Many Arcobacter spp. are free living and are routinely recovered from marine environments.
ABSTRACT
Many Arcobacter spp. are free living and are routinely recovered from marine environments. Arcobacter halophilus was isolated from hypersaline lagoon water in the Hawaiian islands, and it was demonstrated to be an obligate halophile. This study describes the complete whole-genome sequence of the A. halophilus type strain, CCUG 53805 (= LA31BT = ATCC BAA-1022T).
ANNOUNCEMENT
Arcobacter species are often recovered from marine environments. Although many Arcobacter taxa are isolated from shellfish (1–4), others are free living and have been recovered from seawater (5, 6) or marine sediments (7–9). Arcobacter halophilus is an obligate halophile that was cultivated from a water sample collected in October 2000 from the hypersaline Lake Laysan at Laysan Atoll in the Northwestern Hawaiian Islands (10, 11). In this study, we report the first closed genome sequence of the A. halophilus type strain, CCUG 53805 (= LA31BT = ATCC BAA-1022T).
Arcobacter halophilus CCUG 53805T was grown aerobically for 48 h at 30°C on anaerobe basal agar (Oxoid) amended with 5% horse blood and 2% (wt/vol) NaCl. Genomic DNA was prepared from a loop of cells as described previously (12). Shotgun and paired-end Roche GS-FLX+ reads were assembled using Newbler v. 2.6, yielding 91 total contigs and a chromosomal scaffold of 45 unique contigs. Forty-six contigs, representing regions present more than once in the chromosome, were positioned into the scaffold gaps using the custom Perl script contig_extender3 (12). Contig junctions and any remaining small gaps were validated and closed using directed PCR amplification/Sanger sequencing. Joining of the 454 contigs and linking Sanger sequences into a single chromosomal sequence, using the contig order obtained above, was performed using SeqMan v. 8.0.2 (DNASTAR, Madison, WI). The contig order within the 454 sequence was also verified using an optical restriction map (restriction enzyme SpeI; OpGen, Gaithersburg, MD). During closure, a large repetitive region within the chromosome was identified, and PacBio reads that spanned this region were generated. PacBio sequencing was performed as described (12) and generated a single circular sequence that was added to the 454 SeqMan assembly, further confirming the contig order within the 454 scaffold. Illumina HiSeq reads (SeqWright, Houston, TX) independently verified all base calls within the chromosome. These reads were assembled de novo using Newbler, and the resulting contigs were assembled onto the SeqMan 454/PacBio sequence as described (12), with the PacBio sequence corrected with respect to the HiSeq consensus; single-nucleotide polymorphisms (SNPs) in the sequences between the HiSeq contigs were identified using the Geneious v. 11.0.2 (Biomatters Ltd., Auckland, New Zealand) “find variations/SNPs” module with the default minimum variant frequency parameter of 0.3. The final coverage across the genome was 919×.
Genome feature data for A. halophilus strain CCUG 53805T are presented in Table 1. The CCUG 53805T genome is 2,812,536 bp, with an average G+C content of 27.6%. Protein-, rRNA-, and tRNA-encoding genes were identified and annotated as described (13) using the custom Perl script BlastPTrimmer14 (12), with the composite proteome used here for BLAST analyses and comparisons containing proteins from all current complete Campylobacter and Arcobacter genomes. The genome is predicted to encode 2,622 putative protein-coding genes and 19 pseudogenes. Additionally, the CCUG 53805T genome contains 6 rRNA operons and 63 tRNA-encoding genes. A type I-B CRISPR/Cas system and 6 genomic islands ranging from 8.5 kb to 26.9 kb were identified in the CCUG 53805T chromosome; the largest genomic island putatively encodes a type VI secretion system. The CCUG 53805T genome contains no plasmids.
TABLE 1.
