the P335
species (4, 9, 15, 40, 50, 68, 74, 75), whereas only four se-quences are from 936- and c2-like phages, namely, bIL170 andsk1 (936 species) as well as c2 and bIL67 (c2 species) (12, 18,48, 67). The lack of genome sequences from the less frequentlyisolated phage species is probably explained by the higherindustrial incidences of failed fermentations due to the mem-bers of the three above predominant species (37, 39).
According to the proposed classification scheme, lactococcalphages are highly diverse between species but rather homoge-neous within the same species. For example, virulent phageswithin the 936 or c2 species are highly similar at the DNA leveland up to 90% of their genome length can be aligned with over70% nucleotide identity (15, 18, 48). As a result, most of theopen reading frames (ORFs) share more than 80% amino acididentity, some being almost identical. Point mutations andsmall insertion/deletions (indels) were shown to account formost of the differences in the genome sequences of lytic phages(18, 48). Due to the lack of temperate members, homologousrecombination between members of the 936 and c2 species isnot expected to occur frequently, although it may occur duringcoinfection (15). On the other hand, the P335 group of phages,comprising both temperate and lytic members, is less con-served and has a polythetic nature. Indeed, several P335-likephages share conserved modules, but there is no single ORFshared by all members of this group (4, 9, 15, 23, 24, 40).Comparative genomics of these P335-like phages thus revealeda highly mosaic structure, defining functional modules that areexchangeable through homologous recombination, in additionto point mutations and indels (15, 23, 57).
Structural proteins that make up the viral particle have beenstudied for only a few lactococcal phages including c2 (48) andp2/936 species (69) as well as the P335-like phages ul36 (40),r1t (75), BK5-T (50), and TP901-1 (76). Posttranslational mod-ifications of structural proteins, especially the major capsid
* Corresponding author. Mailing address: Groupe de recherche enecologie buccale, Faculte de medecine dentaire, Universite Laval,Quebec, Quebec, Canada G1K 7P4. Phone: (418) 656-3712. Fax: (418)656-2861. E-mail: [email protected].
protein (MCP), have been well documented for a number ofmodel phages such as the coliphage HK97 (29). The MCPfrom the lactococcal phage c2 was also shown to undergoproteolytic cleavage and cross-linking during capsid assembly(48). Although proteolysis of the capsid has been reported fora number of other members, cross-linking among lactococcalphages has been so far reported only for phages c2 and r1t (48,75). In other phages, cross-linking of viral subunits does notappear to be frequent, except perhaps for mycobacteriophages,where this mechanism seems to be more common (28, 34).
Programmed translational frameshifting is well documentedin double-stranded DNA (dsDNA) bacteriophages, includingEscherichia coli phage � (44). Generally, it involves tail pro-teins, more specifically the major tail protein (MTP) and anoverlapping ORF preceding the tape measure protein (79).Most of the frameshift events recognized so far are of the �1type, where a slippery sequence composed of repeated nucle-otides enables the ribosome to slip one nucleotide backwardbefore continuing translation on the corresponding overlap-ping frame. An unusual �2 frameshift was recently demon-strated in tail proteins from phage Mu (79). Only a few exam-ples of �1 frameshifting have been documented in E. coli, withthe well-described examples of release factor 2 (RF2) (17) andan expression system involving human transferrin (Tf) (25). Inthe case of Tf, a “shifty stop” with the sequence CCC.UGAwas found to be responsible for ribosome frameshifting (25).Recently, the first case of programmed �1 translational frame-shifting in dsDNA phages was reported for the Listeria mono-cytogenes temperate phage PSA, where elongated versions ofthe capsid protein and MTP were identified (80). In the MTPframeshift, a CCC.UGA “shifty stop” at the end of the tailgene was also found to be responsible for the event. Notably,a pseudoknot 3� to the slippery signal was probably also in-volved in the mechanism (80). Secondary structures down-stream from slippery signals have been shown to promoteframeshifting (43).
Here, we report the microbiological and molecular charac-terization of a novel lactococcal phage species, for which thelytic phage Q54 is currently the only member. Completegenomic and transcriptional analyses are provided which re-veal an unusual module configuration and shed light on itspeculiar origin. A detailed analysis of the structural proteinsuncovered interesting features, including cross-linking of theMCP and a programmed �1 translational frameshift betweenthe MTP and the receptor-binding protein (RBP). To ourknowledge, this is only the second case of programmed �1translational frameshifting in dsDNA phages and the first caseof a translational coupling between a major tail protein and anRBP, the latter being involved in host recognition.
MATERIALS AND METHODS
Bacterial and phage amplification. Lactococcus lactis SMQ-562 and L. lactisLM0230 were grown at 30°C in M17 broth (72) supplemented with 0.5% glucose(GM17) (Difco) unless otherwise specified. Glycine (0.5%) was also added to thetop agar to increase plaque size and facilitate phage enumeration (45). CaCl2(final concentration of 10 mM) was added whenever phages were used. Whenhigh phage titers were required, lysates were concentrated with polyethyleneglycol and purified by two CsCl gradient ultracentrifugations (14). The firstcentrifugation was performed at 35,000 rpm for 3 h in a discontinuous-step CsClgradient using a Beckman SW41 Ti rotor. The phage band was picked up and
further purified by ultracentrifugation at 60,000 rpm for 18 h in a continuoussingle-phase CsCl gradient using a Beckman NVT65 rotor (66).
Microbiological assays. One-step growth curve assays were performed as re-ported elsewhere (55). The burst size was determined by dividing the averagephage titer after the exponential phase by the average titer before infected cellsbegan to release virions (55). The efficiency of plaque formation (EOP) wascalculated by dividing the phage titer on the tested L. lactis strain by the titer onthe sensitive control L. lactis strain.
Phage DNA analysis. Phage Q54 genomic DNA was isolated using the MaxiLambda DNA purification kit (QIAGEN). Genome sequencing was first per-formed using a shotgun cloning strategy (Integrated Genomics Inc., Chicago,Ill.). Completion of the genome was done by direct sequencing of the phageDNA with primers and using an ABI Prism 3700 apparatus from the genomicplatform at the research center of the Centre Hospitalier de l’Universite Laval.The cos site was determined by direct sequencing of the phage DNA and bysequencing of a PCR product encompassing the T4-ligated cohesive termini.Standard PCR and ligation procedures were used (66).
RNA isolation and Northern blot analysis. Two hundred milliliters of GM17was inoculated (1%) with an overnight culture of L. lactis SMQ-562, and cellswere grown at 30°C until the optical density at 600 nm reached 0.1. CaCl2 wasadded to a final concentration of 5 mM, and incubation was continued for 5 min.Cells were then centrifuged at room temperature for 2 min at 10,000 rpm in aBeckman GSA rotor. The pellet was suspended in 20 ml of GM17 containing 5mM CaCl2 to obtain a final optical density at 600 nm of 1.0. At time zero, asample of 1.5 ml was removed and quickly centrifuged for 15 s, the supernatantwas removed, and the cell pellet was snap frozen in liquid nitrogen. Cells werestored at �80°C until RNA extraction. Phage Q54 was added to the remainingculture at a multiplicity of infection of 10. Aliquots of 1.5 ml were withdrawn atvarious time intervals and processed as described above. Lysis of the remainingphage-infected culture after 45 min was indicative of the completion of the phagelytic cycle.
The next day, cells were thawed on ice and resuspended in a total volume of100 �l of a sucrose solution (0.5 M) containing 120 mg/ml lysozyme. The mixturewas incubated 10 min at 37°C prior to the addition of 1 ml of Trizol (Invitrogen),as recommended by the supplier. This step was necessary to achieve rapid andcomplete cell lysis. Following its isolation, the RNA was treated with 30 unitsRNase-free DNase I (Roche) for 30 min at 37°C in the presence of ProtectorRNase inhibitor (Roche) and quantified by UV spectrometry at 260 nm. North-ern blotting was performed after separation of 5 �g of RNA through a 1%agarose-formaldehyde denaturing gel (66). Probes used to detect mRNAs con-sisted of 32P-labeled oligonucleotides (25- to 35-mer; sequences available uponrequest) complementary to the coding strand of the gene of interest. Labelingreaction mixtures consisted of 2 pmol of oligonucleotide, 1� reaction buffer, 30�Ci of [�-32P]ATP (GE Healthcare), and 20 units of T4 polynucleotide kinase(Roche) in a final volume of 20 �l. Incubation was performed at 37°C for 30 min,after which 50 �l of distilled water was added. Probes were quickly purified usingMicro Bio-Spin 30 columns (Bio-Rad Laboratories). The efficiency of label-ing of the purified probes was determined by liquid scintillation counting. Aminimum specific activity of 2 � 106 cpm/pmol oligonucleotide was our cutoffin order to use the labeled probe in the hybridization experiments. Mem-branes were prehybridized at 42°C for at least 1 h in 5 ml of UltraHyb-Oligohybridization buffer (Ambion) in a 50-ml disposable screw-cap tube. Hybrid-ization was performed overnight at 42°C in the presence of 5 � 106 cpm of thefreshly labeled probe. Membranes were washed twice at 42°C for 30 min in2� SSC (1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate) plus 0.5%sodium dodecyl sulfate (SDS) and then exposed to Kodak XOMAT-AR filmsat �80°C with intensifying screens.
