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Announcement
15 October 2020

Closed Genome Sequence of Aeromonas veronii Strain Hm21, an Isolate from the Medicinal Leech Hirudo verbana

ABSTRACT

Aeromonas veronii strain Hm21 was isolated from the medicinal leech Hirudo verbana and is used for genetic studies. We present here the 4.71-Mbp genome with a 56-kb plasmid and identify the mutations present in strains commonly used for genetic engineering.

ANNOUNCEMENT

Aeromonads are found in many aquatic environments and in pathogenic and beneficial associations with animals (1). One well-studied association is the digestive tract symbioses with leeches (2). Aeromonas veronii Hm21 was cultured from the digestive tract content of Hirudo verbana in 1996 by plating dilutions of the gut content on blood agar plates, incubating this overnight at 30°C, and maintaining it as a frozen stock culture (3). Antibiotic-resistant derivatives of this strain have been used in subsequent studies characterizing the molecular interactions between A. veronii and the host (46).
Previously, we published a genome based on Sanger sequencing, 454 pyrosequencing, and Illumina technology (7). This yielded a 4,684,957-bp genome comprising 75 contigs larger than 2 kb. We extracted the DNA using the MasterPure complete DNA and RNA purification kit (Epicentre, Madison, WI). For Illumina sequencing, NexteraXT libraries were prepared and sequenced on a MiSeq instrument (Illumina, San Diego, CA) (7). For PacBio sequencing, the unsheared DNA was used and the library prepared with the template kit 2.0 (PacBio, Menlo Park, CA), which was size selected using a BluePippin system (Sage Science, Beverly, MA). A single-molecule real-time (SMRT) cell was sequenced on the PacBio RS II platform using P5-C3 chemistry. A total of 60,113 reads with an N50 read length of 12,096 bp and a mean read score of 0.83 were assembled using the Hierarchical Genome Assembly Process (HGAP) 3 assembler with default parameters (8), yielding two contigs (4,710,355 bp [58.7% GC content] and 56,525 bp [59.7% GC content]) that were circularized manually. Using Illumina reads and default parameters, we used Trimmomatic for trimming the reads and adaptor trimming and breseq to polish the assemblies and detect mutations in the antibiotic-resistant derivatives (9, 10). The origin of replication was identified based on its proximity to dnaA, and the genome was rotated using Geneious R10 (Biomatters, Auckland, New Zealand). The genome was annotated using PGAP 4.12 for the NCBI submission (11). The closed genome contained 4,253 coding DNA sequences (CDS), 63 of which were on the plasmid, 10 complete rRNA operons, and 123 tRNAs.
The closed genome allowed us to examine repetitive features such as the rRNA operons. Five of the 10 16S rRNA genes were identical, but the others differed by up to 20 nucleotides. Such differences can lead to potentially misidentifying the species of Aeromonas (12). In this case, three out of seven sequences would have misidentified the species.
Several antibiotic-resistant derivatives of Hm21 that are used for genetic manipulations (36, 13) were analyzed. The genotypes of the rifampicin-resistant derivative, JG84, were rpoB S531F, a Δ21 deletion in an intragenic region at position 1648065, and nrdD K582N; the streptomycin-resistant derivative, JG1002, had the mutation rpsL K88R. Interestingly, JG304 (Hm21RS), a streptomycin-resistant derivative of JG84, lost the cryptic plasmid pHm21 while gaining rpsL K88R.

Data availability.

This whole-genome shotgun project has been deposited at NCBI under the BioProject number PRJNA205862. The accession numbers are CP059396.1 and CP059397.1, and for the raw reads, the accession numbers are SRX8815181, SRX8815182, SRX8815183, SRX8815184, and SRX8815185. Strains are available from the corresponding author upon request.

ACKNOWLEDGMENTS

This work was funded by the USDA-ARS CRIS project 8082-32000-006-00-D to J.G. and J. P. Gogarten and NSF 1710511 to J.G. and V. Cooper.

REFERENCES

1.
Janda JM, Abbott SL. 2010. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 23:35–73.
2.
Marden JN, McClure EA, Beka L, Graf J. 2016. Host matters: medicinal leech digestive-tract symbionts and their pathogenic potential. Front Microbiol 7:1569.
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Graf J. 1999. Symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech: a novel model for digestive tract associations. Infect Immun 67:1–7.
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Maltz M, LeVarge B, Graf J. 2015. Identification of iron and heme utilization genes in Aeromonas and their role in the colonization of the leech digestive tract. Front Microbiol 6:763.
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Silver AC, Rabinowitz NM, Kuffer S, Graf J. 2007. Identification of Aeromonas veronii genes required for colonization of the medicinal leech, Hirudo verbana. J Bacteriol 189:6763–6772.
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Rio RVM, Anderegg M, Graf J. 2007. Characterization of a catalase gene from Aeromonas veronii, the digestive-tract symbiont of the medicinal leech. Microbiology (Reading) 153:1897–1906.
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Bomar L, Stephens WZ, Nelson MC, Velle K, Guillemin K, Graf J. 2013. Draft genome sequence of Aeromonas veronii Hm21, a symbiotic isolate from the medicinal leech digestive tract. Genome Announc 1:e00800-13.
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Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569.
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Deatherage DE, Barrick JE. 2014. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol 1151:165–188.
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Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120.
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Zhao Y, Wu J, Yang J, Sun S, Xiao J, Yu J. 2012. PGAP: pan-genomes analysis pipeline. Bioinformatics 28:416–418.
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Morandi A, Zhaxybayeva O, Gogarten JP, Graf J. 2005. Evolutionary and diagnostic implications of intragenomic heterogeneity in the 16S rRNA gene in Aeromonas strains. J Bacteriol 187:6561–6564.
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Ott BM, Dacks AM, Ryan KJ, Rio RV. 2016. A tale of transmission: Aeromonas veronii activity within leech-exuded mucus. Appl Environ Microbiol 82:2644–2655.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 9Number 4215 October 2020
eLocator: e00922-20
Editor: Julie C. Dunning Hotopp, University of Maryland School of Medicine
PubMed: 33060270

History

Received: 4 August 2020
Accepted: 21 September 2020
Published online: 15 October 2020

Contributors

Authors

University of Connecticut, Department of Molecular and Cell Biology, Storrs, Connecticut, USA
University of Connecticut, Department of Molecular and Cell Biology, Storrs, Connecticut, USA
Present address: Michael C. Nelson, Sema4, Stamford, Connecticut, USA; Sophie M. Colston, Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC, USA.
Sophie M. Colston
University of Connecticut, Department of Molecular and Cell Biology, Storrs, Connecticut, USA
Present address: Michael C. Nelson, Sema4, Stamford, Connecticut, USA; Sophie M. Colston, Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC, USA.

Editor

Julie C. Dunning Hotopp
Editor
University of Maryland School of Medicine

Notes

Address correspondence to Joerg Graf, [email protected].

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