Complete Genome Sequences of Two Rhodobacter Strains Brian P. Anton, a Richard J. Roberts, a Alexey Fomenkov, a Allison Humbert, b Natalie Stoian, b Jill Zeilstra-Ryalls b a New England Biolabs, Ipswich, Massachusetts, USA b Bowling Green State University, Bowling Green, Ohio, USA ABSTRACT We report the complete genome sequences of two strains of the Alpha- proteobacteria genus Rhodobacter, Rhodobacter blasticus 28/5, the source of the com- mercially available enzyme RsaI, and a new isolate of Rhodobacter sphaeroides 2.4.1. Both strains contain multiple restriction-modification systems, and their DNA methyl- ation motifs are included in this report. S everal type II restriction endonucleases (REases) have been isolated from strains of Rhodobacter (formerly known as Rhodopseudomonas) sphaeroides (class Alphapro- teobacteria). RsaI was first purified from strain 28/5 (1); RshI, an isoschizomer of PvuI, was first purified from the type strain 2.4.1 (ATCC 17023) (2); and RsrI, an isoschizomer of EcoRI (3, 4), and RsrII were both purified from the wild-type strain RS630 (5). We have sequenced and characterized the methylomes of Rhodobacter strains 28/5 and 2.4.1, both originally obtained from the laboratory of Samuel Kaplan (University of Texas Health Science Center––Houston), using the PacBio RS II platform. Both strains were cultured aerobically to an optical density at 660 nm (OD 660 ) of 0.8, with strain 2.4.1 cultured in Sistrom’s minimal medium A with succinate (6) and strain 28/5 cultured in LB (7). Total DNA was isolated using the GenElute bacterial genomic DNA kit (Sigma-Aldrich) and fragmented to roughly 10 to 20 kb with g-TUBES (Covaris). Standard 20-kb libraries were prepared and sequenced with P6-C4 chem- istry using two single-molecule real-time (SMRT) cells each, for one of which the library was size selected (9 to 50 kb) using the BluePippin electrophoresis system (Sage), and movie times of 240 or 360 min. Sequencing reads (182,877, mean subread length 6,892 for 2.4.1; 79,991, mean subread length 4,023 for 28/5) were processed, mapped, and assembled de novo with RS_HGAP_Assembly.3 (8) in the SMRT Analysis 2.3.0 environment using default settings. Error correction and closure were performed using RS_BridgeMapper.1, and methylation patterns were deter- mined using RS_Modification_and_Motif_Analysis.1, both also within SMRT Analy- sis. Annotation was performed at the NCBI using PGAP (9). Strain 28/5 assembled as a single chromosome of 3.54 Mb (66.6% GC content) and a single plasmid of 157 kb (62.4% GC content). Consistent with a previous report suggesting that it was not in fact an R. sphaeroides strain (10), we found the 16S rRNA gene sequences of 28/5 to be 99% identical (1,377/1,389) to those of Rhodobacter blasticus DSM 2131 (synonym, ATCC 33485), a type strain. We suggest that the RsaI source strain be renamed R. blasticus strain 28/5. R. sphaeroides 2.4.1 assembled as two chromosomes of 3.19 Mb and 943 kb (both with 69.0% GC content) and four plasmids, A (124 kb), B (114 kb), C (107 kb), and D-E (152 kb). There is substantial agreement between our sequence and the Department of Energy (DOE) reference (GenBank accession numbers CP000143 through CP000147, DQ232586, and DQ232587), although our assembled plasmid D-E is a combination of the DOE reference plasmid E and fragment D (DQ232587), and our plasmid A is a slightly larger, circularized version of the DOE fragment A (DQ232586). Most indels were 1 to 2 bp long and in mononucleotide runs, and most single nucleotide polymorphisms Received 20 August 2018 Accepted 6 September 2018 Published 27 September 2018 Citation Anton BP, Roberts RJ, Fomenkov A, Humbert A, Stoian N, Zeilstra-Ryalls J. 2018. Complete genome sequences of two Rhodobacter strains. Microbiol Resour Announc 7:e01162-18. https://doi.org/10.1128/MRA .01162-18. Editor Irene L. G. Newton, Indiana University Bloomington Copyright © 2018 Anton et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Brian P. Anton, anton@neb.com. GENOME SEQUENCES crossm Volume 7 Issue 12 e01162-18 mra.asm.org 1 on June 7, 2020 by guest http://mra.asm.org/ Downloaded from