Complete Genome Sequence of the Arcobacter halophilus Type Strain CCUG 53805.

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

Sequencing metrics and genomic data for A. halophilus strain CCUG 53805T

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).