SDS-PAGE analysis of structural proteins. Purified phages (�1 � 1011 PFU/ml) were analyzed for structural proteins by standard Tris-glycine 12% SDS-polyacrylamide gel electrophoresis (PAGE) procedures (42). Samples weremixed with 2� sample loading buffer and boiled for 10 min before loading.Proteins were detected after Coomassie blue staining. For phage Q54, furtherconcentration of the phage suspension was required before electrophoresis. Thiswas achieved by centrifuging 1.5 ml of CsCl-purified phage suspension throughNanosep 3k columns (Pall), which led to a further 1-log concentration factor.Protein bands were cut out of the gel, digested with trypsin, and identified byliquid chromatography-tandem mass spectrometry (LC-MS/MS) (Genome Que-bec Innovation Centre, McGill University). For comparison purposes, the struc-tural proteins of the reference phage c2 (48) were also analyzed by SDS-PAGE.
Bioinformatics analysis. DNA sequence analysis, contig assembly, and editingwere done using the Staden package 1.5.3 (available at http://staden.sourceforge.net/). Some editing was also done using BioEdit 7.0.5.2 (33). Protein sequencecomparisons were performed using BLASTP 2.2.13 (2), and conserved domains
were identified using RPS-BLAST 2.2.11 and the Conserved Domains Database(CDD) at the National Center for Biotechnology Information (NCBI) (51, 52).PSI-BLAST was also used to search for more distant homologous proteins whenstandard BLAST hits gave only proteins with putative or unknown functions.InterProScan at EMBL-EBI (http://www.ebi.ac.uk/InterProScan/) was used toidentify conserved domains in some proteins that did not show any significantsimilarity by BLAST searches. Putative RNA secondary structures were deter-mined using Vienna RNA secondary structure prediction v1.5 at http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi and MFOLD at http://bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html (81).
Nucleotide sequence accession number. The sequence data reported in thispaper have been deposited in EMBL/GenBank/DDBJ databases under accessionnumber DQ490056.
RESULTS
Characteristics of Q54 and its lytic cycle. The lytic phageQ54 was previously isolated from a failed sour cream produc-tion, which used Lactococcus lactis SMQ-562 in its starterculture. Transmission electron microscopy analysis revealed amorphology similar to that of phage c2, with a prolate capsid(56 � 43 nm) and a noncontractile tail (109 nm) (Fig. 1) (26).Thus, phage Q54 belongs to the Siphoviridae family of theCaudovirales order. It was recently reported that DNA-DNAhybridization experiments failed to show any homology be-tween Q54 and genomic probes from the nine other knownlactococcal phage species, c2, 936, P335, 1358, P087, 1706, 949,P034, and KSY1 (26). Based on these observations, Q54 wasclassified as a new lactococcal phage species, and thisprompted us to further characterize this phage.
The phage Q54 host range was assessed using 58 L. lactisstrains, and this phage was shown to infect only L. lactis SMQ-562. Of note, this L. lactis strain is also sensitive to the refer-ence phages 1706 and 949, which are members of two otherlactococcal phage species that bear their names. A single-stepgrowth curve assay of phage Q54 was performed at 30°C withL. lactis SMQ-562. The phage has a latent period of 40 min anda burst size of 42 8 PFU. The permissive temperature rangefor propagation of Q54 is 22 to 33°C. Interestingly, at 34°C andabove (up to 38°C), we could not detect a single plaque on L.lactis SMQ-562.
The sensitivity of phage Q54 towards three phage resistancemechanisms, namely, AbiK (32), AbiQ (31), and AbiT (6), wasdetermined. Numerous phage resistance mechanisms are cur-rently used by the dairy industry to control the three mainphage species (936, c2, and P335). Thus, we were interested toknow if the new phage species Q54 could be controlled bythese abortive infection mechanisms (Abis). High-copy-num-ber plasmids expressing the three Abi systems were transferredinto L. lactis strain SMQ-562, and the resulting transformantswere challenged with Q54. AbiQ and AbiT were very effectiveagainst Q54, with mean EOP values of �10�8 and 3.3 � 10�7 2.2 � 10�7, respectively. On the other hand, AbiK was inef-fective against this phage (EOP of 0.81 0.54). AbiK is gen-erally very efficient against the three main phage species (32),which confirms the distinct nature of Q54.
Complete nucleotide sequence of Q54 genome. A shotguncloning strategy was used to sequence �90% of the Q54 ge-nome. Primer walking and direct sequencing of the phageDNA were performed to complete the remaining 10% cover-ing both genome extremities. Runoff sequencing reactions en-abled us to locate the exact ends of the linear phage genome.
No terminal redundancy was observed, suggesting a cos-typephage. Confirmation was made by ligating the cohesive terminiwith T4 DNA ligase followed by sequencing of a PCR fragmentspanning the resulting junction (Fig. 2). The cohesive terminiare composed of single-stranded 10-bp 3� overhangs (Fig. 3A).The complete genome sequence of 26,537 bp has a molar G�Ccontent of 37.1 mol%, which is similar to those of L. lactis andother lactococcal phages (5, 12, 40, 48).
Analysis of Q54 cos region. Analysis of the DNA sequencesurrounding the cos site revealed features common to otherlactococcal phages of the 936 and c2 species, including severaldirect and inverted repeats and G- and C-rich clusters (G andC boxes) as well as an A/T-rich region (16 A � 47 T) (Fig. 3A)(13, 47, 61, 62). However, the cos site of phage Q54 lacks aconserved T in one of the inverted repeats (4 bp, TCAN)typically found within a 15-bp segment spanning the cos site oflactococcal phages (61, 62). The phage Q54 cos region alsocontains nine �-like R consensus sequences (19). In �, the Rsites are 16 nucleotides long whereas in Q54 the consensus wasextended to 19 nucleotides from comparison with those previ-ously reported for phages c2 and sk1 (Fig. 3B) (62). The �-Rsites are involved in terminase recognition and binding as wellas termination of packaging. However, in contrast to phages �and c2, the Q54 �-R sequences are unevenly distributed acrossa 900-bp region. In the former, the �-R sites are regularlyspaced (�250 bp between R sites). Alignment of the R sitesfrom � with the �-like R sites from phages Q54, c2, and sk1(Fig. 3B) highlighted common bases likely involved in thepackaging process.
Comparison of Q54 ORFs and functional assignment. Fur-ther bioinformatics analyses enabled the identification of 47putative ORFs of 35 amino acids or more (Fig. 2A; Table 1) inthe genome of phage Q54. The standard ATG start codon andalso alternative start codons were used to locate ORFs. Thesewere determined on the basis of the presence of a suitableribosome binding site complementary to the 3� end of the 16SrRNA of L. lactis located upstream of the selected start codonas well as the absence of extensive overlapping between ORFs.The longest gap observed was 286 bp between ORFs 9 and 10,and the longest overlap tolerated was 13 bp between ORFs 10and 11 (Table 1). There are two main groups of ORFs in Q54:one group spanning almost the entire genome and which is onthe upper strand and a second group of 10 ORFs (orf35 to
FIG. 1. Electron micrograph of phage Q54. Bar, 50 nm.
VOL. 188, 2006 Q54, A NEW HYBRID LACTOCOCCAL PHAGE SPECIES 6103
orf47) encoded by the lower strand (approximately 3.5 kb) atthe right end of the genome (Fig. 2A).
