Open access
Announcement
28 May 2020

Draft Genome Sequence of Tumebacillus sp. Strain BK434, Isolated from the Roots of Eastern Cottonwood

ABSTRACT

A Gram-positive bacterium was isolated from the root of an eastern cottonwood tree (Populus deltoides) in Georgia and identified as a Tumebacillus species with 99% 16S rRNA nucleotide identity to Tumebacillus avium. The genome is 4.6 Mbp and encodes 4,072 proteins and 251 RNAs.

ANNOUNCEMENT

Tumebacillus is a genus of Gram-positive rod-forming bacteria that was only recently described in the phylum Firmicutes (1). Since its discovery, eight species from this genus have been isolated from diverse environments, such as permafrost (1), soil (2, 3), river water (4), algal scum (5), and the gut of a vulture (6). Here, we report the first draft genome of a Tumebacillus species associated with a plant root.
The roots from a Populus deltoides (genotype WV94) (7) tree growing on a nursery site in Bellville, Georgia, were sampled in 2018. Fine roots (<2 mm) were excised from freshly harvested root samples and processed as described previously (8, 9). Nonsterilized roots were macerated in 10 ml of MgSO4 (10 mM) and serially diluted onto Reasoner’s 2A (R2A) agar (10). Cultures were isolated through three rounds of restreaking onto R2A medium at 28°C. The isolate was inoculated into R2A liquid medium and grown for 2 days at 30°C. Genomic DNA was isolated with the Qiagen DNeasy blood and tissue kit according to the manufacturer’s instructions. An initial identification was carried out using Sanger sequencing of the 16S rRNA amplicon of strain BK434 with primers 27F and 1492R (11). Based on an NCBI BLAST (12) search of the nonredundant/nucleotide database, the 16S rRNA amplicon of strain BK434 was found to have 99.15% nucleotide identity, across 1,414 bp, to Tumebacillus avium. Because the genus Tumebacillus has very few genome-sequenced representatives, we proceeded with genome sequencing to allow greater exploration of the genetic potential of this group. The draft genome of Tumebacillus was generated at the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) using Illumina technology (13). A standard shotgun library was constructed; briefly, DNA was sheared by ultrasonication (Covaris, Woburn, MA) to a 600-bp average fragment size, and an Illumina shotgun library was prepared with the Kapa Biosystems library preparation kit (Roche, Wilmington, MA) and sequenced using the NovaSeq Xp reagent kit v1.0 with 2 × 151-bp reads on the Illumina NovaSeq platform, which generated 9,668,990 reads totaling 1,460,017,490 bp. Default parameters were used for all software unless otherwise specified. Raw Illumina reads were quality filtered to remove known sequencing artifacts and contaminants and read depth was normalized using BBTools (14). The filtered reads were assembled using SPAdes (v3.12.0) (phred-offset, 33; cov-cutoff, auto; t, 16; m, 64; careful; k, 25,55,95) (15), and for quality purposes, contigs of <1 kbp were discarded (BBTools reformat.sh: minlength). The final draft assembly contained 34 contigs (L50, 3; N50, 708,514 bp) in 33 scaffolds, totaling 4,646,936 bp with a GC content of 57.94%. The final assembly was based on 1,431,753,368 bp of Illumina data with a mapped coverage of 305.7×. The genome was annotated using the IMG Microbial Genome Annotation Pipeline (MGAP) (v4.16.4) (16). A total of 4,072 protein-coding genes were predicted, with 251 RNA genes and 7 CRISPR arrays. Protein-coding regions included those involved in spore germination, which is common in Firmicutes, and in flagellar motion. Flagella have been characterized for some species of Tumebacillus (5, 17), including Tumebacillus avium (6), the strain that is 99.15% similar based on 16S rRNA findings.

Data availability.

The assembly has been deposited in GenBank under accession number SLXS00000000 and is also available from the IMG/M database under accession number 2795386103. Raw sequences have been deposited in the NCBI Sequence Read Archive under BioProject number PRJNA520069 and run number SRR8861549.

ACKNOWLEDGMENTS

We thank Kerrie Barry and Nicole Shapiro (JGI) for project management and Tse-Yuan S. Lu (Oak Ridge National Laboratory) for help with strain isolation.
This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the U.S. DOE. Part of this research was funded by the U.S. DOE Office of Biological and Environmental Research, Genomic Science Program, as part of the Plant Microbe Interfaces Scientific Focus Area (http://pmi.ornl.gov). The Bellville field site maintenance was funded by the BioEnergy Science Center project. The BioEnergy Science Center is a U.S. DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the U.S. DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. The work conducted by the U.S. DOE JGI, a DOE Office of Science User Facility, is supported under contract DE-AC02-05CH11231. The data were generated for JGI proposal 503495 under U.C.K.

REFERENCES

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Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 9Number 2228 May 2020
eLocator: 10.1128/mra.00351-20
Editor: Kenneth M. Stedman, Portland State University
PubMed: 32467272

History

Received: 3 April 2020
Accepted: 7 May 2020
Published online: 28 May 2020

Contributors

Authors

Dana L. Carper
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
Christopher W. Schadt
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
Leah H. Burdick
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
Udaya C. Kalluri
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
Dale A. Pelletier
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

Editor

Kenneth M. Stedman
Editor
Portland State University

Notes

Address correspondence to Udaya C. Kalluri, [email protected], or Dale A. Pelletier, [email protected].

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