Genome sequence and global gene expression of Q54, a new phage species linking the 936 and c2 phage species of Lactococcus lactis

Abstract

The lytic lactococcal phage Q54 was previously isolated from a failed sour cream production. Its complete genomic sequence (26,537 bp) is reported here, and the analysis indicated that it represents a new Lactococcus lactis phage species. A striking feature of phage Q54 is the low level of similarity of its proteome (47 open reading frames) with proteins in databases. A global gene expression study confirmed the presence of two early gene modules in Q54. The unusual configuration of these modules, combined with results of comparative analysis with other lactococcal phage genomes, suggests that one of these modules was acquired through recombination events between c2- and 936-like phages. Proteolytic cleavage and cross-linking of the major capsid protein were demonstrated through structural protein analyses. A programmed translational frameshift between the major tail protein (MTP) and the receptor-binding protein (RBP) was also discovered. A "shifty stop" 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 and the first case of translational coupling between an MTP and an RBP. Thus, phage Q54 represents a fascinating member of a new species with unusual characteristics that brings new insights into lactococcal phage evolution.

FIG. 1.

Electron micrograph of phage Q54. Bar, 50 nm.

FIG. 2.

Q54 complete genome (26,537 bp). (A) Genomic organization. A scale (in kilobases) is shown above the genetic map. Each arrow represents a putative ORF, and the numbering refers to Table 1. Gray arrows represent ORFs for which a putative function could be inferred from bioinformatics analyses (indicated above the ORFs). White arrows represent ORFs for which no putative function could be attributed. Hairpins represent putative Rho-independent transcriptional terminators. Arrows with heavy outlines represent gene products detected by LC-MS/MS analysis (Fig. 4). (B) Phage Q54 mRNA transcripts detected in the course of an infection of L. lactis SMQ-562. Short thick lines above the transcripts indicate probes. The size (in kilobases) is indicated on the right or left of each transcript (arrows), and the line thickness is representative of their relative abundance, with the thicker line corresponding to the highest concentration. Arrowheads show the direction of transcription, and the temporal expression (early or late) is indicated below the genome scale.

FIG. 3.

Analysis of Q54 cos region. (A) Analysis of the cos site and flanking regions. Typical features found in the cos vicinity of other phages, such as direct repeats (DR1 and DR2), inverted repeats (IR), A/T-rich region, G- and C-rich segments (G and C box, respectively), and λ-R-like sequences (λ-like), are indicated. Details of the cohesive termini are also shown. The sites of cleavage resulting in 3′-overhang termini are indicated by vertical arrows. Italic characters with reverse highlighting represent nucleotides that are identical to a consensus sequence found in all lactococcal phages for which the cos region has been sequenced (see text for details). (B) Multiple alignment of λ-R-like sequences from Q54, c2, and sk1 with those of λ-R sequences from phage λ. Conserved nucleotides are shaded.

FIG. 4.

LC-MS/MS analysis of structural proteins of phage Q54. (A) Coomassie blue staining of a 10% SDS-polyacrylamide gel showing structural 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. The sizes (in kilodaltons) of the different proteins from the broad-range molecular mass standard (M) are indicated on the left. (B) Identification of Q54 viral proteins from corresponding bands shown in panel A.

FIG. 5.

Programmed +1 translational frameshift. (A) Programmed +1 translational frameshift identified between orf23 and orf24 leading to translation of the 48-kDa protein (band E). A potential hairpin structure, shown above the sequence, was found next to the orf23 stop codon using the MFold program. (B) LC-MS/MS peptide mapping of ORF23-ORF24 fusion protein. The sequence represented here corresponds to that resulting from a +1 translational frameshift. The peptides detected by LC-MS/MS are shaded, and the translated intergenic region between ORF23 and ORF24 is underlined.

FIG. 6.

Comparative genomics between Q54 and phages of the 936 and c2 species. Genome alignments present similarities between proteins of Q54 and phages c2 (accession no. NC_001706) and sk1 (accession no. NC_001835). ORFs from Q54 showing similarity to ORFs from these phages are linked by gray shading. The absence of shading means there was no significant similarity. The percent amino acid identity indicated inside the shading is representative of the aligned region only, whereas values in parentheses represent the percent amino acid identity over the entire length of the smaller of the two aligned proteins. Phages bIL67 (accession no. L33769) and bIL170 (accession no. NC_001909) were added into the alignment to show the extensive homology existing between phages of the same species. Color shading was used when two genomes from phages of the same species were aligned to discriminate between similarity of >80% amino acid identity (dark color) and similarity of ≤79% amino acid identity (light color).