The 47 deduced proteins were compared with protein data-bases using BLASTP in order to assign a putative function.Since there was only limited similarity between phage Q54proteins and other proteins in the databases, the assignment ofputative functions was further reinforced by position-basedcomparison with the genomes of other known phages, by thepresence of conserved domains and/or secondary structures,and by experimental identification of structural proteins. Func-tional assignments and the best matches in databases are pre-sented in Table 1, and further details are given below, but onlyfor some of the ORFs.
(i) ORF11. The best BLAST result for ORF11 is a proteinwith no predicted function from L. lactis SK11 (39% identity;77/197) (accession no. ZP_00382242). Similarities were alsofound with proteins of the essential recombination factor(ERF) family, including Sak2 from phage P335 (44% identity;67/152) (8). A conserved ERF domain (pfam04404) was alsoidentified in ORF11. This domain is present in DNA single-stranded annealing proteins of the ERF superfamily of pro-teins, which function in RecA-dependent and RecA-indepen-dent DNA recombination pathways, such as RecT, Red-, andRad52. In phages with cohesive ends (such as Q54), the role ofsuch a recombination protein was proposed to be similar tothat of Red from phage �, which increases the amount ofpackageable DNA (67).
(ii) ORF12. ORF12 shows similarity with several bacterialsingle-stranded binding (SSB) proteins including those of L.lactis strains IL1403 (5) and SK11 (Table 1). Similarity was alsoobserved with phage-encoded SSB proteins such as ORF9from Streptococcus thermophilus phage 7201 (59% identity;
97/153) (70) and ORF1 from Streptococcus pneumoniae phageVO1 (58) (55% identity; 98/180). SSB proteins are involved inprotection of single-stranded DNA segments generated duringDNA replication (60, 78).
(iii) ORF16. CDD searches revealed the presence of con-served domains within ORF16, namely, S1 (ribosomal proteinS1-like RNA-binding domain: smart00316 and pfam00575),RNB (RNB-like protein: pfam00773), and VacB (exoribo-nuclease R: COG0557). These domains are found in RNA-binding proteins involved in RNA metabolism (e.g., transcrip-tion). Comparison with protein databases showed similaritywith several chromosome-encoded RNases and exoribonucle-ases from diverse bacterial origins (Table 1).
(iv) ORF17. The deduced protein ORF17 is similar to gp80from Listeria phage P100 (Table 1) (11) and ORF98 fromStaphylococcus aureus phage K (29% identity; 75/251) (59),which have unknown functions. After PSI-BLAST analysis(four iterations), we also found homologues for which a DNApolymerase function was predicted. The most significant hitsincluded a DNA-directed DNA polymerase from the uncul-tured archaeon GZfos19C8 (15% identity; 49/315, E � 1E-49)and a DNA polymerase delta small subunit from Methanother-mobacter thermautotrophicus strain Delta H (14% identity; 47/314, E � 3E-47) (accession no. AAU82757 and AAB85882,respectively). An InterProScan analysis also revealed the pres-ence of a metallophosphoesterase domain (IPR004843) foundin a large number of proteins involved in phosphorylation,including DNA polymerases, exonucleases, and other phos-phatases.
(v) ORF18. ORF18 is similar to a putative protein fromStreptococcus pyogenes prophage 315.3 (29% identity; 47/158)(3) and also to a conserved phage-associated protein from
FIG. 2. Q54 complete genome (26,537 bp). (A) Genomic organization. A scale (in kilobases) is shown above the genetic map. Each arrowrepresents a putative ORF, and the numbering refers to Table 1. Gray arrows represent ORFs for which a putative function could be inferred frombioinformatics analyses (indicated above the ORFs). White arrows represent ORFs for which no putative function could be attributed. Hairpinsrepresent putative Rho-independent transcriptional terminators. Arrows with heavy outlines represent gene products detected by LC-MS/MSanalysis (Fig. 4). (B) Phage Q54 mRNA transcripts detected in the course of an infection of L. lactis SMQ-562. Short thick lines above thetranscripts indicate probes. The size (in kilobases) is indicated on the right or left of each transcript (arrows), and the line thickness isrepresentative of their relative abundance, with the thicker line corresponding to the highest concentration. Arrowheads show the direction oftranscription, and the temporal expression (early or late) is indicated below the genome scale.
Streptococcus suis 89/1591 (24% identity; 39/157) (accessionno. ZP_00875013). There is also similarity with a crossoverjunction endodeoxyribonuclease, RuvC from Chlorobium limi-cola DSM 245 (25% identity; 34/131, E � 0.016) (accession no.ZP_00511492). After two iterations of PSI-BLAST analysis,further hits were obtained with RuvC-like Holliday junctionresolvases, including ORFm3 from lactococcal phages bIL170(936 species, 25% identity; 40/157) (18) and ORF53 fromphage sk1 (936 species, 25% identity; 40/158) (12). No hitswere found with gene products from phages of the c2 species.However, BLAST analyses using ORF55 from phage sk1 re-vealed significant similarity with ORFl2 from phages c2 (44%identity; 71/161) and ORF23 from phage bIL67 (42% identity;69/161) (67), which was recently demonstrated to be a RuvC-like resolvase (21).
Structural genes and morphogenesis. Phage Q54 morphol-ogy is similar to that of the prolate-headed phages of the c2species, and compared using BLAST analyses, most of thestructural proteins were related to phage c2 or bIL67, or othermembers of the c2 species for which partial sequences areavailable.
(i) ORF20. The only significant hits are ORFl4 from phagec2 (21% identity; 46/210), ORF25 from phage bIL67 (20%identity; 37/177), and a putative portal protein from a Strepto-coccus agalactiae prophage (17% identity; 28/159, E � 0.006)(accession no. AAN00717). The similarity with the latter pro-tein and its location upstream from the capsid protein suggestthat ORF20 from Q54 might be the putative portal protein.Also, a protein corresponding to this ORF was identified in thevirion structure by SDS-PAGE mass spectrometry analysis(Fig. 4). It was previously suggested that ORFl4 of phage c2might be a portal protein (22).
(ii) ORF21. The ORF21 protein was also detected in thestructure of phage Q54 (Fig. 4). The deduced protein hassimilarity with the major capsid proteins (pfam05065) ofphages of the lactococcal c2 species, including ORFl5 fromphage c2 (27% identity; 117/428) (48) and ORF26 from phagebIL67 (25% identity; 134/518) (67). However, the percentageof identity was very low, as opposed to the 93 to 97% identityobserved between the MCPs of c2-like phages such as c2,bIL67, Q38, Q44, and eb1.
(iii) ORF23. ORF23 is most likely the MTP, because it issimilar to homologues in phages c2 (ORFl7) and bIL67(ORF28) (29% identity; 61/205 and 62/211, respectively) (48,67). Again, a very low percentage of amino acid identity wasobserved, contrasting with the 88% identity (182/205) foundbetween ORFl7 and ORF28 from phages c2 and bIL67, re-spectively. We also detected the corresponding protein in thestructure of the virion (Fig. 4).
(iv) ORF24. This protein is the third most conserved in Q54,showing similarity with several tail host specificity proteins andaccessory tail fibers from diverse phages infecting both L. lactisand S. thermophilus. We noted 59% identity (120/201) withORF45 from the lytic lactococcal phage 4268 of the P335species (74). Similarity was also observed, although withslightly lower E values, with ORFl15 from L. lactis phage c2(29% identity; 62/211) (48). InterProScan analyses also re-vealed the presence of a galactose-binding-like domain(IPR008979) within ORF24 of phage Q54. This domain isinvolved in binding to cell surface-attached galactose-contain-
FIG
.3.
AnalysisofQ
54cosregion.(A
)Analysisofthe
cossiteand
flankingregions.T
ypicalfeaturesfoundin
thecosvicinity
ofotherphages,suchasdirectrepeats(D
R1
andD
R2),inverted
repeats(IR
),A/T
-richregion,G
-andC
-richsegm
ents(G
andC
box,respectively),and�-R
-likesequences
(�-like),areindicated.D
etailsofthe
cohesiveterm
iniarealso
shown.T
hesites
ofcleavageresulting
in3�-overhang
terminiare
indicatedby
verticalarrows.Italic
characterswith
reversehighlighting
representnucleotidesthatareidenticalto
aconsensussequence
foundin
alllactococcalphagesforwhich
thecosregion
hasbeensequenced
(seetextfordetails).(B
)Multiple
alignmentof�-R
-likesequencesfrom
Q54,c2,and
sk1w
iththose
of�-Rsequencesfrom
phage�.C
onservednucleotidesare
shaded.
