Genome organization and characterization of the virulent lactococcal phage 1358 and its similarities to Listeria phages

Abstract

Virulent phage 1358 is the reference member of a rare group of phages infecting Lactococcus lactis. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the Siphoviridae family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two Listeria monocytogenes phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.

FIG. 1.

Electron micrograph of phage 1358. Scale bar, 100 nm.

FIG. 2.

Comparison of the GC content of phage 1358 with that of other lactococcal phages. The GC contents of these phages are in relation to their genome positions. pb, base pairs.

FIG. 3.

Genomic organizations of virulent lactococcal phage 1358 and L. monocytogenes phages P40 and P35. The scale above the map is in base pairs. Each arrow represents a putative ORF, and the numbering refers to Table 1. The putative functions inferred from bioinformatic or structural analyses are indicated above the ORFs. For the phage 1358 genome, arrows with thick outlines represent gene products detected by LC-MS/MS analyses. Gray shadows linking ORFs with the same color indicate more than 23% amino acid identity. White arrows represent ORFs for which no putative function can be attributed. Red arrows represent ORFs sharing identity with at least one phage shown here, and green arrows represent ORFs that do not share identity. Blue arrows represent ORFs sharing identity with proteins for which a function was attributed.

FIG. 4.

LC-MS/MS analysis of phage 1358 structural proteins. (Left) Coomassie blue staining of a 12% SDS-polyacrylamide gel showing phage 1358 structural proteins. Letters on the right indicate bands cut out of the gel and identified by LC-MS/MS. The sizes (in kDa) of the proteins in the broad-range molecular mass standard (M) are indicated on the left. (Right) Identification of phage 1358 proteins from corresponding bands shown in the left panel. Numbers at the right correspond to the numbers indicated in the left panel. ^aCalculated from the gene sequence.