Feature | Dataa |
---|---|
Sequencing metric | |
Platform | |
454 (shotgun) | |
No. of reads | 158,389 |
No. of bases | 53,780,918 |
Average length (bases) | 340 |
Coverage (×) | 19.1 |
454 (paired end) | |
No. of reads | 670,813 |
No. of bases | 215,687,983 |
Average length (bases) | 322 |
Coverage (×) | 95.8 |
Illumina HiSeq 2000 | |
No. of reads | 18,199,888 |
No. of bases | 1,838,188,068 |
Average length (bases) | 101 |
Coverage (×) | 653.6 |
PacBio | |
No. of reads | 125,464 |
No. of bases | 476,556,540 |
Average length (bases) | 3,798.4b |
Coverage (×) | 169.4 |
Genomic data | |
Chromosome | |
Size (bp) | 2,812,536 |
G+C content (%) | 27.61 |
No. of CDSc | 2,622 |
Assigned function (% CDS) | 1,009 (38.5) |
General function annotation (% CDS) | 1,025 (39.1) |
Domain/family annotation only (% CDS) | 173 (6.6) |
Hypothetical (% CDS) | 415 (15.8) |
No. of pseudogenes | 19 |
Genomic islands/CRISPR | |
No. of genetic islands | 6 |
No. of CDS in genetic islands | 106 [1] |
CRISPR/Cas loci | I-B |
Gene content/pathways | |
Signal transduction | |
Che proteins | cheABCDRVW(Y)3 |
No. of methyl-accepting chemotaxis proteins | 29 |
No. of response regulators | 60 |
No. of histidine kinases | 76 |
No. of response regulator/histidine kinase fusions | 3 |
No. of diguanylate cyclases | 26 |
No. of diguanylate phosphodiesterases (HD-GYP, EAL) | 5, 5 |
No. of diguanylate cyclase/phosphodiesterases | 11 [1] |
No. of others | 13 |
Motility | |
Flagellin genes | fla1, fla2, fla3, fla4 |
Restriction/modification | |
No. of type I (hsd) systems | 2 |
No. of type II systems | 1 |
No. of type III systems | 0 |
Transcription/translation | |
No. of transcriptional regulatory proteins | 60 |
Non-ECF σ factorsd | σ70 |
No. of ECF σ factors | 1 |
No. of tRNAs | 63 |
No. of ribosomal loci | 6 |
Nitrogen fixation (nif) | No |
Osmoprotection | BCCT4, cai/fix, betA, ectABCD, proABCVWX |
Pyruvate → acetyl-CoAe | |
Pyruvate dehydrogenase (E1/E2/E3) | Yes |
Pyruvate:ferredoxin oxidoreductase | por |
Urease | No |
Vitamin B12 biosynthesis | No |
Numbers in brackets indicate pseudogenes/fragments.
Maximum length, 23,873 bp.
Numbers do not include pseudogenes. CDS, coding DNA sequences.
ECF, extracytoplasmic function.
CoA, coenzyme A.
Consistent with its description as an obligate halophile, the genome of strain CCUG 53805T contains several genes associated with the biosynthesis and uptake of osmolytes. These include genes for the biosynthesis of ectoine (ectABC), 5-hydroxyectoine (ectD), proline (proABC), glycine betaine aldehyde (betA), and γ-butyrobetaine (cai/fix). Additionally, the CCUG 53805T genome encodes the ProVWX proline/glycine betaine ABC transporter and four BCCT (betaine/carnitine/choline transporter) family proteins.
Data availability.
The complete genome sequence of A. halophilus strain CCUG 53805T has been deposited in GenBank under the accession number CP031218. The 454, HiSeq, and PacBio sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP155008. The source codes for contig_extender3 and BlastPTrimmer14 are available through GitHub (https://github.com/Arcobacter/Genome_perl).
ACKNOWLEDGMENTS
This work was funded by the United States Department of Agriculture, Agricultural Research Service, CRIS projects 2030-42000-230-047, 2030-42000-230-051, and 3040-42000-015-00D.
We thank Stephen On for providing A. halophilus strain CCUG 53805T.