VOL. 188, 2006 Q54, A NEW HYBRID LACTOCOCCAL PHAGE SPECIES 6105
ing carbohydrates of prokaryotes and eukaryotes (36). More-over, ORF24 was found to be similar to the variable region 1(VR1) of S. thermophilus phages Sfi11, Sfi19, Sfi21, MD2, andDT2, which were previously shown to be involved in hostrecognition (30). These findings further support the predictedfunction of ORF24 as the receptor-binding protein (73).
(v) ORF26. ORF26 shares similarity with several c2-likephage tail proteins, and the best match was with the tapemeasure protein (ORFl10) of phage c2 (34% identity; 193/567). Other matches included the industrial lactococcalphage isolates 5469 (35% identity; 180/507) and 5440 (28%identity; 148/528) (64). ORF26 was also found in the struc-ture of Q54 as revealed by SDS-PAGE and mass spectrom-etry analyses (Fig. 4).
(vi) ORF28. The ORF28 gene product has similarities withORF32 of phage bIL67 (34% identity; 172/505) and ORFl12 ofphage c2 (33% identity; 171/505), both predicted to be termi-nases (48, 67). A conserved Terminase_1 domain (pfam03354;COG4626) was also identified in ORF28 while CDD searcheswere being performed. The size of the gene product also sug-gests that it is the large terminase subunit, although no putativesmall terminase subunit could be identified by bioinformaticanalysis. The presence of only one identifiable subunit of theterminase is not unusual, as it was observed for various lacto-coccal phages such as c2 (48), 4268 (74), and �LC3 (4) as wellas for the Lactobacillus phage �adh (1).
Lysis module of phage Q54. (i) ORF33. No significantBLAST hit was obtained with the deduced protein ORF33.However, the size and the location of this ORF within thegenome suggest that it may encode the phage holin. UsingInterProScan, we found three transmembrane-spanning seg-ments (amino acids [aa] 10 to 30, 45 to 65, and 77 to 99) and14 charged amino acids (including six lysine residues) withinthe 26 C-terminal residues of ORF33. These two elementsare characteristics of holins, and the presence of three trans-membrane segments would place this protein into class I ofthe holins (41, 77). Similar to the holins of the c2-like phages, nodual-start motif could be identified in the N terminus of ORF33,as opposed to the holin from phages of the 936 species (41).
(ii) ORF34. This protein is predicted to be the endolysin,and it is one of two gene products of phage Q54 showing anamino acid identity greater than 60%. CDD searches revealedthe presence of an N-acetylmuramoyl-L-alanine amidase con-served domain (smart00644, pfam01510). BLAST searches re-vealed several hits with the endolysins from various phages,especially those of the lactococcal phages of the 936 species,including bIL170 (ORFl22, 61% identity; 154/250) (18) andsk1 (ORF20, 59% identity; 125/211) (12). Notably, a pairwisealignment of the orf34 of Q54 with orfl22 of phage bIL170showed 60% nucleotide identity over the entire gene, whichmakes this gene the most conserved at the nucleotide level.
(iii) ORF35, ORF40, and ORF41. BLAST searches withORF35 gave no significant result, at least based on a thresholdE value of 1.0 or below. Nevertheless, a single hit was foundwith ORFe25 from phage bIL170 when a lower stringency wasused for the search (28% identity; 14/50, E � 1.8) (18). ORF40was found to be similar to ORFe24 from phage bIL170 (44%identity; 45/102) (18) and ORFe26 from phage sk1 (38% iden-tity; 39/102) (12). Finally, ORF41 is similar to early gene prod-ucts from 936-like phages, including ORF41 from phage sk132
2106
221
679
205
23.1
6.7
AT
AT
TT
GA
GG
ggga
aatt
ttat
aAT
G—
3321
754
2213
112
513
.86.
5A
GG
AG
Gga
caaa
aaaA
TG
Hol
ine
3422
146
2292
225
629
.07.
7A
AA
AA
GG
AG
GT
attc
aAT
GE
ndol
ysin
OR
F12
2L
.lac
tisph
age
bIL
170
(154
/250
;61%
)1E
-83
233
AA
R26
447
3523
116
2295
553
6.5
4.7
AG
AA
AG
GA
aaaa
aaA
TG
—O
RF
e25
L.l
actis
phag
ebI
L17
0(1
4/50
;28%
)1.
853
AA
R26
450
3623
444
2313
010
411
.710
.4A
AA
GA
GT
AT
Gac
ccta
gacc
gtcc
gcaA
TG
—37
2368
323
570
374.
39.
2A
AA
TG
AT
Gac
aaaa
acag
aaA
TT
—38
2390
923
676
779.
59.
7A
AA
CG
AG
Ggt
taaa
aaaA
TG
—39
2422
124
108
374.
37.
9A
GA
AT
GG
GA
Ggg
taaa
aaA
TG
—40
2450
524
218
9511
.38.
7A
TT
AA
TG
GG
Gaa
aata
caaa
AT
G—
OR
Fe2
4L
.lac
tisph
age
bIL
170
(45/
102;
44%
)1E
-14
102
NP_
0471
49
4124
758
2449
288
10.7
9.4
AA
GT
AT
Gat
aaaa
taga
attA
TG
—O
RF
41L
.lac
tisph
age
sk1
(26/
78;3
3%)
5E-6
88N
P_04
4987
4225
042
2475
595
11.5
9.0
AG
AA
AG
GA
AG
attt
acgg
gtgg
acta
aAT
G—
4325
212
2503
957
6.6
9.6
AG
AA
AT
GA
TT
Tgt
tctA
TG
—44
2544
425
202
809.
89.
3A
AG
AG
GT
aaaa
aaA
TG
—45
2561
425
441
576.
84.
3A
AG
GA
Gta
aaaa
aaaA
TG
—46
2579
725
621
586.
99.
6A
AG
GA
GG
GT
atga
aAT
G—
4726
179
2585
610
712
.04.
9A
CT
AA
GG
AG
GT
acat
actA
TG
—
aR
BS,
ribo
som
albi
ndin
gsi
te.
bU
nder
linin
gin
dica
tes
nucl
eotid
esid
entic
alto
the
RB
Sco
nsen
sus;
low
erca
sein
dica
tes
spac
ernu
cleo
tides
betw
een
the
RB
San
dst
art
codo
n;bo
ldfa
cein
dica
tes
the
star
tco
don.
c—
,no
pred
icte
dfu
nctio
n.d
HA
D,h
aloa
cid
deha
loge
nase
-like
hydr
olas
e.e
Iden
tified
byse
cond
ary
stru
ctur
ean
alys
esus
ing
Inte
rPro
Scan
atE
MB
L-E
BI
(htt
p://w
ww
.ebi
.ac.
uk/in
terp
ro).
fSi
zeof
the
alig
ned
prot
ein.
VOL. 188, 2006 Q54, A NEW HYBRID LACTOCOCCAL PHAGE SPECIES 6107
(33% identity; 26/78) and ORFe7 from phages bIL66M1 (27)and bIL170 (both 36% identity; 27/74). Despite these similar-ities, no function could be assigned to ORF35, ORF40, andORF41.
Comparative genomics of Q54 versus phages of the 936 andc2 species. To better visualize the similarities between phageQ54 and the lactococcal phages from the c2 and 936 speciesdescribed above, a whole-genome comparison was performed.Phages c2 and sk1 are closer to Q54 because several BLASThits (although not always the best) were associated with thesephages. As shown in Fig. 6, most of the ORFs present in themorphogenesis module of phage Q54 could be aligned withthose of phage c2 (blue arrows). The order, number, and sizeof ORFs 20 to 29 were well conserved between the two phages.However, other ORFs are more closely related to phage sk1.For example, the protein similarity of Q54 endolysin (ORF34)with that of sk1 (ORF20) suggests that it might originate froma phage of the 936 species such as sk1.
