Genome Sequences of Gordonia Bacteriophages Obliviate, UmaThurman, and Guacamole

We describe three newly isolated phages—Obliviate, UmaThurman, and Guacamole—that infect Gordonia terrae 3612. The three genomes are related to one another but are not closely related to other previously sequenced phages or prophages. The three phages are predicted to use integration-dependent immunity systems as described in several mycobacteriophages.

members of the taxonomic order Corynebacteriales. The hundreds of sequenced mycobacteriophages display considerable genetic diversity (1), but few Gordonia phage genome sequences are available, and their diversity and relationships to the mycobacteriophages are ill defined (2)(3)(4)(5)(6)(7). Isolation and characterization of Gordonia phages in the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program assists in addressing these questions (1,8).
Phages Obliviate, UmaThurman, and Guacamole were isolated by direct plating of soil samples from Pittsburgh, Pennsylvania, USA, on lawns of G. terrae 3612. They were then plaquepurified and amplified, and their DNA was extracted. All three phages have similar virion morphologies with 50-nm diameter isometric heads and long flexible tails, approximately 250 nm long. Each genome was sequenced using the Illumina MiSeq platform, and 140-bp single-end reads were assembled into major single contigs with lengths of 49,286 bp, 50,127 bp, and 49,894 bp with 619-fold, 1,434-fold, and 809-fold coverage for Obliviate, UmaThurman, and Guacamole, respectively. All have defined ends with 10-base 3= extensions (Obliviate and Guacamole: 5=-TCGCCGGTGA; UmaThurman: 5=-TCTCCGGTGA). The GC contents of the genomes are 67.2, 67.5, and 67.0%, similar to G. terrae (67.8%). The three phages share extensive nucleotide sequence similarity with pairwise 96% nucleotide sequence identity spanning 72 to 82% of their genome lengths. The greatest similarities are within the virion structural and assembly genes, with interspersed segments of similarity within the nonstructural genes. The phages do not share extensive nucleotide sequence similarity with other phages or predicted prophages, although there are two small segments (~1.5 kb) with similarity to putative capsid assembly protease and lysis genes of a potential prophage in Gordonia sp. KTR9 (9).
Using GeneMark and GLIMMER (10, 11) we identified 80, 83, and 78 protein-coding genes in Obliviate, UmaThurman, and Guacamole, respectively; none of the genomes encode tRNAs. All the predicted genes are transcribed rightward, with the exception of five to seven leftward-transcribed genes near the genome centers that include putative immunity repressors and integrase genes. The genome left arms contain the virion structure and assembly genes, and the right arms contain nonstructural genes, including recET recombinases. The lysis genes are unusually located amid the phage tail gene cluster, and two genes, Obliviate 19 and 20, encode endolysin functions-the peptidase and glycoside hydrolase domains, respectively.
Obliviate is predicted to use an integration-dependent immunity system as described for some mycobacteriophages (12,13), characterized by the location of the phage attachment site (attP) within the repressor gene (38), and a degradation tag (-DAA) at the C-terminus of gp38. Obliviate is predicted to integrate at an attB site overlapping a Gordonia tRNA thr gene (KRT9_RS04270). Guacamole encodes a distantly related repressor (gp40; 41% amino acid [aa] identity with Obliviate gp38), although it contains the same attP core and is predicted to integrate at the same attB site. The UmaThurman repressor (gp36) is more distantly related to the Obliviate/Guacamole repressors (Ͻ40% aa identity) and contains a different attP corresponding to an attB site overlapping a tRNA arg gene conserved in many actinobacterial strains.
Nucleotide sequence accession numbers. The Obliviate, UmaThurman, and Guacamole genomes are available from GenBank under the accession numbers KU963254, KU963251, and KU963259.

FUNDING INFORMATION
This work, including the efforts of Graham F. Hatfull, was funded by Howard Hughes Medical Institute (HHMI) (54308198).