Genomic diversity of bacteriophages infecting Microbacterium spp

Abstract

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Fig 1. Geographical distribution of Microbacterium phages.

Geographic distribution of the isolation sites of Microbacterium phages with completely sequenced genomes.

Fig 2. Microbacterium phage virion morphologies.

Representative virion particles of each Microbacterium phage cluster show a predominance of siphoviridae morphologies; Cluster EK and singleton Burro are podoviridae. All have isometric capsids with the exception of Count, which has a prolate capsid.

Fig 3. Heatmap of Microbacterium phage average nucleotide identities (ANIs).

Pairwise average nucleotide identities were calculated for 116 Microbacterium phages using DNA Master with default settings. The heatmap was generated using R and the ‘heatmap2’ function, which determines distances between each genome and calculates the optimal genome order for representation, using distance parameter and clustering methods of ‘maximum’ and ‘single’, respectively. Genome clusters are shown on the axes, colored according to cluster, with singletons show in black; subclusters are indicated with numbers alongside their cluster designations. Phage vB_MosX-ISF9 genome is not included in these analyses [30].

Fig 4. Relationships among Microbacterium phages and their bacterial hosts.

A. A network phylogeny of Microbacterium phages. The predicted proteins of all 116 Microbacterium phage were sorted into 1975 phamilies (phams) according to shared amino acid sequence similarities using Phamerator [34]. Each genome was then assigned a value reflecting the presence or absence of a pham member; the genomes were compared and displayed using Splitstree [35]. The clusters and subclusters derived from dotplot and ANI comparisons are indicated with larger colored circles. Smaller colored circles at the nodes indicate the bacterium host used for isolation, as noted in the key. Phages isolated on M. foliorum NRRL B-24224 have no small colored circle at the node. The scale bar indicates 0.01 substitutions/site. B. Evolutionary relationships of bacterial host taxa, using MEGA7 [–38]. The number of phages isolated on each host is shown in parentheses.

Fig 5. Pairwise alignment of Microbacterium phage Cluster EA genomes.

A representative genome from each EA subcluster phage is shown. The sole Subcluster EA7 phage Theresita is shown at the bottom as it is substantially different from the others in the right halves of the genomes. Pairwise nucleotide sequence similarities are displayed with spectrum-coloring between genomes, with violet representing greatest similarity and red the least similar, above a threshold E value of 10⁻⁵. Genes are represented as boxes above or below the genomes reflecting rightwards- and leftwards-transcription respectively. Genes are colored according to their phamily designations using Phamerator [34] and database Actinobacteriophage_2422. White boxes represent ‘orphams’, genes with no close relatives in this dataset.

Fig 6. Genome organization of Microbacterium Subcluster EA1 phage TeddyBear.

A. The genome of Microbacterium phage TeddyBear is shown with predicted genes represented as boxes above or below the genome reflecting rightwards- and leftwards-transcription respectively. Genes are colored according to their phamily designations using Phamerator [34] and database Actinobacteriophage_2422. The phamily numbers shown above each gene with the number of phamily members in parentheses. B. Pairwise alignment of Microbacterium phages Golden, Theresita, Goodman, and Johann genomes. See Fig 5 for map details. Theresita shares 45.2% and 23.6% average gene content with Golden and Goodman, respectively.

Fig 7. Genome organization of Microbacterium Cluster EB phage Dismas.

See Fig 6A for details.

Fig 8. Genome organization of Microbacterium cluster EC phage Quahwah.

See Fig 6A for details. Vertical arrows show the positions of 18 bp repeated motifs corresponding to the consensus 5’-TAGaCTCTaGGTgTaAgC, where upper case letters indicate complete conservation, and lower case letters are those appearing in at least 9 of the 12 instances. Each instance of the motif has no more than eight departures from the 18 bp consensus sequence and they are numbered as listed in S10A Fig.

Fig 9. Genome organization of Microbacterium Subcluster ED1 phage Hortus1.

See Fig 6A for details.

Fig 10. Genome organization of Microbacterium Cluster EE phage BurtonThePup.

See Fig 6A for details.

Fig 11. Genome organization of Microbacterium cluster EF phage AnnaSerena, See Fig 6A for details.

The small red vertical arrows indicate the locations containing both conserved SAS and ESAS sequences; black arrows indicate the locations with an ESAS motif that lack an associated SAS. The SAS motifs are numbered as listed in S10B and S10C Fig.

Fig 12. Genome organization of Microbacterium cluster EG phage hyperion.

See Fig 6A for details. Vertical arrows indicate the positions of conserved short inverted repeat sequences and numbered as listed in S10D Fig.

Fig 13. Genome organization of Microbacterium cluster EH phage percival.

See Fig 6A for details.

Fig 14. Genome organization of Microbacterium cluster EI phage MementoMori.

See Fig 6A for details. Vertical arrows indicate the locations of 30 bp repeated sequences in intergenic regions, numbered as shown in S10E.

Fig 15. Genome organization of Microbacterium Cluster EJ phage Goodman.

See Fig 6A for details.

Fig 16. Microbacterium cluster EK phages and singleton burro.

A. Genome organization of phages ArMaWen, Akoni and Burro, clusters EK1, EK2, and a singleton, respectively. See Fig 5 for details. B. SDS-PAGE of Burro virions. Lanes 1 and 2 correspond to Burro virions purified through one and two CsCl density gradients, respectively. M, marker with protein sizes indicated in kDa.

Fig 17. Genome organization of Microbacterium Cluster EL phages, Camille and Count.

See Fig 5 for details.

Fig 18. Genome organization of Microbacterium Singleton phage Appa.

See Fig 6A for details.

Fig 19. Genome organization of Microbacterium Singleton phage Hendrix.

See Fig 6A for details.

Fig 20. Genome organization of Microbacterium Singleton phage Triscuit.

See Fig 6A for details.

Fig 21. Genome organization of Microbacterium Singleton phage ValentiniPuff.

See Fig 6A for details.

Fig 22. Genome organization of Microbacterium Singleton phage Zeta1847.

See Fig 6A for details.

Fig 23. Comparison of phage genomic diversity by host genera.

A. Genomic similarity plot comparing gene content dissimilarity (gcd) to whole genome nucleotide distance, as previously reported [49]. Each data point reflects a genome comparison involving one phage that infects the indicated host genus and (orange) a second phage that infects the indicated host genus or (black) one phage that infects a different host genus. n = number of phages that infect the indicated host genus. B. Box plot comparing the MaxGCDGap of phages from different host genera. Each data point is a phage genome, the box depicts the middle 50% of data, and the black bar represents the median. Number of phages per host genus as in panel A. C. Three representative genome networks highlight genomic relationships of Microbacterium phages to phages of other host genera. Each node represents a phage genome and is colored according to host genus. Edges between nodes represent pairs of phages that exhibit ‘intra-cluster’ genomic similarities (as measured by gene content dissimilarity and nucleotide distance from panel A). Selected phage names or cluster designations are highlighted for reference.