Transcriptome analysis. Temporal gene expression of phageQ54 was assessed by standard Northern blotting. Twenty ra-diolabeled probes were used to cover the genome and ORFswith predicted functions. Transcript sizes ranged from 0.5 to 10kb and were detected over the entire genome (Fig. 2B). Thelevel of mRNA transcripts was variable; some were detected asfaint signals (thin arrows), and others were highly expressed(thick arrows) (Fig. 2B). According to our results, all ORFs areencoded by at least one mRNA, but most of them are encodedby more than one mRNA species, generally of different lengthand abundance. Transcripts that were detected at 5 min afterthe infection were classified as early mRNAs (orf1 to orf19 andorf35 to orf47) whereas transcripts that were detected after 10to 15 min were considered late mRNAs (ORFs 20 to 34). Theabundance and relative decay of the early mRNAs were highlyvariable. For example, the 8.0-kb transcript (covering ORFs 7to 19) was detected at 5 min but almost completely disap-peared after 15 min, whereas other transcripts like the 4.3-kb
(covering ORFs 15 to 19) and 3.6-kb (covering ORFs 35 to 47)transcripts decreased gradually from 5 min to 30 min but werestill easily detectable after 30 min (data not shown). Con-versely, all late transcripts were detected after only 10 to 15min and the signal continued to increase until the end of theassay. Based on the above transcriptional analysis and furtherbioinformatics analyses, three putative rho-independent termi-nators were identified (Fig. 2B). The first is located betweenORFs 19 and 20 on the upper strand, and the two others arelocated between ORFs 34 and 35 on both strands and wouldprobably function as a bidirectional terminator. A similar bi-directional terminator was also found between the early andlate gene clusters in lactococcal phage sk1 (12).
SDS-PAGE analysis of Q54 structural proteins. The proteincomposition of Q54 virions was characterized by SDS-PAGEcoupled with LC-MS/MS. Since phage Q54 and the referencephage c2 share morphological and some protein sequence sim-ilarities, we included a purified sample from phage c2 forcomparison purposes. Strikingly, the protein profiles fromthe two phages were highly similar (Fig. 4A). Mass spec-trometry analysis of the nine bands detected after Coomas-sie blue staining of the gel enabled us to identify the corre-sponding coding gene in the genome of Q54 and make thecorrelation with the predicted functions (Fig. 4B). The mo-lecular masses estimated by SDS-PAGE were in agreementwith the masses calculated from the gene sequence for sixproteins. Discrepancies were observed for proteins detectedin bands A, B, D, and E (Fig. 4B).
Three bands of different sizes (A, B, and D) corresponded tothe MCP. Additionally, the vast majority of peptides detectedby LC-MS/MS from these three bands were mapped within thelast 273 aa of the protein. These data strongly suggest that theN-terminal amino acids (250 aa) of the MCP are cleaved offduring capsid assembly, as observed for phage c2 (48). Surpris-ingly, the molecular masses of bands A, B, and D were wellover the molecular mass calculated from the gene sequence of
FIG. 4. LC-MS/MS analysis of structural proteins of phage Q54. (A) Coomassie blue staining of a 10% SDS-polyacrylamide gel showingstructural proteins from phages Q54 and c2. Arrows and letters on the right indicate bands cut out from the gel and identified by LC-MS/MS. Thesizes (in kilodaltons) of the different proteins from the broad-range molecular mass standard (M) are indicated on the left. (B) Identification ofQ54 viral proteins from corresponding bands shown in panel A.
the orf21 product, which is 56 kDa. However, inclusion of onlythe last 273 aa of the protein, as suggested by the LC-MS/MSanalyses, would lead to a calculated mass of 29 kDa. Thus,dimers, trimers, and hexamers of this protein would corre-spond to masses of 59, 88, and 176 kDa, respectively, andwould reasonably fit with the observed masses for bands A, B,and D on SDS-PAGE (Fig. 4A). In phage c2, two major bandsof 175 kDa and 90 kDa comigrated with bands A and B fromQ54 and were previously proposed to be hexamers and trimersof the MCP, respectively (48). Additionally, there was a high-molecular-mass protein complex that barely penetrated the gelin the lanes for Q54 and c2. Although we did not try to identifythis protein complex here, it was previously shown for phage c2that this band contains a higher multimeric form of the MCP(48). According to the above data, the MCP subunits of Q54are most likely processed and cross-linked during capsid as-sembly.
The fourth striking difference was the LC-MS/MS analysis ofband E (Fig. 4), which revealed the presence of two proteins,
namely, the major tail protein (ORF23/MTP) and receptor-binding protein (ORF24/RBP). An equivalent number of pep-tides covering the two proteins were detected, suggesting anequimolar ratio. This finding was rather surprising as band I(Fig. 4A) also corresponded to ORF23 and the molecular massobserved on SDS-PAGE was close to the mass calculated fromthe orf23 sequence. Since orf23 is translated from frame 3 andorf24 from frame 1, we investigated the possibility of a trans-lational frameshift occurring between these two ORFs.
A detailed analysis of the intergenic region between orf23and orf24 revealed the presence of a CCC.UAA codon at theend of orf23 which could act as a putative �1 “shifty stop” (Fig.5A). Forward slippage (�1) of the translational machineryfrom this location would cause translation to occur on frame 1,the same as orf24. The frame 1 translational product coveringthe intergenic region starting from the CCU codon was foundto be highly similar to a segment upstream from the region ofORF45 from lactococcal phage 4268 that matched Q54 RBP(Table 1). Importantly, a �2 frameshift would also lead to the
FIG. 5. Programmed �1 translational frameshift. (A) Programmed �1 translational frameshift identified between orf23 and orf24 leading totranslation of the 48-kDa protein (band E). A potential hairpin structure, shown above the sequence, was found next to the orf23 stop codon usingthe MFold program. (B) LC-MS/MS peptide mapping of ORF23-ORF24 fusion protein. The sequence represented here corresponds to thatresulting from a �1 translational frameshift. The peptides detected by LC-MS/MS are shaded, and the translated intergenic region betweenORF23 and ORF24 is underlined.
VOL. 188, 2006 Q54, A NEW HYBRID LACTOCOCCAL PHAGE SPECIES 6109
same result, i.e., translation in frame 1, but with the addition ofa supplementary amino acid (a tyrosine) between the isoleu-cine (I) and the last proline (P) residue from ORF23. Obvi-ously, this additional amino acid would not be observed in a �1frameshift. Thus, to confirm the occurrence of a frameshiftevent and to discriminate between the two possible frameshiftmechanisms (�1 or �2), we searched for a matching peptidein band E. Analysis of the LC-MS/MS data enabled the iden-tification of specific peptides mapping to this region and lack-ing a tyrosine residue, thus corresponding to the translationalproduct resulting from a �1 frameshift (Fig. 5B). The result isan ORF23-ORF24 fusion protein with a calculated mass of 48kDa, which fits well with the mass observed on SDS-PAGE forband E (Fig. 4A).
In L. lactis, the CCC codon, which encodes a proline residue,is �5 times less frequent than the CCU codon, also encodinga proline (http://www.kazusa.or.jp/codon/index.html). Accord-ing to the proposed models, the translation machinery wouldmake a pause at the CCC codon, which eventually would forcea certain proportion of the ribosomes to slip forward (�1) tothe next CCU codon. Translation would then continue down-stream in the overlapping frame until the next stop codon.The presence of a two-stem-loop secondary structure next tothe stop codon at the end of orf23 of phage Q54 (Fig. 5A)probably favors ribosome stalling as proposed by others (43,80). Notably, the second stem-loop structure overlaps theribosome-binding site upstream of orf24 and this could alsoregulate its translation. Accordingly, we did not detectORF24 alone on SDS-PAGE (Fig. 4A), suggesting thattranslation initiation upstream of ORF24 is probably infre-quent or may not occur at all.
DISCUSSION
This paper describes the microbiological and molecularcharacterization of Q54, a prolate-headed virulent lactococcalphage that was recently proposed to form a new lactococcalphage species (26). So far, phage Q54 is unique, and it wasisolated after causing a fermentation failure during the man-ufacture of sour cream. Its very narrow host spectrum com-bined with its relative sensitivity to temperature (inability topropagate above 34°C) and to natural phage resistance mech-anisms (AbiQ and AbiT) probably explains why this phage hasbeen rarely isolated in fermentation facilities.
Determination of the complete genomic sequence of Q54confirmed that this phage clearly belongs to a different group,despite its morphological relatedness to the prolate-headed c2species. The 26,537-bp dsDNA genome was shown to havecohesive termini (cos-type phage) with several features com-mon to other lactococcal phages such as direct and invertedrepeats, as well as an A/T-rich region close to the cos site (13,19, 20, 61, 62). Nonetheless, the cos site [CCAA(N10)AACT]of phage Q54 is atypical. To our knowledge, this is the first L.lactis phage to deviate from the lactococcal phage consensuscos site [TCAN(N4–7)NACT] (62).
