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X Marks the Spot:

Exploring Repetitive Sequences in Cluster Q Mycobacteriophages

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Phage Lab II: Rachel Martindale and Emma Topping

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Why do we analyze bacteriophage genomes?

  • Complex relationships and evolution of phages�and bacteria�

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  • FDA approval for phage therapy against antibiotic �resistant bacteria
    • Absence of integrase gene
    • Absence of genes encoding toxins, antimicrobial�resistance, virulence factors, etc.

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Using Phamerator to compare phage genomes

  • Grouped into clusters (50% sequence similarity) and subclusters �(75% sequence similarity)�
  • Exhibit synteny (conservation of gene order)

Purple ≈ identical (E-value = 0)

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Red = significant similarity

(E-value = 1e-4 )�

White = no similarity

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Phage genomes are highly mosaic

  • Mosaicism = patchy sequence similarity when phages are compared

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  • Horizontal gene transfer

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Cluster Q Mycobacteriophages

  • 20 members�
  • Generally temperate
    • Integrase
    • Repressor
    • Excise protein

Gancho

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Cluster Q phages exhibit a unique pattern of sequence repeats

Amochick (Q)

Giles (Q)

Amymech (Q)

Webster2 (Q)

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Repetitive sequences occur in the tail proteins of some other phage clusters

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These repetitive sequences are not the same between different clusters

BeesKnees (A1)

Grizzly (G1)

Giles (Q)

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Non-coding region upstream of the cluster Q integrase gene

has bacterial BLAST alignments

Giles (Q)

Amymech

(Q)

Cluster Q

phages

Mycobacteria spp.

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BLAST results of Amymech’s gene 25-29:

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Dot plot comparison of cluster Q major capsid protein

Amymech

Daegal

Hail

Kinbote

Amymech

Daegal

Hail

Kinbote

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Dotplot comparison of cluster Q minor tail protein has sequence inversions

Amymech

Daegal

Hail

Kinbote

Green = forward alignment Red = reverse alignment

Amymech

Daegal

Hail

Kinbote

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Dot plot comparison of cluster G1 minor tail protein does not have sequence inversions

Annihilator

Grizzly

Hotshotbaby7

Annihilator

Grizzly

Hotshotbaby7

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Cluster Q repetitive sequences may�be related to inversion mutations

  • Phage are highly mutable�and evolve quickly

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  • DNA can recombine after breaking

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  • Phage encoded recombinases may be more effective

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Evidence of modular genetics in �tail proteins

  • Modular = interchangeable

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  • Research into cryophage suggests “homologous genes present in the pan-genome interchange to create new phage variants”

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  • Interchangeable tail proteins could benefit phage pathogeny

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Conclusions:

  • Bacterial alignments ahead of integrase gene suggest possible attP site
  • Possible sequence inversions are unique to cluster Q minor tail proteins

Future Directions:

  • Remove possible attP site → is the phage unable to form a lysogen
  • Genetically replace cluster Q minor tail protein with related minor tail protein lacking repeats and inversions

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Acknowledgments

  • Dr Fillman and Dr Guild
  • Dr Chuong and Alex Hirano
  • Our Phage Lab II classmates
  • Howard Hughes Medical Institute
  • MCDB Department

References�Bellas CM, Schroeder DC, Edwards A, Barker G, Anesio AM. 2020. Flexible genes establish widespread bacteriophage pan-genomes in cryoconite hole ecosystems. Nat Commun. 11(1):4403. doi:10.1038/s41467-020-18236-8.