ABSTRACT
Cluster EC ClearAsMud and cluster EA4 Kauala are lytic Siphoviridae bacteriophages that were isolated from soil in southern California using Microbacterium foliorum NRRL B-24224 as the host. The ClearAsMud and Kauala genomes are 52,987 bp and 39,378 bp, respectively, and contain 92 and 56 predicted protein-coding genes, respectively.
ANNOUNCEMENT
In order to characterize viral diversity and evolution, ClearAsMud and Kauala were isolated as part of the La Sierra University 2020 Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course (1). ClearAsMud was isolated from a mud sample from Riverside, California, while Kauala was isolated from a potted plant in Rancho Cucamonga, California. Phages were purified and amplified in Microbacterium foliorum NRRL B-24224 grown in peptone-yeast-calcium agar (PYCa) medium at 30°C, with shaking at 250 rpm. Both phages produced clear plaques and lacked integrase genes. Negative-staining transmission electron microscopy showed that both phages had icosahedral capsids with flexible, noncontractile tails (Fig. 1).
Transmission electron micrographs of Microbacterium phages ClearAsMud (A) and Kauala (B). Phage lysates were negatively stained with 1% uranyl acetate.
After two rounds of plaque purification, DNA was purified from high-titer lysates using the Wizard DNA clean-up kit (A7280; Promega). Sequencing libraries were prepared using the NEBNext Ultra II FS DNA library prep kit with dual-indexed barcoding. The libraries were run on an Illumina MiSeq instrument, yielding 2.2 million and 925,630 paired-end 150-base reads for Kauala and ClearAsMud, respectively. This was sufficient to provide 27-fold and 7,821-fold coverage for ClearAsMud and Kauala, respectively. Raw reads were assembled with Newbler (v2.9) with default settings, yielding a single phage contig for each, and results were checked for completeness, accuracy, and genome termini using Consed. The genomes were manually annotated using the Phage Evidence Collection and Annotation Network (PECAAN) (http://pecaan.kbrinsgd.org), which collects information from Starterator (v1.2), Glimmer (v3.02) (2), and GeneMark (v2.5) (3) to identify start sites and coding capacity and from PhagesDB with BLAST (4), HHpred (v3.2) (5), NCBI BLAST (6), the Conserved Domain Database (7), TMHMM (v2.0) (8), TOPCONS (v2.0) (9), and tRNAscan-SE (v2.0) (10) for evidence of gene function. Default parameters were used for all software unless otherwise specified.
ClearAsMud and Kauala were placed in cluster EC and subcluster EA4, respectively, based on criteria described previously (11, 12). Both have circularly permuted genome ends, as the genomes had different starting and ending locations and were slightly longer than one genome length (13). The ClearAsMud genome is 52,987 bp long, with a G+C content of 69%. Putative functions were assigned for 37 of the 92 predicted protein-coding genes. As is the case for all cluster EC phages, all of the ClearAsMud open reading frames are transcribed in the forward direction. Characteristic of cluster EC phages, ClearAsMud contains 9 copies of a unique 14-bp sequence (CTATAGGTGTAAGC) found 19 to 30 bp upstream of transcriptional start sites. Interestingly, ClearAsMud and KaiHaiDragon, another cluster EC phage discovered on the campus of La Sierra University, are 91.5% identical based on average nucleotide identity (ANI) as determined by OrthoANI (14).
The Kauala genome has a G+C content of 64.4% and is 39,378 bp long. Of a total of 56 genes, 26 genes were assigned a function; no tRNA was identified. Kauala showed genomewide nucleotide similarity to other phages in its subcluster, with 90.27% and 90.26% identities to its closest relatives Sinatra (GenBank accession number MK937602) and PrincePhergus (GenBank accession number MK620901), respectively. Additional details on ClearAsMud and Kauala can be found in the Actinobacteriophage Database (https://phagesdb.org/phages/ClearAsMud/ for ClearAsMud and https://phagesdb.org/phages/Kauala/ for Kauala) (4).
Data availability.GenBank and SRA accession numbers are MT657336 and SRX8516562, respectively, for ClearAsMud and MT657344 and SRX8516563, respectively, for Kauala.
ACKNOWLEDGMENTS
This work was made possible by the La Sierra University Biology Department and the Howard Hughes Medical Institute SEA-PHAGES program.
FOOTNOTES
- Received 3 September 2020.
- Accepted 22 October 2020.
- Published 12 November 2020.
- Copyright © 2020 Lin et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