Another distinctive feature of phage Q54 is the very lowdegree of conservation of its deduced ORFs with proteins indatabases. Globally, only limited protein similarity was ob-served with structural proteins of lactococcal c2-like phages (22to 34% amino acid identity). Interestingly, a few early gene
products from Q54 (ORF1, ORF35, ORF40, and ORF41)were similar to early expressed proteins from lactococcalphages of the 936 species. A whole-genome comparison be-tween Q54 and c2 (Fig. 6), clearly suggests the presence of anextra module at the end of the late gene cluster of Q54. Tran-scriptional analyses demonstrated that this extra module istranscribed early after infection. Two diverging early modulesseparated by the cos site are thus present in the phage Q54genome. This is, to our knowledge, the first report of a lacticacid bacterial phage with such a genomic organization. It istempting to propose that phage Q54 is the result of past re-combination events involving 936- and c2-like phages. Geneticdrift and recombination events would have then modified thegenome of Q54 to its present form. A notable supportingobservation was the presence of a conserved DNA region be-tween Q54 and a 936-like phage that is located at the boundaryof the late and early module and falls within the endolysin gene(orf34). This region might have been a point of homologousrecombination. Phage Q54 is clearly not adapted for optimumpropagation, as suggested by its relatively long latent period(for a lactococcal phage) and small burst size. It is possible thatQ54 is currently evolving to gain more fitness in order toreplicate more efficiently.
Three of the putative ORFs encoded by Q54 (ORF4,ORF11, and ORF24) were more closely related to proteinsfrom P335-like phages than to those from any other phages.ORF131 and Sak2 from the type phage P335, which are ho-mologous to ORF4 and ORF11 of phage Q54, respectively,were previously shown to be involved in homologous recom-bination events with the host chromosome (7). In phage P335,sak2 is flanked by orf131 on one side and a gene encoding anSSB protein on the other side. In phage Q54, six genes showingno relatedness to any lactococcal phage or bacterial strainseparate orf4 and orf11. However, a gene coding for an SSBprotein from a lactococcal host immediately follows orf11 inthe genome of phage Q54. These observations strongly suggestthat Q54 acquired these genes through homologous recombi-nation with prophages from the chromosome of an L. lactishost. Whether these three genes were acquired simultaneouslyas a functional module, followed by genetic drift and insertionof six additional genes, or whether they were acquired sepa-rately through two or more recombination events is unknown.
For strict lytic phages such as members of the 936 and c2lactococcal phage groups, homologous recombination is lesslikely to occur due to the absence of temperate members (15).Consequently, finding Q54 genes homologous to bacterial orprophage sequences is intriguing. Some authors suggested thattemperate/lytic phages evolve by horizontal gene transfer withprophages residing in the host chromosome, whereas strictlyvirulent phages evolve by point mutations and small indels butrarely exchange DNA outside their group (15, 35). It is notexcluded that Q54 could have had a temperate lifestyle beforelosing its lysogeny module to become strictly virulent. How-ever, no remnant of a lysogeny module could be found in Q54,which could support this hypothesis.
Posttranslational modifications of structural proteins. TheSDS-PAGE and LC-MS/MS analyses strongly suggest that theMCP of phage Q54 is processed. Cleavage of the N-terminalpart of the protein, in addition to cross-linking, appears nec-essary for building the mature viral capsid. A similar observa-
tion was made for the type phage c2 where N-terminal se-quencing showed that the MCP was lacking its 205 N-terminalresidues, in addition to being cross-linked (48). Cross-linkingand proteolytic cleavage of the MCP subunits during assemblyhave been reported for a number of other phages (1, 16, 29, 34,46, 53, 63).
Programmed �1 translational frameshifting in tail struc-tural genes. Programmed translational frameshifting (�1 type)seems to be relatively common among dsDNA phages, as re-vealed by a recent study (79), which identified potential signalsfor such frameshifting in about 70% of the analyzed phagegenomes. Typical frameshift signals were found within the ma-jor tail protein and an overlapping gene. Only one example ofa natural �2 frameshift event was demonstrated for phage Mu(79). Two examples of �1 translational frameshifting havebeen confirmed in E. coli, which includes RF2 (17), and forrecombinant human transferrin (25). In the latter case, frame-shifting occurred at a rate of 2 to 4% and was due to thepresence of a CCC.UGA “shifty stop” at the end of the gene.This frequency was much lower than the 50% rate observed forRF2 (17). In phages, however, �1 translational frameshiftshave been reported only for the temperate phage PSA from L.monocytogenes (80). In this case, the major capsid protein andmajor tail protein were both extended in the C-terminal endsat an estimated frequency of 25%. The CCC.UGA andCCC.UCA slippery sequences were shown to be involved inthis mechanism (80). Our LC-MS/MS analysis revealed a pro-grammed � 1 translational frameshifting event involving a“shifty stop” and secondary structures and occurring betweenthe major tail protein (ORF23) and the receptor-binding pro-tein (ORF24) of phage Q54. According to the recent study byXu et al. (2004), this would represent the first case of transla-tional coupling between an MTP and an RBP (79). Also, basedon the relative band intensities on SDS-PAGE, we estimatethis event to occur at a frequency of approximately 25%. Sincethe RBP (ORF24) alone was not detected in the virion struc-ture, it suggests that only a fraction of the MTP is fused to theRBP. This chimeric protein would be most likely located at thedistal part of the tail for host recognition, while the other singleunits of the MTP would probably be associated with the chi-meric MTP/RBP for completion of the tail.
In summary, we have shown that the lytic phage Q54 repre-sents a new lactococcal phage species. Despite a familiar mor-phology, phage Q54 has a novel genome architecture and adistinctive transcriptional map, both of which suggest past re-combination events with the genomes of members of the threemost common L. lactis phage groups. However, the degree ofprotein relatedness is very low, suggesting that the breakpointlikely occurred a long time ago. Such DNA shuffling throughevolution may have led to the unusual �1 translational frame-shifting in the tail structural genes of phage Q54.
ACKNOWLEDGMENTS
We thank D. Tremblay for phage DNA preparation and J. Garneaufor host range assays.
This study was funded by a strategic grant from the Natural Sciencesand Engineering Research Council (NSERC) of Canada.
REFERENCES
1. Altermann, E., J. R. Klein, and B. Henrich. 1999. Primary structure andfeatures of the genome of the Lactobacillus gasseri temperate bacteriophage(phi)adh. Gene 236:333–346.
2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.
3. Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella,M.-Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, D. S. Campbell, T. M.Smith, J. K. McCormick, D. Y. M. Leung, P. M. Schlievert, and J. M.Musser. 2002. Genome sequence of a serotype M3 strain of group A Strep-tococcus: phage-encoded toxins, the high-virulence phenotype, and cloneemergence. Proc. Natl. Acad. Sci. USA 99:10078–10083.
4. Blatny, J. M., L. Godager, M. Lunde, and I. F. Nes. 2004. Complete genomesequence of the Lactococcus lactis temperate phage phiLC3: comparativeanalysis of phiLC3 and its relatives in lactococci and streptococci. Virology318:231–244.
5. Bolotin, A., P. Wincker, S. Mauger, O. Jaillon, K. Malarme, J. Weissenbach,S. D. Ehrlich, and A. Sorokin. 2001. The complete genome sequence of thelactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res.11:731–753.
6. Bouchard, J. D., E. Dion, F. Bissonnette, and S. Moineau. 2002. Character-ization of the two-component abortive phage infection mechanism AbiTfrom Lactococcus lactis. J. Bacteriol. 184:6325–6332.
7. Bouchard, J. D., and S. Moineau. 2000. Homologous recombination betweena lactococcal bacteriophage and the chromosome of its host strain. Virology270:65–75.
8. Bouchard, J. D., and S. Moineau. 2004. Lactococcal phage genes involved insensitivity to AbiK and their relation to single-strand annealing proteins. J.Bacteriol. 186:3649–3652.
9. Brondsted, L., S. Ostergaard, M. Pedersen, K. Hammer, and F. K.Vogensen. 2001. Analysis of the complete DNA sequence of the temperatebacteriophage TP901-1: evolution, structure, and genome organization oflactococcal bacteriophages. Virology 283:93–109.
10. Brussow, H., and F. Desiere. 2001. Comparative phage genomics and theevolution of Siphoviridae: insights from dairy phages. Mol. Microbiol. 39:213–222.
11. Carlton, R. M., W. H. Noordman, B. Biswas, E. D. de Meester, and M. J.Loessner. 2005. Bacteriophage P100 for control of Listeria monocytogenes infoods: genome sequence, bioinformatic analyses, oral toxicity study, andapplication. Regul. Toxicol. Pharmacol. 43:301–312.
