Complete Genome Sequences of the Arcobacter cryaerophilus Strains ATCC 43158T and ATCC 49615.

Arcobacter cryaerophilus was originally recovered from aborted bovine and porcine fetuses, but it has been subsequently isolated from meat, water, and human clinical samples. This study describes the complete whole-genome sequences of two A. cryaerophilus strains, ATCC 43158T (=A 169/BT =LMG 24291T) and ATCC 49615 (=CDC D2610 =LMG 10829).

ANNOUNCEMENT

Arcobacter cryaerophilus (formerly Campylobacter cryaerophila [1]) was originally recovered from aborted bovine and porcine fetuses (1–3), from the milk of cows with mastitis (4), and from bovine and porcine feces (1). Subsequent studies reported the recovery of A. cryaerophilus isolates from meat (5, 6), water (7), and human clinical samples (8, 9). Additionally, based on the results of whole-cell protein and rRNA restriction fragment length polymorphism analyses (10, 11), A. cryaerophilus strains have been divided into two subgroups, 1A and 1B. In this study, we report the first closed genome sequences of two A. cryaerophilus strains, the type strain ATCC 43158 (subgroup 1A; =A 169/BT [1] =LMG 24291T), isolated in the United Kingdom from the brain of an aborted bovine fetus, and strain ATCC 49615 (subgroup 1B; =CDC D2610 [12] =LMG 10829), isolated in Illinois from human blood. A draft genome sequence (91 scaffolds) was deposited previously for A. cryaerophilus strain ATCC 43158T (GenBank accession number NXGK01000000). Additionally, draft genome sequences for 12 other A. cryaerophilus strains have been deposited previously in GenBank (https://www.ncbi.nlm.nih.gov/genome/genomes/11530).

Both A. cryaerophilus strains were grown aerobically at 30°C for 48 h on anaerobe basal agar (Oxoid) amended with 5% horse blood. Cells were separately removed from each plate using sterile 5-μl inoculating loops, and genomic DNA was prepared from each loopful of cells using the Promega Wizard genomic DNA purification kit (Madison, WI). Roche 454 shotgun and paired-end libraries were constructed for both strains, using the manufacturer’s standard protocols, and sequenced on a GS-FLX+ instrument using the Titanium chemistry. For each strain, the shotgun and paired-end reads were assembled together, using Newbler v. 2.6 (Roche) and default parameters; 454 reads were quality controlled within Newbler. Sequencing metrics for both strains are presented in Table 1. Two scaffolds were obtained for strain ATCC 43158T, one chromosomal scaffold of 20 contigs and a megaplasmid scaffold of 4 contigs. One chromosomal scaffold of 16 contigs was obtained for strain ATCC 49615. Each scaffold was closed by using the custom Perl script contig_extender3 (13) to place within the scaffold gaps unique, nonscaffolded contigs and/or contigs representing sequences present at two or more locations. The two chromosomes and the megaplasmid were assembled manually within SeqMan Pro v. 8.0 (DNASTAR, Madison, WI), using the contig order defined above and contig-spanning 454 reads. Assembly of both genomes was verified using an optical restriction map (restriction enzyme XbaI; OpGen, Gaithersburg, MD). Illumina HiSeq reads were obtained from SeqWright (Houston, TX) and assembled and quality controlled within Newbler v. 2.6, as above. The Illumina contigs were assembled automatically (unique contigs) or positioned manually (repeated contigs) within the 454 SeqMan assembly. The Illumina contigs and reads were used to verify the 454 base calls, as described previously (14). A repeat region within the type strain chromosome required long reads for validation. Therefore, a 20-kb PacBio library was constructed, using standard methods (15), and sequenced on an RS II instrument, using standard protocols, as described previously (14). PacBio reads were assembled with RS_HGAP_Assembly v. 3 (Pacific Biosciences) and default settings, which yielded single chromosomal and megaplasmid contigs. The PacBio chromosomal contig was manually placed within the SeqMan assembly to confirm the 454 contig order across the repeat region.

TABLE 1

Sequencing metrics and genomic data for Arcobacter cryaerophilus strains ATCC 43158T and ATCC 49615

