ABSTRACT
We report the genome sequences of 24 newly discovered bacteriophages that infect Rhodobacter capsulatus, a model for photosynthesis and horizontal gene transfer studies. All have substantial relatedness to previously reported siphovirus bacteriophages. Most are categorized in known clusters (RcB, RcC, RcD, and RcF), with one forming a new cluster, RcG.
ANNOUNCEMENT
Rhodobacter capsulatus, a freshwater alphaproteobacterium, was the first microorganism found to perform horizontal gene transfer with a bacteriophage-like gene transfer agent (RcGTA) (1). Recent studies found that many bacteriophages that infect this host share genes with RcGTA (2–6). Prior to this announcement, 29 R. capsulatus phage genomes were organized into six distinct clusters (RcA to RcF), with four remaining as singletons (2). To advance our understanding of these phages and RcGTA, we have expanded this collection with 24 genomes from isolates collected throughout the state of Illinois.
Phages were discovered using previously described enrichment protocols, where 10-mL environmentally collected water samples were combined with 10 mL of fresh yeast extract-peptone medium and 10 mL of R. capsulatus strain YW1 culture grown to an optical density at 600 nm (OD600) of ~1.0 (2). After 3 to 5 of days shaking at 30°C, samples were filtered through 0.22-μm filters and plated with a 0.4% top agar overlay embedded with R. capsulatus cells. Plates were examined for plaques after a 1- to 3-day 30°C incubation. After 2 to 4 single-plaque isolations, DNA was extracted from high-titer (>5.0 × 107 PFU/mL) lysates with the Wizard DNA prep kit (Promega), and Illumina sequencing was conducted using either the MiSeq or NextSeq 2000 platform (Table 1). Libraries were prepared using the TruSeq Nano DNA library kit (Illumina), with Covaris shearing. Resulting 150-bp single-end reads were assembled using PATRICBRC.org (7) or Newbler v.2.9 and assessed for quality and completeness using Consed v.29 (8). Annotations used DNAMaster v. 5.23.6 (9) and Pecaan v. 20210526 (https://blog.kbrinsgd.org/), with GeneMark v. 3.25 (10), GLIMMER v.3.02 (11), NCBI BLAST v.2.9.0 (12), tRNAscan-SE v.2.0 (13), ARAGORN v.1.2.38 (14), HHpred v.3.2.0 (15), and Phamerator v.326 (16), all with default parameters.
TABLE 1.
Genometrics of 24 additional R. capsulatus bacteriophages
| Phage name | GPS coordinates (°) | Collection date (mo/day/yr) | Sequencing platform | Cluster | Genome length (bp) | G+C content (%) | Coverage (no. of reads) | No. of CDSsa | GenBank accession no. | SRA accession no. |
|---|---|---|---|---|---|---|---|---|---|---|
| RcMotherGoose | 40.533539, −88.994855 | 5/19/2022 | Illumina NextSeq 2000 | RcB | 44,429 | 55.2 | 7,664 | 63 | OP009282 | SRX17116970 |
| RcKai | 40.546383, −88.956529 | 9/15/2020 | Illumina NextSeq 2000 | RcC | 41,645 | 64.2 | 3,524 | 68 | OP009272 | SRX17116951 |
| RcKvothe | 40.496051, −88.990428 | 10/10/2019 | Illumina MiSeq | RcC | 41,447 | 64.2 | 1,712 | 67 | OP009275 | SRX17116967 |
| RcMamaDuck | 40.468213, −88.970813 | 5/19/2022 | Illumina NextSeq 2000 | RcC | 41,838 | 64 | 7,202 | 69 | OP009278 | SRX17116950 |
| RcMeacham | 41.962013, −88.072160 | 9/28/2019 | Illumina MiSeq | RcC | 41,198 | 64.2 | 1,518 | 67 | OP009280 | SRX17116968 |
| RcSwan | 40.503560, −88.926892 | 9/17/2021 | Illumina NextSeq 2000 | RcC | 41,417 | 64.2 | 7691 | 68 | OP009286 | SRX17116962 |
| RcAqua | 40.503066, −88.930833 | 9/17/2021 | Illumina NextSeq 2000 | RcD | 66,472 | 60.2 | 124 | 95 | OP009265 | SRX17116953 |
| RcAquaphina | 40.326284, −88.570188 | 9/19/2020 | Illumina NextSeq 2000 | RcD | 68,972 | 59.9 | 7,459 | 102 | OP009266 | SRX17116964 |
| RcBigEagle | 40.531708, −88.932522 | 5/23/2022 | Illumina NextSeq 2000 | RcD | 68,138 | 60.2 | 5,069 | 100 | OP009267 | SRX17116954 |
| RcCWillis | 40.495769, −88.989552 | 9/14/2021 | Illumina NextSeq 2000 | RcD | 68,804 | 61.2 | 5,671 | 97 | OP009268 | SRX17116973 |
| RcDora | 40.494485, −89.012498 | 9/16/2021 | Illumina NextSeq 2000 | RcD | 67,639 | 60.2 | 7,620 | 100 | OP009269 | SRX17116965 |
| RcExplorer | 40.459407, −88.948256 | 9/11/2021 | Illumina NextSeq 2000 | RcD | 68,400 | 60.1 | 6,986 | 102 | OP009270 | SRX17116959 |
