ABSTRACT

CaiB is a DR cluster actinobacteriophage that was isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540 as the host. The genome is 61,620 bp, has a GC content of 68.6%, and contains 85 predicted protein coding genes. CaiB has several putative operons and has repeated intergenic regions that may be involved in gene regulation.

ANNOUNCEMENT

There are an estimated 1031 phage particles in the world, and 4,000 actinobacteriophage genomes have been annotated (1). Since approximately 70% of annotated protein coding genes do not have known function (2), the isolation of evolutionarily diverse actinobacteriophages helps advance the understanding of phage genomics and evolutionary science.
CaiB was isolated from a moist soil sample from Tampa, Florida (28.086388N, 82.384166W), using Gordonia rubripertincta NRRL B-16540 as the host. Bacterial infections were performed at 30°C utilizing peptone-yeast calcium agar (PYCa). Genomic DNA was isolated after three rounds of plaque purification using the Wizard DNA cleanup kit (A7280; Promega). Genomic DNA was used to create sequencing libraries with NEBNext Ultra II library preparation kit v3 reagents. Sequencing was performed by the Pittsburgh Bacteriophage Institute, and the libraries were run on an Illumina MiSeq instrument, yielding 295,653 paired-end 150-base reads with 625-fold average coverage. Raw reads were assembled with Newbler v2.9 (3), yielding a single phage contig. The results were checked for completeness, accuracy, and genome termini using Consed (4). Default parameters were used for all software unless otherwise specified. CaiB is circularly permuted based on a lack of defined genome ends (5) and was bioinformatically linearized such that base 1 is assigned in accord with other Gordonia phages (5). CaiB was autoannotated using DNA Master v5.23.6 (6), and all of the genes were then manually validated for correct starts and functional calls. GeneMark v2.5 (7) and Glimmer v3.02 (8) were utilized to assess start sites and coding potential, and Starterator v1.2 (2) was used to summarize the starts across each family of phage genes. To collect evidence for gene function and the validity of each gene product, HHpred v3.2 (9), NCBI BLAST (10), the Conserved Domain Database (CDD) (11), TMHMM v2.0 (12), and SOSUI (13) were utilized. tRNAscan-SE v2.0 (14) and ARAGORN v1.2.41 (15) were utilized to identify putative tRNAs and transfer-messenger RNAs.
Negative-staining transmission electron microscopy shows that CaiB has a 310-nm tail and icosahedral capsid of 62 nm (Fig. 1). CaiB has a 61,620-bp genome, has a GC content of 68.6%, and contains 85 predicted protein coding genes. CaiB is one of 11 phages in the DR cluster (Table 1). DR phages have similar genome organizations and morphologies. The CaiB genome shows between 65 and 97% nucleotide identity to the other DR members (Table 1) (16). Genes 1 to 5 precede the terminase, have 4-bp overlaps, and encode a series of nucleotide modification enzymes, including ParB-like nuclease, TET/JBP oxidoreductase, pyrophosphorylase, and adenylate kinase, which may be involved in evasion of host restriction systems (17). CaiB contains several repeated intergenic regions upstream of genes 42, 71, 77, 80, 81, and 85. These regions contain 100% identity in positions −4 to −11 (5′-GAGAGGAC-3′), −14 to −21 (5′-ACCCGCTC-3′), −25 to −30 (5′-GCGGGA-3′), and −35 to −40 (5′-TGTTGT-3′) and may serve a function in regulation.
FIG 1
FIG 1 Transmission electron micrograph of Gordonia phage CaiB (https://phagesdb.org/phages/CaiB). Phage lysates were negatively stained with 1% uranyl acetate. Scale bar = 120 nm.
TABLE 1
TABLE 1 DR cluster phage characteristics and nucleotide identity values
PhageGenBank accession no.Genome size (bp)GC content (%)Nucleotide identity (%) with:
NHagosCaiBAnarQueCloverMinnieMariokartAxumiteLigmaSourBiggityBassAnClarYago84
NHagosMN36975859,58068.2100          
CaiBON10864461,62068.675.88100         
AnarQueOK21687961,82268.876.1996.96100        
CloverMinnieMN23419661,09868.776.2396.9497.98100       
MariokartMT65733560,76270.567.2966.4466.7266.81100      
AxumiteON08133361,71470.267.4465.9966.3166.4381.79100     
LigmaOM10588661,71470.267.4465.9966.3166.4381.7999.99100    
SourMH15381061,67068.064.1164.7064.8664.9771.0171.6271.62100   
BiggityBassON26081363,20269.466.1665.2365.3065.4373.4774.7674.7672.74100  
AnClarMN90869361,85669.866.0365.1465.2265.2973.3174.7574.7673.0488.56100 
Yago84MK80172561,89070.066.0065.1865.3065.3773.0674.5574.5573.9789.3098.49100

Data availability.

This whole-genome shotgun project has been deposited in DDB/ENA/GenBank under the accession numbers ON108644 and SRX14597705. The version described in this paper is the first version. Data for CaiB are archived in Phamerator (18) and the Actinobacteriophage Database at PhagesDB.org (2) (https://phagesdb.org/phages/CaiB).

ACKNOWLEDGMENTS

This work was made possible by the Howard Hughes Medical Institute (HHMI) Science Education Alliance and the University of Pittsburgh.
R. A. Garlena, D. A. Russell, D. Jacobs-Sera, and G. H. Hatfull are gratefully acknowledged for their help with genome sequencing and quality control, and we also acknowledge support of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (19).

REFERENCES

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Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 11Number 721 July 2022
eLocator: e00376-22
Editor: John J. Dennehy, Queens College CUNY
PubMed: 35758688

History

Received: 21 April 2022
Accepted: 4 June 2022
Published online: 27 June 2022

Contributors

Authors

Bienna Welsh
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Nader M. Abdalla
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Esteban Aldana
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Veronica M. Alvarado Fernandez
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Bruna Arenales Salgado de Oliveira
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Diane Fakhre
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Amelia J. Haymond
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Katelyn M. Helton
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Aditi Kanchibhatta
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Jahwanza Knight
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Sydney Marshall
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Maomi Laine N. Martinez
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Arielle Merkher
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Savannah E. Morrow
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Katie P. Nguyen
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Jahanvi J. Patel
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Somesh R. Patel
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Pravalika Rayala
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Kira M. Ruiz-Houston
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Aarya P. Satardekar
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Shifa M. Shaikh
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Adrian E. Terron Osorio
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Rachel C. Weitz
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Louis Otero
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, USA

Editor

John J. Dennehy
Editor
Queens College CUNY

Notes

The authors declare no conflict of interest.

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