ABSTRACT
Acinetobacter calcoaceticus 65 is the original source strain for the restriction enzyme Acc65I. Its complete sequence and full methylome were determined using single-molecule real-time (SMRT) sequencing.
GENOME ANNOUNCEMENT
The bacterial strain Acinetobacter calcoaceticus 65 (recently renamed Acinetobacter junii 65) was originally isolated from a limnetic water sample and is now housed in the New England BioLabs culture collection (NEB1023). It is the original source of the Type II restriction enzyme Acc65I, identified as a neoschizomer of KpnI. The Acc65 restriction enzyme recognizes and cleaves the DNA sequence G↓GTACC, producing 5′ cohesive ends, in contrast to KpnI, which cleaves the same DNA sequence but leaves a 3′ extension GGTAC↓C (1, 2).
Genomic DNA from a culture of A. calcoaceticus 65 was purified using a modified Qiagen method and the genome sequenced using the Pacific Biosciences (PacBio) RSII sequencing platform. Briefly, SMRTbell libraries were constructed from a genomic DNA sample sheared to an average size of ~10 to 20 kb using the G-tubes protocol (Covaris, Woburn, MA, USA), end repaired, and ligated to hairpin adapters. Incompletely formed SMRTbell templates were digested with a combination of exonuclease III and exonuclease VII (New England BioLabs; Ipswich, MA, USA). Genomic DNA fragments and SMRTbell library qualification and quantification were performed using a Qubit fluorimeter (Invitrogen, Eugene, OR) and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). One 20-kb SMRTbell library was prepared according to the modified PacBio sample preparation protocols, including additional separation on a BluePippin, and sequenced using C4-P6 chemistry and three single-molecule real-time (SMRT) cells with a 240-min collection time. Sequencing reads were processed, mapped, and assembled by the Pacific Biosciences SMRT Analysis pipeline, using the HGAP3 protocol, and polished using Quiver (3) to yield 632.2 Mb of sequence data, which were assembled into a single closed circular genome of 3,377,773 bp with 134-fold coverage. The assembled sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) and has been deposited at DDBJ/EMBL/GenBank (Table 1).
TABLE 1 .
Summary of methyltransferases identified in Acinetobacter calcoaceticus 65
| Motifa | Assigned or predicted | Methylation type |
Restriction modification type |
|---|---|---|---|
| GGTACC | M.Acc65I | m6A | II beta |
| CTAGNNNNNNNTGAA | M.Acc65II | m6A | I |
| CNNTAYNNNNNNTCTT | M.Acc65III | m6A | I gamma |
| GAGNNNNNTCG | M.Acc65IV | m6A | I gamma |
| GACGCA | M.Acc65V | m6A | II G, S gamma |
| CTKMAG | M.Acc65VI | m6A | II gamma |
Modified bases are highlighted in bold.
The advantage of the PacBio sequencing platform is its ability to detect the epigenetic state of sequenced DNA, which allows for the identification of modified nucleotides and their corresponding motifs. Epigenetic modification at each nucleotide position was measured as kinetic variations (KVs) in the nucleotide incorporation rates, and methylated motifs were deduced from the KV data (4–6). Six DNA methyltransferase recognition motifs were detected, each containing m6A modifications. The motifs were then matched with methyltransferase genes in the genome, and the results are shown in the table below. They have also been deposited in REBASE (7).
Accession number(s).
The complete genome sequence of the Acinetobacter calcoaceticus 65 is available in GenBank under the accession number CP019041 (Acinetobacter junii 65).
ACKNOWLEDGMENTS
This project was supported by the Small Business Innovation Research Program (NIGMS) of the National Institutes of Health under award R44GM105125 to R.J.R. R.J.R., A.F., and T.V. work for New England BioLabs, and S.K.D. works at Sibenzyme Ltd., companies that sell research reagents, including restriction enzymes and DNA methylases to the scientific community.
Footnotes
Citation Fomenkov A, Vincze T, Degtyarev SK, Roberts RJ. 2017. Complete genome sequence and methylome analysis of Acinetobacter calcoaceticus 65. Genome Announc 5:e00060-17. https://doi.org/10.1128/genomeA.00060-17.
REFERENCES
- 1.Prichodko GG, Rechnukova NI, Repin VE, Degtyarev SK. 1991. Determination of substrate specificity of restriction endonuclease Acc65I. Sib Biol J 1:59–60. (In Russian.) [Google Scholar]
- 2.Tomassini J, Roychoudhury R, Wu R, Roberts RJ. 1978. Recognition sequence of restriction endonuclease KpnI from Klebsiella pneumoniae. Nucleic Acids Res 5:4055–4064. doi: 10.1093/nar/5.11.4055 C342733. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.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. doi: 10.1038/nmeth.2474. [DOI] [PubMed] [Google Scholar]
- 4.Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Korlach J, Turner SW. 2010. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7:461–465. doi: 10.1038/nmeth.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Clark TA, Murray IA, Morgan RD, Kislyuk AO, Spittle KE, Boitano M, Fomenkov A, Roberts RJ, Korlach J. 2012. Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing. Nucleic Acids Res 40:e29. doi: 10.1093/nar/gkr1146. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Korlach J, Turner SW. 2012. Going beyond five bases in DNA sequencing. Curr Opin Struct Biol 22:251–261. doi: 10.1016/j.sbi.2012.04.002. [DOI] [PubMed] [Google Scholar]
- 7.Roberts RJ, Vincze T, Posfai J, Macelis D. 2010. REBASE–a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res 38:D234–D236. doi: 10.1093/nar/gku1046. [DOI] [PMC free article] [PubMed] [Google Scholar]