Expanding the Diversity of Myoviridae Phages Infecting Lactobacillus plantarum-A Novel Lineage of Lactobacillus Phages Comprising Five New Members

Abstract

Lactobacillus plantarum is a bacterium with probiotic properties and promising applications in the food industry and agriculture. So far, bacteriophages of this bacterium have been moderately addressed. We examined the diversity of five new L. plantarum phages via whole genome shotgun sequencing and in silico protein predictions. Moreover, we looked into their phylogeny and their potential genomic similarities to other complete phage genome records through extensive nucleotide and protein comparisons. These analyses revealed a high degree of similarity among the five phages, which extended to the vast majority of predicted virion-associated proteins. Based on these, we selected one of the phages as a representative and performed transmission electron microscopy and structural protein sequencing tests. Overall, the results suggested that the five phages belong to the family Myoviridae, they have a long genome of 137,973-141,344 bp, a G/C content of 36.3-36.6% that is quite distinct from their host's, and surprisingly, 7 to 15 tRNAs. Only an average 41/174 of their predicted genes were assigned a function. The comparative analyses unraveled considerable genetic diversity for the five L. plantarum phages in this study. Hence, the new genus "Semelevirus" was proposed, comprising exclusively of the five phages. This novel lineage of Lactobacillus phages provides further insight into the genetic heterogeneity of phages infecting Lactobacillus sp. The five new Lactobacillus phages have potential value for the development of more robust starters through, for example, the selection of mutants insensitive to phage infections. The five phages could also form part of phage cocktails, which producers would apply in different stages of L. plantarum fermentations in order to create a range of organoleptic outputs.

Keywords: Lactobacillus plantarum; annotation; comparative genomics; diversity; isolation; new genus; phage; phylogenetics.

Figure 1

Transmission electron micrographs of L. plantarum phage Dionysus negatively stained with 2% (w/v) uranyl acetate. Triangles and arrows indicate the terminal baseplate structure and representative flexible appendages with terminal globular structures, respectively, attached underneath them. The single distal tail fibers terminating with three distinct knob-like structures are indicated by the star symbol. Phage Dionysus is shown with extended tail sheath (a) and contracted tail sheath (b), substantiating that it is a Myoviridae phage.

Figure 2

BLASTn heatplot generated using Gegenees. All-against-all comparisons were run with fragment length 50 bp, step size 25 bp, and threshold 0%. The studied phages (25–29) form a separate group.

Figure 3

Phylogenetic trees for phages Semele, Bacchae, Dionysus, Iacchus and Bromius and other Lactobacillus phages yielding average similarity of at least 0.05 or higher with Gegenees. (A) Tree constructed using the major capsid proteins, (B) tree constructed using the large subunit terminases. The amino acid sequences were compared with the “One Click mode” (http://phylogeny.Lirmm.fr/). Bacillus phage Bastille proteins were used as outliers.

Figure 4

BLASTp comparison between and within proteomes of the five phages and their closest phage relative LpeD using CMG-biotools system. Phage pairs with >50% proteome homology are shown in green and those with <50% are depicted in grey. Red signifies the presence of one or more groups of paralogous proteins within a phage proteome.

Figure 5

BLASTn comparisons of the genomes of the five phages with distant relatives LpeD and LP65 using Easyfig. Arrows represent the locations of genes and lines represent the level of homology between each tandemly placed group of phages. In the genomes of the five phages, genes are coloured according to the assigned function of the protein each gene encodes. These are: turquoise for DNA packaging, yellow for cell wall and membrane degradation, green for transcription and translation takeover, and blue for selfish genetic elements. Genes in red are part of the morphogenesis module and genes in deep purple are part of the DNA metabolism, replication, recombination and repair module. Black colour denotes genes that encode either hypothetical proteins or other predicted proteins with unclear functions. Figures S1–S5 follow the same colour scheme.