Featureb	Value(s)a
	ATCC 43158T	ATCC 49615
Sequencing metrics
454 (shotgun) platform
No. of reads	215,129	87,759
No. of bases	90,272,116	36,134,765
Avg length (bases)	419.6	411.7
Coverage (×)	45.0	17.6
454 (paired-end) platform
No. of reads	118,785	161,571
No. of bases	38,763,675	49,505,772
Avg length (bases)	326.3	306.4
Coverage (×)	19.3	24.1
llumina HiSeq 2000 platform
No. of reads	18,366,932	18,769,966
No. of bases	1,836,693,200	1,895,766,566
Avg length (bases)	100	101
Coverage (×)	915.2	922.1
PacBio platform
No. of reads	117,659	NA
No. of bases	437,690,629	NA
Avg length (bases)	3,720c	NA
Coverage (×)	218.1	NA
Newbler metricsd
Total no. of contigs	45	32
N50ContigSize (454)	195,432	346,474
Q40PlusBases (454) (%)	99.90	99.87
N50ContigSize (HiSeq pool 1)	195,198	301,090
Q40PlusBases (HiSeq pool 1) (%)	99.98	99.96
N50ContigSize (HiSeq pool 2)	195,524	165,874
Q40PlusBases (HiSeq pool 2) (%)	99.96	99.96
N50ContigSize (HiSeq pool 3)	195,196	301,081
Q40PlusBases (HiSeq pool 3) (%)	99.97	99.99
Genomic data
Size (bp)	2,006,909; 101,431	2,055,914
G+C content (%)	27.46; 24.63	27.51
No. of CDSe	1,951; 94	1,994
Assigned function (% CDS)	847 (43.4); 3 (3.2)	857 (43.0)
General function annotation (% CDS)	675 (34.6); 34 (36.2)	694 (34.8)
Domain/family annotation only (% CDS)	131 (6.7); 4 (4.3)	136 (6.8)
Hypothetical (% CDS)	298 (15.3); 53 (56.4)	307 (15.4)
Pseudogenes	23; 6	8
Genomic islands/CRISPR
No. of genetic islands	8; 0	5
No. of CDS in genetic islands	108 [2]; 0	85 [2]
No. of CRISPR/Cas loci	0	0
Gene content/pathways
IS elements, mobile elements, or transposases	2; 1	2
Signal transduction
No. of Che proteins	9; 0	9
No. of methyl-accepting chemotaxis proteins	20; 0	23
No. of response regulators	23; 2	25
No. of histidine kinases	25; 1	27
No. of response regulator/histidine kinase fusions	0	3
No. of diguanylate cyclases	14; 0	15
No. of diguanylate phosphodiesterases (HD-GYP, EAL)	4, 1; 0	5, 1
No. of diguanylate cyclase/phosphodiesterases	9; 0	9
No. of other	10; 1	10
Motility
Flagellin genes	flaAB	flaAB
Restriction/modification
No. of type I systems (hsd)	[2]; 0	2
No. of type II systems	1; 0	3
No. of type III systems	0	2
Transcription/translation
No. of transcriptional regulatory proteins	32; 4	32
Non-ECF σ factors	σ70	σ70
No. of ECF σ factors	0	0
No. of tRNAs	49; 0	51
No. of ribosomal loci	5; 0	5
CO dehydrogenase (coxSLF)	No	No
Ethanolamine utilization (eutBCH)	No	No
Nitrogen fixation (nif)	No	No
Osmoprotection	ectABC	No
Pyruvate → acetyl-CoA
Pyruvate dehydrogenase (E1/E2/E3)	Yes	Yes
Pyruvate:ferredoxin oxidoreductase	No	No
Urease	No	No
Vitamin B12 biosynthesis	No	No

Numbers in square brackets indicate pseudogenes or fragments. Strain ATCC 43158T values before a semicolon are for the chromosome, while values after the semicolon are for the pACRY43158 plasmid. NA, not applicable.

CDS, coding sequences; ECF, extracytoplasmic function; acetyl-CoA, acetyl coenzyme A.

Maximum length, 25,867 bases.

Features and values taken from largeContigMetrics within 454NewblerMetrics.txt for each assembly. Large contigs were defined as having ≥500 bases. Due to the large number of HiSeq reads, the total reads were split into three pools and assembled independently. N50ContigSize value is in number of bases.

Numbers do not include pseudogenes.

Genomic data for the two A. cryaerophilus strains are presented in Table 1. The average genome size for the two strains is approximately 2 Mbp, with an average G+C content of 27.5%. Protein-, rRNA-, and tRNA-encoding genes were identified using GeneMark, RNAmmer, and ARAGORN (16–18), respectively, and annotated as described previously (14). The ATCC 43158T genome also contains the 101,431-bp megaplasmid pACRY43158.

Data availability.

The complete genome sequence of A. cryaerophilus strain ATCC 43158T has been deposited in GenBank under the accession numbers CP032823 (chromosome) and CP032824 (pACRY43158), and the complete genome sequence of A. cryaerophilus strain ATCC 49615 has been deposited in GenBank under the accession number CP032825. All 454, HiSeq, and PacBio sequencing reads for strain ATCC 43158T and 454 and HiSeq reads for strain ATCC 49615 have been deposited in the NCBI Sequence Read Archive (SRA) under the accession numbers SRP164716 and SRP164722, respectively.