Complete Genome Sequence of the Arcobacter bivalviorum Type Strain LMG 26154.

Arcobacters are routinely recovered from marine environments, and multiple Arcobacter species have been isolated from shellfish. Arcobacter bivalviorum was recovered from mussels collected in the Ebro Delta in northeastern Spain. This report describes the complete whole-genome sequence of the A. bivalviorum type strain LMG 26154 (= F4T = CECT 7835T).

ANNOUNCEMENT

Members of the genus Arcobacter have been isolated from a wide range of hosts and environments (1–3). While many Arcobacter species are routinely isolated from terrestrial food animals, at least seven Arcobacter species have been recovered from shellfish (4–13). Arcobacter bivalviorum is a catalase-positive, urease-negative arcobacter that was isolated originally from mussels collected in the Ebro Delta in Catalonia, Spain (6). The A. bivalviorum type strain LMG 26154 (= F4T = CECT 7835T) was isolated in March 2007 from mussels of the Ebro Delta. In this study, we report the first closed genome sequence of the A. bivalviorum type strain LMG 26154.

Arcobacter bivalviorum was grown at 28°C aerobically for 48 h on anaerobe basal agar (Oxoid) amended with 5% horse blood, and genomic DNA was extracted as described (14) from a 5-μl loop of cells. Shotgun and paired-end Roche 454 reads were generated as described (14) and assembled into 40 contigs using Newbler (version 2.6) with default parameters. A single chromosomal scaffold was produced from 21 unique contigs and closed into a circular, contiguous sequence by using the 19 remaining contigs that represent repeat regions, the Perl script contig_extender3 (14), and directed PCR amplification followed by Sanger sequencing. PacBio sequencing was performed as described (14), generating a single circular chromosomal sequence which was inserted into a SeqMan version 8.0.2 (DNASTAR, Madison, WI) assembly comprised of 454 and Sanger reads, creating a composite 454/PacBio chromosomal sequence and assembly, and additionally verifying the order of the 454 contigs within the original assembly. Base calls within this composite 454/PacBio chromosomal sequence were verified using Illumina HiSeq reads (SeqWright, Houston, TX), as follows. The HiSeq reads were also assembled within Newbler as described above; the resulting trimmed contigs, with a per-base quality score of ≥40, were assembled onto the SeqMan 454/PacBio assembly. Sequences between the Illumina contigs were queried for single nucleotide polymorphisms (SNPs) using the HiSeq reads and the “find variations/SNPs” module within Geneious version 11.0.2 (Biomatters Ltd., Auckland, New Zealand). The final coverage across the closed genome (i.e., a single circular chromosome with no gaps or ambiguous bases) was 1,327×. Chromosomal assembly was also validated using an optical restriction map (restriction enzyme XbaI; OpGen, Gaithersburg, MD).

A summary of the LMG 26154T genome features is presented in Table 1. A. bivalviorum strain LMG 26154T has a circular genome of 2,684,688 bp with an average GC content of 28.1%. Protein-, rRNA-, and tRNA-encoding genes were identified and annotated as described (15), with the exception that the composite proteome used here for comparison included proteins derived from all currently completed Campylobacter and Arcobacter genomes. The genome is predicted to encode 2,584 putative protein-coding genes and 16 pseudogenes. Additionally, the LMG 26154T genome contains 4 rRNA operons and 47 tRNA-encoding genes. Four genomic islands were identified in the LMG 26154T chromosome. Two of these genomic islands are approximately 10 kb in size, and two are approximately 15 kb in size. No plasmids were identified in strain LMG 26154T.

TABLE 1

Sequencing metrics and genomic data for A. bivalviorum strain LMG 26154T

Feature	Value(s)a
Sequencing metrics
454 (shotgun) platform
No. of reads	117,700
No. of bases	65,973,662
Average length (bases)	560.5
Coverage (×)	24.6
454 (paired end) platform
No. of reads	89,211
No. of bases	28,498,539
Average length (bases)	319.5
Coverage (×)	10.6
Illumina HiSeq platform
No. of reads	18,785,810
No. of bases	1,878,581,000
Average length (bases)	100
Coverage (×)	699.8
PacBio platform
No. of reads	356,337
No. of bases	1,588,024,540
Average length (bases)	4,456.5b
Coverage (×)	591.5
Genomic data
Chromosome
Size (bp)	2,684,688
G+C content (%)	28.11
No. of CDSc	2,584
Assigned function (% CDS)	961 (37.2)
General function annotation (% CDS)	1,018 (39.4)
Domain/family annotation only (% CDS)	180 (7.0)
Hypothetical (% CDS)	425 (16.4)
Pseudogenes	16
Genomic islands/CRISPRs
No. of genetic islands	4
No. of CDS in genetic islands	49
No. of CRISPR-Cas loci	0
Gene content/pathways
Signal transduction
Che proteins	cheABCDRVW(Y)2
No. of methyl-accepting chemotaxis proteins	23
No. of response regulators	55
No. of histidine kinases	67, [1]
No. of response regulator/histidine kinase fusions	6
No. of diguanylate cyclases	30, [1]
No. of diguanylate phosphodiesterases (HD-GYP, EAL)	9, 5
No. of diguanylate cyclase/phosphodiesterases	12
No. of other	10
Motility
Flagellin genes	flaAB
Restriction/modification
No. of type I systems (hsd)	0
No. of type II systems	1
No. of type III systems	0
Transcription/translation
No. of transcriptional regulatory proteins	64, [1]
Non-ECFd σ factor	σ70
No. of ECF σ factors	2
No. of tRNAs	47
No. of ribosomal loci	4
Nitrogen fixation (nif)	No
Osmoprotection	ectABC, proVWX
Pyruvate → acetyl-CoA
Pyruvate dehydrogenase (E1/E2/E3)	Yes
Pyruvate:ferredoxin oxidoreductase	por
Urease	No
Vitamin B12 biosynthesis	Yes

Numbers in brackets indicate pseudogenes or fragments.

Maximum length, 29,273 bp.

Numbers do not include pseudogenes. CDS, coding sequences.

ECF, extracytoplasmic function.

Strain LMG 26154T contains 25 of the 30 genes necessary for de novo biosynthesis of adenosylcobalamin (coenzyme B12) via the oxygen-independent (anaerobic) pathway (16). Orthologs of cbiJ, cobA, cobC, fre, and pduX were not identified; however, these enzymatic functions could be provided by other proteins or by orthologs with low similarity to proteins currently in the NCBI nr database.

Data availability.

The complete genome sequence of A. bivalviorum strain LMG 26154T has been deposited in GenBank under the accession number CP031217. Illumina HiSeq, 454, and PacBio sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP154993.