Complete Genome Sequence of Curtobacterium sp. Strain MR_MD2014, Isolated from Topsoil in Woods Hole, Massachusetts

Members of the genus Curtobacterium, first defined in 1972 by Yamada and Komagata (1), belong to the family Microbacteriaceae, within the phylum Actinobacteria. Several Curtobacterium isolates associate with plants as either pathogens or nonpathogens. Their cells are generally Gram-positive irregularly shaped motile rods. Here, Curtobacterium sp. strain MR_MD2014 was cocultured with Streptomyces sp. strain CCM_MD2014 from the topsoil collected near a rusted fire hydrant in Woods Hole, MA (41°31′44.65″N 70°40′21.5″W) on 7 July 2014, on starch-arginine-tryptophan (SAT) medium using protocols modified from El-Nakeeb and Lechevalier (2). This strain was then isolated from the coculture by selective culture, using antibiotic discs on a modified LB agar. The identity of each of the isolates was verified using the 16S rRNA gene sequence. This strain was cultivated as part of a student-led microbial isolation and sequencing initiative at the 2014 Microbial Diversity Summer Program at the Marine Biological Laboratory in Woods Hole, MA.

From the original coculture, total DNA extraction was carried out by 1 h of lysozyme digestion, followed by use of the Promega Wizard genomic DNA purification kit. The isolated DNA was quantified using the Promega QuantiFluor double-stranded DNA (dsDNA) system and then size selected for a minimum length of 4 kb. The size-selected DNA was sequenced on the Pacific Biosciences RSII sequencing platform with P5C3 chemistry. The sequenced fragments were assembled using HGAP3 on the SMRT Portal (3). The final assembled circular genome had a size of 3,443,800 bp, with 89× sequencing coverage and a G+C composition of 71.95%.

The genome was annotated using NCBI Prokaryotic Genome Annotation Pipeline version 2.8 (rev. 449627) (4, 5). The identified genes were composed of 2,675 coding sequences (CDSs), 4 rRNA operons, 48 tRNAs, and 1 noncoding RNA (ncRNA) gene. CRISPRFinder (6) identified one definite clustered regularly interspaced short palindromic repeat (CRISPR) and 13 potential CRISPRs in the genome. The PHAST server (7) predicted three prophages, two of which were incomplete, and one of which was of questionable quality. REBASE (8) identified 3 candidate methylase genes and one methylated motif, GG^m6AGGC, which was found using Pacific Biosciences SMRT Portal analysis. This was unambiguously assigned to the type IIG restriction enzyme Csp2014I (see organism number 14032 on the REBASE website for details).

Phylogenetic analysis of the 16S rRNA gene sequence using SSU-align (version 0.1) (9) and RAxML (version 8.2.3) (10) revealed that strain MR_MD2014 belongs to the genus Curtobacterium, in the family Microbacteriaceae. The 16S rRNA gene-based analysis did not provide sufficient data for a refined classification of this strain at the species level. A phylogenetic reconstruction based upon 42 conserved single-copy-marker genes using CheckM (11) identified Curtobacterium sp. YR515 as being the most similar to the strain MR_MD2014 (see https://doi.org/10.6084/m9.figshare.1574022). An average nucleotide identity (ANI) calculation using IMG (12) confirmed that the closest available genome to this strain is Curtobacterium sp. YR515, with only 85.3% average nucleotide identity across shared genes.

The isolation and culturing of this organism were carried out as part of the 2014 Microbial Diversity Summer Program at the Marine Biological Laboratory in Woods Hole, MA. The course was supported by generous grants from the Simons Foundation (grant 309981 to the Marine Biological Laboratory), the Beckman Foundation, and the Howard Hughes Medical Institute. This material is based upon work supported by the National Aeronautics and Space Administration (grant NNA13AA92A), the National Science Foundation (grant MCB-1417876), and the U.S. Department of Energy (grant DE-FG02-85ER13361). R.M.M. was supported by a fellowship from Alabama EPSCoR GRSP, Auburn University's Cell and Molecular Biosciences (CMB) Summer Fellowship, Selman A. Waksman Endowed Scholarship in Microbial Diversity, the Bernard Davis Endowed Scholarship (grant 47802012050), the Auburn Graduate School Travel Award, and the Joseph Kirby Farrington Endowed Fund for Excellence. These funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

Promega Corporation donated the molecular reagents used in this project. The sequencing for this organism was supported and carried out by Pacific Biosciences. We thank George O’Toole, Alison Butler, Emil Ruff, Arpita Bose, Suzanne Kern, Louis Kerr, Kurt Dahlstrom, Alice Michel, and Cristian Salgado for their assistance and advice.

R.J.R. works for New England BioLabs, a company that sells research reagents, including restriction enzymes and DNA methylases, to the scientific community. J.K. and M.B. are full-time employees at Pacific Biosciences, a company that develops single-molecule real-time sequencing technologies, including the sequencing platform used in this project.

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