REFERENCES
- 1.Dieguez AL, Balboa S, Magnesen T, Romalde JL. 2017. Arcobacter lekithochrous sp. nov., isolated from a molluscan hatchery. Int J Syst Evol Microbiol 67:1327–1332. doi: 10.1099/ijsem.0.001809. [DOI] [PubMed] [Google Scholar]
- 2.Figueras MJ, Collado L, Levican A, Perez J, Solsona MJ, Yustes C. 2011. Arcobacter molluscorum sp. nov., a new species isolated from shellfish. Syst Appl Microbiol 34:105–109. doi: 10.1016/j.syapm.2010.10.001. [DOI] [PubMed] [Google Scholar]
- 3.Figueras MJ, Levican A, Collado L, Inza MI, Yustes C. 2011. Arcobacter ellisii sp. nov., isolated from mussels. Syst Appl Microbiol 34:414–418. doi: 10.1016/j.syapm.2011.04.004. [DOI] [PubMed] [Google Scholar]
- 4.Levican A, Collado L, Aguilar C, Yustes C, Dieguez AL, Romalde JL, Figueras MJ. 2012. Arcobacter bivalviorum sp. nov. and Arcobacter venerupis sp. nov., new species isolated from shellfish. Syst Appl Microbiol 35:133–138. doi: 10.1016/j.syapm.2012.01.002. [DOI] [PubMed] [Google Scholar]
- 5.Fera MT, Maugeri TL, Gugliandolo C, Beninati C, Giannone M, La Camera E, Carbone M. 2004. Detection of Arcobacter spp. in the coastal environment of the Mediterranean Sea. Appl Environ Microbiol 70:1271–1276. doi: 10.1128/AEM.70.3.1271-1276.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Levican A, Rubio-Arcos S, Martinez-Murcia A, Collado L, Figueras MJ. 2015. Arcobacter ebronensis sp. nov. and Arcobacter aquimarinus sp. nov., two new species isolated from marine environment. Syst Appl Microbiol 38:30–35. doi: 10.1016/j.syapm.2014.10.011. [DOI] [PubMed] [Google Scholar]
- 7.Llobet-Brossa E, Rosselló-Mora R, Amann R. 1998. Microbial community composition of Wadden Sea sediments as revealed by fluorescence in situ hybridization. Appl Environ Microbiol 64:2691–2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Bowman JP, McCuaig RD. 2003. Biodiversity, community structural shifts, and biogeography of prokaryotes within Antarctic continental shelf sediment. Appl Environ Microbiol 69:2463–2483. doi: 10.1128/AEM.69.5.2463-2483.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Sasi Jyothsna TS, Rahul K, Ramaprasad EVV, Sasikala C, Ramana CV. 2013. Arcobacter anaerophilus sp. nov., isolated from an estuarine sediment and emended description of the genus Arcobacter. Int J Syst Evol Microbiol 63:4619–4625. doi: 10.1099/ijs.0.054155-0. [DOI] [PubMed] [Google Scholar]
- 10.Donachie SP, Bowman JP, On SL, Alam M. 2005. Arcobacter halophilus sp. nov., the first obligate halophile in the genus arcobacter. Int J Syst Evol Microbiol 55:1271–1277. doi: 10.1099/ijs.0.63581-0. [DOI] [PubMed] [Google Scholar]
- 11.Donachie SP, Hou S, Lee KS, Riley CW, Pikina A, Belisle C, Kempe S, Gregory TS, Bossuyt A, Boerema J, Liu J, Freitas TA, Malahoff A, Alam M. 2004. The Hawaiian Archipelago: a microbial diversity hotspot. Microb Ecol 48:509–520. doi: 10.1007/s00248-004-0217-1. [DOI] [PubMed] [Google Scholar]
- 12.Miller WG, Yee E, Lopes BS, Chapman MH, Huynh S, Bono JL, Parker CT, Strachan NJ, Forbes KJ. 2017. Comparative genomic analysis identifies a Campylobacter clade deficient in selenium metabolism. Genome Biol Evol 9:1843–1858. doi: 10.1093/gbe/evx093. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Miller WG, Yee E, Chapman MH, Smith TP, Bono JL, Huynh S, Parker CT, Vandamme P, Luong K, Korlach J. 2014. Comparative genomics of the Campylobacter lari group. Genome Biol Evol 6:3252–3266. doi: 10.1093/gbe/evu249. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
The complete genome sequence of A. halophilus strain CCUG 53805T has been deposited in GenBank under the accession number CP031218. The 454, HiSeq, and PacBio sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP155008. The source codes for contig_extender3 and BlastPTrimmer14 are available through GitHub (https://github.com/Arcobacter/Genome_perl).