12. Chandry, P. S., S. C. Moore, J. D. Boyce, B. E. Davidson, and A. J. Hillier.1997. Analysis of the DNA sequence, gene expression, origin of replicationand modular structure of the Lactococcus lactis lytic bacteriophage sk1. Mol.Microbiol. 26:49–64.
13. Chandry, P. S., S. C. Moore, B. E. Davidson, and A. J. Hillier. 1994. Analysisof the cos region of the Lactococcus lactis bacteriophage sk1. Gene 138:123–126.
14. Chibani Azaiez, S. R., I. Fliss, R. E. Simard, and S. Moineau. 1998. Mono-clonal antibodies raised against native major capsid proteins of lactococcalc2-like bacteriophages. Appl. Environ. Microbiol. 64:4255–4259.
15. Chopin, A., A. Bolotin, A. Sorokin, S. D. Ehrlich, and M. Chopin. 2001.Analysis of six prophages in Lactococcus lactis IL1403: different geneticstructure of temperate and virulent phage populations. Nucleic Acids Res.29:644–651.
16. Conway, J. F., R. L. Duda, N. Cheng, R. W. Hendrix, and A. C. Steven. 1995.Proteolytic and conformational control of virus capsid maturation: the bac-teriophage HK97 system. J. Mol. Biol. 253:86–99.
17. Craigen, W. J., and C. T. Caskey. 1986. Expression of peptide chain releasefactor 2 requires high-efficiency frameshift. Nature 322:273–275.
18. Crutz-Le Coq, A. M., B. Cesselin, J. Commissaire, and J. Anba. 2002.Sequence analysis of the lactococcal bacteriophage bIL170: insights intostructural proteins and HNH endonucleases in dairy phages. Microbiology148:985–1001.
19. Cue, D., and M. Feiss. 1993. The role of cosB, the binding site for terminase,the DNA packaging enzyme of bacteriophage lambda, in the nicking reac-tion. J. Mol. Biol. 234:594–609.
20. Cue, D., and M. Feiss. 1993. A site required for termination of packaging ofthe phage lambda chromosome. Proc. Natl. Acad. Sci. USA 90:9290–9294.
21. Curtis, F. A., P. Reed, and G. J. Sharples. 2005. Evolution of a phage RuvCendonuclease for resolution of both Holliday and branched DNA junctions.Mol. Microbiol. 55:1332–1345.
22. Desiere, F., S. Lucchini, and H. Brussow. 1999. Comparative sequence anal-ysis of the DNA packaging, head, and tail morphogenesis modules in thetemperate cos-site Streptococcus thermophilus bacteriophage Sfi21. Virology260:244–253.
23. Desiere, F., S. Lucchini, C. Canchaya, M. Ventura, and H. Brussow. 2002.Comparative genomics of phages and prophages in lactic acid bacteria.Antonie Leeuwenhoek 82:73–91.
24. Desiere, F., C. Mahanivong, A. J. Hillier, P. S. Chandry, B. E. Davidson, andH. Brussow. 2001. Comparative genomics of lactococcal phages: insight fromthe complete genome sequence of Lactococcus lactis phage BK5-T. Virology283:240–252.
25. de Smit, M. H., J. van Duin, P. H. van Knippenberg, and H. G. van Eijk.1994. CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli.Gene 143:43–47.
26. Deveau, H., S. J. Labrie, M. C. Chopin, and S. Moineau. 2006. Biodiversityand classification of lactococcal phages. Appl. Environ. Microbiol. 72:4338–4346.
27. Domingues, S., A. Chopin, S. D. Ehrlich, and M.-C. Chopin. 2004. Thelactococcal abortive phage infection system AbiP prevents both phage DNAreplication and temporal transcription switch. J. Bacteriol. 186:713–721.
28. Duda, R. L. 1998. Protein chainmail: catenated protein in viral capsids. Cell94:55–60.
29. Duda, R. L., K. Martincic, Z. Xie, and R. W. Hendrix. 1995. BacteriophageHK97 head assembly. FEMS Microbiol. Rev. 17:41–46.
30. Duplessis, M., and S. Moineau. 2001. Identification of a genetic determinantresponsible for host specificity in Streptococcus thermophilus bacteriophages.Mol. Microbiol. 41:325–336.
31. Emond, E., E. Dion, S. A. Walker, E. R. Vedamuthu, J. K. Kondo, and S.Moineau. 1998. AbiQ, an abortive infection mechanism from Lactococcuslactis. Appl. Environ. Microbiol. 64:4718–4756.
32. Emond, E., B. J. Holler, I. Boucher, P. A. Vandenbergh, E. R. Vedamuthu,J. K. Kondo, and S. Moineau. 1997. Phenotypic and genetic characterizationof the bacteriophage abortive infection mechanism AbiK from Lactococcuslactis. Appl. Environ. Microbiol. 63:1274–1283.
33. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignmenteditor and analysis program for Windows 95/98/NT. Nucleic Acids Symp.Ser. 41:95–98.
34. Hatfull, G. F., and G. J. Sarkis. 1993. DNA sequence, structure and geneexpression of mycobacteriophage L5: a phage system for mycobacterial ge-netics. Mol. Microbiol. 7:395–405.
35. Hendrix, R. W., M. C. M. Smith, R. N. Burns, M. E. Ford, and G. F. Hatfull.1999. Evolutionary relationships among diverse bacteriophages and pro-phages: all the world’s a phage. Proc. Natl. Acad. Sci. USA 96:2192–2197.
36. Ito, N., S. E. V. Phillips, C. Stevens, Z. B. Ogel, M. J. McPherson, J. N. Keen,K. D. S. Yadav, and P. F. Knowles. 1991. Novel thioether bond revealed bya 1.7 Å crystal structure of galactose oxidase. Nature 350:87–90.
37. Jarvis, A. W. 1984. Differentiation of lactic streptococcal phages into phagespecies by DNA-DNA homology. Appl. Environ. Microbiol. 47:343–349.
38. Jarvis, A. W., G. F. Fitzgerald, M. Mata, A. Mercenier, H. Neve, I. B. Powell,C. Ronda, M. Saxelin, and M. Teuber. 1991. Species and type phages oflactococcal bacteriophages. Intervirology 32:2–9.
39. Josephsen, J., N. Andersen, H. Behrndt, E. Brandsborg, G. Christiansen,M. B. Hansen, S. Hansen, E. W. Nielsen, and F. K. Vogensen. 1994. Anecological study of lytic bacteriophages of Lactococcus lactis subsp. cremorisisolated in a cheese plant over a five year period. Int. Dairy J. 4:123–140.
40. Labrie, S., and S. Moineau. 2002. Complete genomic sequence of bacterio-phage ul36: demonstration of phage heterogeneity within the P335 quasi-species of lactococcal phages. Virology 296:308–320.
41. Labrie, S., N. Vukov, M. J. Loessner, and S. Moineau. 2004. Distribution andcomposition of the lysis cassette of Lactococcus lactis phages and functionalanalysis of bacteriophage ul36 holin. FEMS Microbiol. Lett. 233:37–43.
42. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.
43. Larsen, B., R. F. Gesteland, and J. F. Atkins. 1997. Structural probing andmutagenic analysis of the stem-loop required for Escherichia coli dnaXribosomal frameshifting: programmed efficiency of 50%. J. Mol. Biol. 271:47–60.
44. Levin, M. E., R. W. Hendrix, and S. R. Casjens. 1993. A programmedtranslational frameshift is required for the synthesis of a bacteriophagelambda tail assembly protein. J. Mol. Biol. 234:124–139.
45. Lillehaug, D. 1997. An improved plaque assay for poor plaque-producingtemperate lactococcal bacteriophages. J. Appl. Microbiol. 83:85–90.
46. Liu, J., and A. Mushegian. 2004. Displacements of prohead protease genesin the late operons of double-stranded-DNA bacteriophages. J. Bacteriol.186:4369–4375.
47. Lubbers, M. W., L. J. Ward, T. P. Beresford, B. D. Jarvis, and A. W. Jarvis.1994. Sequencing and analysis of the cos region of the lactococcal bacterio-phage c2. Mol. Gen. Genet. 245:160–166.
48. Lubbers, M. W., N. R. Waterfield, T. P. Beresford, R. W. Le Page, and A. W.Jarvis. 1995. Sequencing and analysis of the prolate-headed lactococcalbacteriophage c2 genome and identification of the structural genes. Appl.Environ. Microbiol. 61:4348–4356.
