Complete Genome Sequence of the Arcobacter suis Type Strain LMG 26152.

Arcobacter species are prevalent in pigs, and strains have been isolated from pig feces and pork meat; some Arcobacter strains may be porcine abortifacients. Arcobacter suis was recovered from pork meat in Spain. This study describes the whole-genome sequence of the A. suis type strain LMG 26152 (=F41T =CECT 7833T).

ANNOUNCEMENT

Arcobacter species are often isolated from swine, pig manure, and pork meat (1–6), and some species or strains are possible porcine abortifacients (7, 8). Arcobacter suis, represented by a single strain (the type strain), was originally isolated from retail market pork (9). Subsequent to the initial description, A. suis was also recovered from water buffalo milk (10), operational taxonomic units similar to A. suis were identified in samples from a spinach-processing plant (11), and A. suis was potentially identified in sewage (12). In this study, we report the first closed genome sequence of the A. suis type strain LMG 26152 (=F41T =CECT 7833T), isolated in 2008 from pork meat in Catalonia, Spain (9).

Arcobacter suis strain LMG 26152T was grown at 30°C for 48 h aerobically on anaerobe basal agar (Oxoid) plus 5% horse blood. A loop of cells (∼5 μl) was taken from the plate, and genomic DNA was isolated with the Wizard genomic DNA purification kit (Promega, Madison, WI). Shotgun and paired-end Roche 454 libraries were constructed following the manufacturer’s protocols and with standard methods. PacBio SMRTbell libraries were prepared from 10 μg of genomic DNA with the standard 20-kb PacBio protocol (13). Shotgun and paired-end 454 libraries were sequenced on a GS-FLX+ instrument with Titanium chemistry and standard protocols. The resulting reads were assembled into 79 total contigs and a chromosomal scaffold of 33 contigs with Newbler v. 2.6 (Roche); Roche standard flowgram format (SFF) files were not processed before assembly, and 454 reads were quality controlled within the Newbler assembler. Low-quality contigs were deleted, and the remaining 25 contigs were positioned at one or more locations within the scaffold gaps with the Perl script contig_extender3 (14). PacBio sequencing was performed as described previously (14); the single chromosomal contig was assembled along with the 454 contigs with SeqMan v. 8.0 (DNASTAR, Madison, WI). Chromosomal assembly was also validated with an optical restriction map (restriction enzyme XbaI; OpGen, Gaithersburg, MD). Illumina HiSeq reads were obtained from SeqWright (Houston, TX) and assembled with Newbler v. 2.6. HiSeq reads were not processed before assembly, and any quality trimming of the reads was performed within Newbler. The HiSeq reads and contigs were used to verify and error correct the 454 and PacBio base calls, as described previously (15). The final coverage across the genome was 917×.

Sequencing metrics and genomic data for A. suis strain LMG 26152T are presented in Table 1. A. suis strain LMG 26152T has a circular genome of 2,639,269 bp, with an average G+C content of 27.4%. Putative coding sequences (CDSs), tRNA/transfer-messenger RNA (tmRNA) genes, and rRNA loci were identified with GeneMark, ARAGORN, and RNAmmer, respectively (16–18). A preliminary GenBank-formatted file was created with the genome sequence and the GeneMark-derived CDS coordinates. Identification of putative pseudogenes and genes missed in the original GeneMark analysis and manual curation of each putative CDS were performed with the GenBank-formatted file and Artemis v. 16 (19). Annotation was accomplished with BLASTP to compare the proteome of strain LMG 26152T to proteins in both the NCBI nonredundant (nr) database and a custom protein database constructed from the proteomes of all current completed Arcobacter and Campylobacter genomes. Annotation was further refined with an analysis of Pfam motifs (20).