| RcJoli | 39.867163, −89.621677 | 8/9/2019 | Illumina MiSeq | RcD | 68,547 | 60.1 | 1,233 | 101 | OP009271 | SRX17116958 |
| RcKeef | 40.496629, −88.991761 | 9/1/2021 | Illumina NextSeq 2000 | RcD | 68,260 | 60 | 7,366 | 101 | OP009273 | SRX17116963 |
| RcLkye | 40.494807, −88.987354 | 9/13/2021 | Illumina NextSeq 2000 | RcD | 68,442 | 60 | 6,818 | 101 | OP009276 | SRX17116961 |
| RcMaeve | 41.630898, −88.293465 | 10/1/2016 | Illumina MiSeq | RcD | 65,779 | 60.2 | 1,775 | 94 | OP009277 | SRX17116966 |
| RcMcLean | 40.364950, −88.964090 | 9/25/2021 | Illumina NextSeq 2000 | RcD | 67,427 | 60.2 | 6,477 | 94 | OP009279 | SRX17116957 |
| RcPacific | 40.198975, −88.393187 | 9/12/2017 | Illumina MiSeq | RcD | 67,036 | 60.2 | 376 | 98 | OP009283 | SRX17116952 |
| RcPeripeteia | 40.494456, −89.012508 | 10/13/2019 | Illumina MiSeq | RcD | 66,287 | 60.1 | 331 | 96 | OP009284 | SRX17116960 |
| RcWata | 41.873038, −87.826485 | 9/5/2016 | Illumina MiSeq | RcD | 67,804 | 60.2 | 2,566 | 97 | OP009287 | SRX17116955 |
| RcWhiteOak | 40.495642, −89.012511 | 9/22/2020 | Illumina NextSeq 2000 | RcD | 68,141 | 60.4 | 7,353 | 101 | OP009288 | SRX17116956 |
| RcKickapoo | 40.364950, −88.964090 | 9/25/2021 | Illumina NextSeq 2000 | RcF | 94,139 | 57.7 | 4,337 | 136 | OP009274 | SRX17116971 |
| RcMenchie | 41.928985, −87.844700 | 8/22/2017 | Illumina NextSeq 2000 | RcF | 94,440 | 57.8 | 3,674 | 141 | OP009281 | SRX17116972 |
| RcRudolph | 40.468213, −88.970813 | 6/1/2021 | Illumina NextSeq 2000 | RcG | 64,892 | 65.3 | 4,032 | 85 | OP009285 | SRX17116969 |
CDSs, coding sequences.
All 24 phages share substantial similarity with previously reported isolates, with all sharing at least 77% average nucleotide identity (ANI) (17) with at least one other isolate (Table 1; Fig. 1). RcD phages continue to be the most represented, with all but one of the 15 assigned to cluster RcD and sharing >90% ANI with other RcD phages. RcCWillis shares only 77 to 79% ANI with any other RcD phage. Of its 97 genes, 19 are unique.
FIG 1.
Network phylogeny of R. capsulatus bacteriophages. The predicted proteins of all 53 sequenced R. capsulatus phages and those found in the 14,087-bp RcGTA structural gene region were sorted into 885 families (phams) according to shared amino acid sequence similarities using Phamerator (16). Each genome was then assigned values reflecting the presence or absence of members of each pham; the genomes were compared and displayed using SplitsTree (18). Clusters are indicated with colored ovals. Phage names in bold indicate phages added to this collection as part of this study.
None of the five phages assigned to cluster RcC show such divergence. They share 98 to 99% ANI with 5 previously identified members, but RcKemmy continues to be an outlier, sharing 85 to 86% ANI with the 10 other members. RcMotherGoose is now the most divergent of the 5 known RcB phages, with 89 to 91% ANI to any other member and 4 unique genes. RcF phages RcKickapoo and RcMenchie cluster tightly with the other two members, sharing 98 to 99% ANI.
Last, RcRudolph forms the new cluster, RcG, with RcSimone-Håstad. The ANI between them is 82%. They share 72 genes, with 14 in RcRudolph and 8 in RcSimone-Håstad being unique. RcSimone-Håstad previously stood out as a singleton isolated outside the United States. The discovery of RcRudolph from an Illinois pond demonstrates that this type is not unique to Sweden.
Data availability.
Sequence reads have been deposited at NCBI under BioProject accession number PRJNA865772 and SRA accession numbers SRX17116950 to SRX17116973 (Table 1).
ACKNOWLEDGMENT
This work was supported by the Biology Department at Illinois Wesleyan University with additional funding from the Bruce and Norma Criley Summer Research Fellowship.
Contributor Information
Richard M. Alvey, Email: ralvey@iwu.edu.
Kenneth M. Stedman, Portland State University
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Associated Data
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Data Availability Statement
Sequence reads have been deposited at NCBI under BioProject accession number PRJNA865772 and SRA accession numbers SRX17116950 to SRX17116973 (Table 1).