49. Madera, C., C. Monjardin, and J. E. Suarez. 2004. Milk contamination andresistance to processing conditions determine the fate of Lactococcus lactisbacteriophages in dairies. Appl. Environ. Microbiol. 70:7365–7371.
50. Mahanivong, C., J. D. Boyce, B. E. Davidson, and A. J. Hillier. 2001. Se-quence analysis and molecular characterization of the Lactococcus lactistemperate bacteriophage BK5-T. Appl. Environ. Microbiol. 67:3564–3576.
51. Marchler-Bauer, A., J. B. Anderson, P. F. Cherukuri, C. DeWeese-Scott,L. Y. Geer, M. Gwadz, S. He, D. I. Hurwitz, J. D. Jackson, Z. Ke, C. J.Lanczycki, C. A. Liebert, C. Liu, F. Lu, G. H. Marchler, M. Mullokandov,B. A. Shoemaker, V. Simonyan, J. S. Song, P. A. Thiessen, R. A. Yamashita,J. J. Yin, D. Zhang, and S. H. Bryant. 2005. CDD: a Conserved DomainDatabase for protein classification. Nucleic Acids Res. 33:192–196.
52. Marchler-Bauer, A., and S. H. Bryant. 2004. CD-Search: protein domainannotations on the fly. Nucleic Acids Res. 32:327–331.
53. Martin, A. C., R. Lopez, and P. Garcia. 1998. Pneumococcal bacteriophageCp-1 encodes its own protease essential for phage maturation. J. Virol.72:3491–3494.
54. Moineau, S. 1999. Applications of phage resistance in lactic acid bacteria.Antonie Leeuwenhoek 76:377–382.
55. Moineau, S., E. Durmaz, S. Pandian, and T. R. Klaenhammer. 1993. Dif-ferentiation of two abortive mechanisms by using monoclonal antibodiesdirected toward lactococcal bacteriophage capsid proteins. Appl. Environ.Microbiol. 59:202–218.
56. Moineau, S., and C. Levesque. 2005. Control of bacteriophages in industrialfermentations, p. 285–296. In E. Kutter and A. Sulakvelidze (ed.), Bacterio-phages: biology and applications. CRC Press, Boca Raton, Fla.
57. Moineau, S., S. Pandian, and T. R. Klaenhammer. 1994. Evolution of a lyticbacteriophage via DNA acquisition from the Lactococcus lactis chromosome.Appl. Environ. Microbiol. 60:1832–1841.
58. Obregon, V., P. Garcia, R. Lopez, and J. L. Garcia. 2003. VO1, a temperatebacteriophage of the type 19A multiresistant epidemic 8249 strain of Strep-tococcus pneumoniae: analysis of variability of lytic and putative C5 methyl-transferase genes. Microb. Drug Resist. 9:7–15.
59. O’Flaherty, S., A. Coffey, R. Edwards, W. Meaney, G. F. Fitzgerald, and R. P.Ross. 2004. Genome of staphylococcal phage K: a new lineage of Myoviridaeinfecting gram-positive bacteria with a low G�C content. J. Bacteriol. 186:2862–2871.
60. Ostergaard, S., L. Brondsted, and F. K. Vogensen. 2001. Identification of areplication protein and repeats essential for DNA replication of the temper-ate lactococcal bacteriophage TP901-1. Appl. Environ. Microbiol. 67:774–781.
61. Parreira, R., R. Valyasevi, A. L. Lerayer, S. D. Ehrlich, and M. C. Chopin.1996. Gene organization and transcription of a late-expressed region of aLactococcus lactis phage. J. Bacteriol. 178:6158–6165.
62. Perrin, R., P. Billard, and C. Branlant. 1997. Comparative analysis of thegenomic DNA terminal regions of the lactococcal bacteriophages from spe-cies c2. Res. Microbiol. 148:573–583.
63. Popa, M. P., T. A. McKelvey, J. Hempel, and R. W. Hendrix. 1991. Bacte-riophage HK97 structure: wholesale covalent cross-linking between the ma-jor head shell subunits. J. Virol. 65:3227–3237.
64. Rakonjac, J., P. W. O’Toole, and M. Lubbers. 2005. Isolation of lactococcalprolate phage-phage recombinants by an enrichment strategy reveals twonovel host range determinants. J. Bacteriol. 187:3110–3121.
65. Rohwer, F., and R. Edwards. 2002. The phage proteomic tree: a genome-based taxonomy for phage. J. Bacteriol. 184:4529–4535.
66. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratorymanual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.
67. Schouler, C., S. D. Ehrlich, and M. C. Chopin. 1994. Sequence and organi-zation of the lactococcal prolate-headed bIL67 phage genome. Microbiology140:3061–3069.
68. Seegers, J. F., S. McGrath, M. O’Connell-Motherway, E. K. Arendt, M. vande Guchte, M. Creaven, G. F. Fitzgerald, and D. van Sinderen. 2004. Mo-lecular and transcriptional analysis of the temperate lactococcal bacterio-phage Tuc2009. Virology 329:40–52.
69. Spinelli, S., V. Campanacci, S. Blangy, S. Moineau, M. Tegoni, and C.Cambillau. 2006. Modular structure of the receptor binding proteins ofLactococcus lactis phages: the RBP structure of the temperate phageTP901-1. J. Biol. Chem. 281:14256–14262.
70. Stanley, E., L. Walsh, A. van der Zwet, G. F. Fitzgerald, and D. van Sinderen.2000. Identification of four loci isolated from two Streptococcus thermophilusphage genomes responsible for mediating bacteriophage resistance. FEMSMicrobiol. Lett. 182:271–277.
71. Sturino, J. M., and T. R. Klaenhammer. 2004. Bacteriophage defense sys-tems and strategies for lactic acid bacteria. Adv. Appl. Microbiol. 56:331–378.
72. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lacticstreptococci and their bacteriophages. Appl. Microbiol. 29:807–813.
73. Tremblay, D. M., M. Tegoni, S. Spinelli, V. Campanacci, S. Blangy, C.Huyghe, A. Desmyter, S. Labrie, S. Moineau, and C. Cambillau. 2006.Receptor-binding protein of Lactococcus lactis phages: identification andcharacterization of the saccharide receptor-binding site. J. Bacteriol. 188:2400–2410.
74. Trotter, M., O. McAuliffe, M. Callanan, R. Edwards, G. F. Fitzgerald, A.Coffey, and R. P. Ross. 2006. Genome analysis of the obligately lytic bacte-riophage 4268 of Lactococcus lactis provides insight into its adaptable nature.Gene 366:189–199.
75. van Sinderen, D., H. Karsens, J. Kok, P. Terpstra, M. H. Ruiters, G. Venema,and A. Nauta. 1996. Sequence analysis and molecular characterization of thetemperate lactococcal bacteriophage r1t. Mol. Microbiol. 19:1343–1355.
76. Vegge, C. S., F. K. Vogensen, S. McGrath, H. Neve, D. van Sinderen, and L.Brondsted. 2006. Identification of the lower baseplate protein as the antire-ceptor of the temperate lactococcal bacteriophages TP901-1 and Tuc2009. J.Bacteriol. 188:55–63.
77. Wang, I. N., D. L. Smith, and R. Young. 2000. Holins: the protein clocks ofbacteriophage infections. Annu. Rev. Microbiol. 54:799–825.
VOL. 188, 2006 Q54, A NEW HYBRID LACTOCOCCAL PHAGE SPECIES 6113
78. Weigel, C., and H. Seitz. 2006. Bacteriophage replication modules. FEMSMicrobiol. Rev. 30:321–381.
79. Xu, J., R. W. Hendrix, and R. L. Duda. 2004. Conserved translational frame-shift in dsDNA bacteriophage tail assembly genes. Mol. Cell 16:11–21.
80. Zimmer, M., E. Sattelberger, R. B. Inman, R. Calendar, and M. J. Loessner.2003. Genome and proteome of Listeria monocytogenes phage PSA: an un-
usual case for programmed �1 translational frameshifting in structural pro-tein synthesis. Mol. Microbiol. 50:303–317.
81. Zuker, M., D. H. Mathews, and D. H. Turner. 1999. Algorithms and ther-modynamics for RNA secondary structure prediction: a practical guide inRNA biochemistry and biotechnology. Kluwer Academic Publishers, Dor-drecht, The Netherlands.
