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. 2010 Oct;192(20):5441-53.
doi: 10.1128/JB.00709-10. Epub 2010 Aug 13.

Brochothrix thermosphacta bacteriophages feature heterogeneous and highly mosaic genomes and utilize unique prophage insertion sites

Samuel Kilcher  1 , Martin J Loessner, Jochen Klumpp
Affiliations

Brochothrix thermosphacta bacteriophages feature heterogeneous and highly mosaic genomes and utilize unique prophage insertion sites

Samuel Kilcher et al. J Bacteriol. 2010 Oct.

Abstract

Brochothrix belongs to the low-GC branch of Gram-positive bacteria (Firmicutes), closely related to Listeria, Staphylococcus, Clostridium, and Bacillus. Brochothrix thermosphacta is a nonproteolytic food spoilage organism, adapted to growth in vacuum-packaged meats. We report the first genome sequences and characterization of Brochothrix bacteriophages. Phage A9 is a myovirus with an 89-nm capsid diameter and a 171-nm contractile tail; it belongs to the Spounavirinae subfamily and shares significant homologies with Listeria phage A511, Staphylococcus phage Twort, and others. The A9 unit genome is 127 kb long with 11-kb terminal redundancy; it encodes 198 proteins and 6 tRNAs. Phages BL3 and NF5 are temperate siphoviruses with a head diameter of 56 to 59 nm. The BL3 tail is 270 nm long, whereas NF5 features a short tail of only 94 nm. The NF5 genome (36.95 kb) encodes 57 gene products, BL3 (41.52 kb) encodes 65 products, and both are arranged in life cycle-specific modules. Surprisingly, BL3 and NF5 show little relatedness to Listeria phages but rather demonstrate relatedness to lactococcal phages. Peptide mass fingerprinting of viral proteins indicate programmed -1 translational frameshifts in the NF5 capsid and the BL3 major tail protein. Both NF5 and BL3 feature circularly permuted, terminally redundant genomes, packaged by a headful mechanism, and integrases of the serine (BL3) and tyrosine (NF5) types. They utilize unique target sequences not previously described: BL3 inserts into the 3' end of a RNA methyltransferase, whereas NF5 integrates into the 5'-terminal part of a putative histidinol-phosphatase. Interestingly, both genes are reconstituted by phage sequence.

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Figures

FIG. 1.
FIG. 1.
Electron micrographs of A9, NF5, and BL3. (A) A9 virions with contracted tails show double baseplate structures. Head symmetry is hexagonal; the darker heads are empty. Samples are negatively stained with phosphotungstic acid (PTA). (B) Close-up views of phage A9 with uncontracted (left panel, uranyl acetate [UA] stain) and contracted (right panel, PTA stain) tails; the thin whiskers are visible in the contracted state. Ammonium molybdate (AM) (C)- and PTA (D)-stained NF5 virions reveal a siphovirus morphology, with noncontractile tails and complex baseplates with appendages. (E) Phage BL3 (AM stained) is a siphovirus with a long flexible tail. (F) A single tail fiber with three characteristic nodes is visible in PTA-stained BL3 samples (indicated with arrows).
FIG. 2.
FIG. 2.
Genome map alignments of Brochothrix phage A9, Listeria phage A511, and Enterococcus phage EF24C (A) and Brochothrix phages BL3 and NF5, Lactococcus phage Tuc2009, and prophage SK11_4 (B). Arrows indicate identified ORFs and are drawn to scale. Gene products with sequence identities ≥20% are linked by shaded areas. Color codes for functional clusters apply to Brochothrix bacteriophages only. Putative functions of selected gene products are indicated (see Tables S3 to S5 in the supplemental material). Abbreviations: terS and terL, small and large terminase subunits; scf, scaffold; cps, major capsid; tsh, tail sheath; tmp, tape measure; int, integrase; rep, repressor; ssb, single strand binding.
FIG. 3.
FIG. 3.
Structure analysis of the DNA molecules of phage A9, NF5, and BL3. (A) PFGE of full-length phage DNA. Size markers: λmix, lambda mix marker 19 (Fermentas); M1 and M2, PFG midrange markers I and II (NEB). BL3 and NF5 genomic DNAs run at slightly larger sizes than their unit genome lengths of 41.3 and 37 kb. A9 DNA exhibits approximately the same physical size as Listeria phage A511 DNA (137.62 kb) (37). (B) Gel electrophoresis of PacI-digested A9 DNA after Bal31 nuclease treatment for the indicated time intervals. Terminal fragments of the nonpermuted genomes disappear over time (indicated by arrows). (C) Restriction enzyme profiles of PstI-digested NF5 DNA and SwaI-digested BL3 DNA; the putative pac fragments are indicated by black arrowheads. (D and E) EcoRI (NF5)- and Van91I (BL3)-digested genomic DNA after Bal31 nuclease treatment for the indicated time intervals. Arrows with square heads indicate restriction fragments which were not predicted in silico from a circular molecule (pac fragment, 4.2 kb; smeary band, >10 kb). The “submolar” pac fragment disappears after 10 min of Bal31 treatment. Arrows with pointed heads indicate restriction fragments “downstream” from the putative pac site-containing fragment. Their consecutive appearance within the respective genome is indicated by numbering and correlates with the order of disappearance, indicating incomplete permutation of the DNA molecules based on the limited number of genomes in each replication concatemer.
FIG. 4.
FIG. 4.
Phylogenetic relationships of the large terminase proteins of the three sequenced Brochothrix phages, including other phages with known packaging mechanisms (13). Groups with similar packaging strategies are indicated. The tree is based upon neighbor joining with 1,000 bootstrap replicates, calculated from an alignment with the following parameters: gap open cost, 10; gap extension cost, 1; and end gap cost, free (CLC Genomics Workbench).
FIG. 5.
FIG. 5.
Analysis of A9, BL3, and NF5 protein composition. (A) Identification of virus structural proteins by peptide fingerprinting. Protein molecular mass marker masses (in kilodaltons) and identified gene products are indicated. Abbreviations: Tsh, tail sheath protein; Cps, major capsid protein; Tmp, tape measure protein; gp, gene product; -L, longer protein variants produced by programmed translational frameshifting. (B) Proposed locations of frameshifts in structural protein genes of Brochothrix bacteriophages. The type of frameshift, location within the genome, and slippery sequences (lines) are indicated. The putative frameshifts in phages NF5 and A9 were experimentally verified by peptide mass fingerprinting (see Table S6 in the supplemental material).
FIG. 6.
FIG. 6.
Detail view of BL3 (A) and NF5 (B) integration site in the HER1187 and HER1188 genome, respectively. Regions of identity between phage and host sequence (core sequences) are boxed.
FIG. 7.
FIG. 7.
Identification of intervening sequences (IVS) in the A9 genome. (A) Splicing variants producing the most significant database hits were annotated. The putative intein was identified by Blastp. RNR, ribonucleotide reductase. (B) Alignment of the spliced proteins with homologous sequences which do not contain IVS (a, RNRα subunit of Granulicatella elegans ATCC 700633; b, RNRβ subunit of Enterococcus casseliflavus EC30; A511 TerL, large terminase of Listeria phage A511). Amino acid sequence identities and gaps in the alignment are indicated. Small triangles mark the putative processing sites.
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