TABLE 1

Sequencing metrics and genomic data for Arcobacter suis strain LMG 26152T

Feature	Value(s)a
Sequencing metrics
454 (shotgun) platform
No. of reads	185,325
No. of bases	103,818,564
Average length (bases)	560.2
Coverage (×)	39.3
454 (paired-end) platform
No. of reads	76,546
No. of bases	24,718,237
Average length (bases)	322.9
Coverage (×)	9.4
Illumina HiSeq 2000 platform
No. of reads	17,658,830
No. of bases	1,765,883,000
Average length (bases)	100
Coverage (×)	669.1
PacBio platform
No. of reads	174,492
No. of bases	524,997,072
Average length (bases)	3,008.7b
Coverage (×)	198.9
Newbler metricsc
N50ContigSize (454) (bases)	142,251
Q40PlusBases (454) (%)	99.84
N50ContigSize (HiSeq pool 1) (bases)	142,383
Q40PlusBases (HiSeq pool 1) (%)	99.99
N50ContigSize (HiSeq pool 2) (bases)	142,376
Q40PlusBases (HiSeq pool 2) (%)	99.99
N50ContigSize (HiSeq pool 3) (bases)	142,376
Q40PlusBases (HiSeq pool 3) (%)	99.99
Genomic data
Chromosome
Size (bp)	2,639,269
G+C content (%)	27.39
No. of CDSsd	2,523
Assigned function (% CDSs)	976 (38.7)
General function annotation (% CDSs)	923 (36.6)
Domain/family annotation only (% CDSs)	173 (6.9)
Hypothetical (% CDSs)	451 (17.9)
Pseudogenes	34
Genomic islands/CRISPR
No. of genetic islands	9
No. of CDSs in genetic islands	157, [7]
No. of CRISPR-Cas loci	0
Gene content/pathways
IS elements, mobile elements, or tranposases	3, [2] (IS3)
Signal transduction
Che proteins	cheABCDRVW(Y)2
No. of methyl-accepting chemotaxis proteins	25
No. of response regulators	42, [1]
No. of histidine kinases	53, [1]
No. of response regulator/histidine kinase fusions	6
No. of diguanylate cyclases	17
No. of diguanylate phosphodiesterases (HD-GYP, EAL)	7, 22
No. of diguanylate cyclase/phosphodiesterases	13
No. of other	8, [1]
Motility
Flagellin genes	fla
Restriction/modification
No. of type I systems (hsd)	0
No. of type II systems	1
No. of type III systems	1
Transcription/translation
No. of transcriptional regulatory proteins	54
Non-ECFe σ factors	σ54, σ70
No. of ECF σ factors	3
No. of tRNAs	59
No. of ribosomal loci	5
CO dehydrogenase (coxSLF)	Yes
Ethanolamine utilization (eutBCH)	Yes
Nitrogen fixation (nif)	Yes
Osmoprotection	betA
Pyruvate → acetyl-CoAf
Pyruvate dehydrogenase (E1/E2/E3)	Yes
Pyruvate:ferredoxin oxidoreductase	porABDG
Urease	ureAB
Vitamin B12 biosynthesis	Yes

Numbers in square brackets indicate pseudogenes or fragments.

Maximum length, 24,428 bases.

Features and values taken from largeContigMetrics within 454NewblerMetrics.txt for each assembly. Large contigs were defined as those ≥500 bases. Due to the large number of HiSeq reads, the total reads were split into 3 pools and assembled independently.

Numbers do not include pseudogenes. CDSs, coding sequences.

ECF, extracytoplasmic function.

CoA, coenzyme A.

The LMG 26152T genome is predicted to encode 2,523 putative protein-coding genes, 34 pseudogenes, 5 rRNA operons, and 59 tRNA-encoding genes, and it contains 9 genomic islands ranging from 5.5 to 34.3 kb in size. The LMG 26152T genome contains a nif/rpoN nitrogen fixation gene cluster (21; GenBank accession number CP001999) and the same set of adenosylcobalamin biosynthesis genes identified previously in A. bivalviorum (22). The A. suis genome also encodes the B12-dependent EutBC ethanolamine ammonia-lyase and the EutH ethanolamine permease. The acetaldehyde produced by EutBC would presumably be converted to ethanol and acetyl-coenzyme A by a putative AdhE dehydrogenase (Asuis2568). Two large genes encoding T1SS repeat domain-containing proteins were identified in the A. suis genome: asuis0242 (9,252 bp) and asuis0243 (16,326 bp). Similarly to A. mytili (23), these genes contain tandemly repeated internal motifs (5 × 639 bp, asuis0242; 39 × 647 bp, asuis0243). A 34,282-bp gene with no internal repeats encoding a putative repeats-in-toxin (RTX) family calcium-binding protein was also identified.

Data availability.

The complete genome sequence of A. suis strain LMG 26152T has been deposited in GenBank under the accession number CP032100. 454, HiSeq, and PacBio sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP155204.