Complete Genome Sequences of the Campylobacter fetus subsp. venerealis, Campylobacter lari subsp. concheus, Campylobacter sputorum bv. sputorum, and Campylobacter volucris Type Strains.

Campylobacter spp. are recovered from a wide variety of sources, including birds, livestock, shellfish, and human clinical samples. We present here the complete genomic data for the type strains of Campylobacter fetus subsp. venerealis, Campylobacter lari subsp. concheus, Campylobacter sputorum bv. sputorum, and Campylobacter volucris.

ANNOUNCEMENT

The genus Campylobacter currently contains 37 taxa comprising 31 validly described species, with five of these species further divided into 11 subspecies. Genomic data exist for the type strains of 34 Campylobacter taxa (e.g., see references 1–8). Such information would be valuable as baseline reference data for each species. Although genomic data exist for strains of the remaining three taxa (i.e., Campylobacter lari subsp. concheus, Campylobacter sputorum, and Campylobacter volucris [7, 9]), no genomic data, draft or complete, exist for their type strains. Additionally, while the type genome of Campylobacter fetus subsp. venerealis has been sequenced (10), a complete genome (one gapless contig) is recommended due to the presence of both mobile elements and a suite of S-layer genes with repeated motifs in Campylobacter fetus subsp. venerealis strains (11, 12). In this study, we report the closed genome sequences of Campylobacter lari subsp. concheus LMG 21009T, Campylobacter sputorum bv. sputorum LMG 7795T, Campylobacter volucris LMG 24380T, and Campylobacter fetus subsp. venerealis NCTC 10354T.

The four type strains were obtained from the NCTC (C. fetus subsp. venerealis) or BCCM/LMG (C. lari subsp. concheus, C. sputorum bv. sputorum, and C. volucris) culture collections. All strains were grown, both initially and for one subculture, microaerobically, at 37°C for 48 h on brain heart infusion agar (Thermo Fisher Scientific, Waltham, MA) amended with 5% laked horse blood. Genomic DNAs (gDNAs) were prepared from loopfuls (∼5 μl) of cells using the Promega Wizard genomic DNA purification kit. For each strain, a single preparation of gDNA was used to construct the Illumina and PacBio libraries. Each Illumina library was prepared using the Illumina Nextera DNA Flex library prep kit with 20 ng of gDNA. Pooled libraries were sequenced on a MiSeq instrument at 8.0 pM with dual-index paired-end reads using the MiSeq reagent kit v2 (300 cycles). Each 20-kb PacBio library was prepared using 15 μg of gDNA, the PacBio SMRTbell template prep kit v1.0, and the manufacturer’s protocols. Single-molecule real-time (SMRT) sequencing was performed on an RS II sequencer. PacBio reads were assembled using the Hierarchical Genome Assembly Process (HGAP) v3.0 in SMRT Analysis software v2.3.0. Default parameters were used for all software unless otherwise specified. The sequencing metrics are presented in Table 1. SMRT sequencing resulted in a single chromosomal contig for each of the four strains; for two strains (NCTC 10354T and LMG 24380T), single plasmid contigs were also present. Almost all of the low-quality (Q < 40) bases in these contigs were at the contig ends. Therefore, contig circularization removed these low-quality bases; all contigs were circularized manually with Geneious Prime v2019.1.3 (Biomatters Ltd., Auckland, New Zealand). Base calling was further improved by mapping the quality-trimmed MiSeq reads onto the circularized PacBio contigs within Geneious Prime. Using the Geneious “find variations/SNPs” module with a default minimum variation of 0.3, variations in each genome were identified and corrected to the MiSeq consensus sequence. The final coverages ranged from 280 to 1,233×.

TABLE 1

Sequencing metrics and genomic data

Featurea	Value(s) forb:
	C. fetus subsp. venerealis NCTC 10354T	C. lari subsp. concheus LMG 21009T	C. sputorum bv. sputorum LMG 7795T	C. volucris LMG 24380T
Source	Heifer, vaginal	Shellfish	Human, oral	Black-headed gull
Sequencing metrics
Illumina MiSeq platform
No. of reads	1,460,738	1,699,532	1,651,458	1,702,578
No. of bases	216,732,966	253,024,312	243,920,520	252,662,467
Avg length (bases)	148	149	148	148
Coverage (×)	115	170	139	166
PacBio platform
No. of reads	188,065	328,084	45,750	198,092
No. of bases	1,331,563,178	1,582,521,489	246,714,448	1,189,853,205
Avg length (bases)	7,080.3	4,823.5	5,392.7	6,006.6
Coverage (×)	706	1,063	141	780
Accession no.
GenBank (chromosome)	CP043435	CP043426	CP043427	CP043428
GenBank (plasmid)	CP043436	NA	NA	CP043429
NCBI SRA	SRP219907	SRP219934	SRP219932	SRP219757
Genomic data
Chromosome
Size (kbp)c	1,885.7	1,488.5	1,753.4	1,524.9
G+C content (%)	33.29	29.77	29.71	28.57
No. of CDSd	1,808	1,443	1,662	1,482
Assigned function (% CDS)	915 (50.6)	865 (59.9)	914 (55.0)	873 (58.9)
General function annotation (% CDS)	510 (28.2)	345 (23.9)	430 (25.9)	357 (24.1)
Domain/family annotation only (% CDS)	106 (5.9)	96 (6.7)	99 (6.0)	87 (5.9)
Hypothetical (% CDS)	277 (15.3)	137 (9.5)	219 (13.2)	165 (11.1)
No. of pseudogenes	50	18	52	19
No. of GC tracts ≥8 bp (no. hypervariable)	33 (28)	13 (13)	17 (11)	17 (17)
Plasmid (size [bp])	p3226 (27,915)	No	No	p9726 (7,145)
Genomic island(s)/CRISPR
No. of genetic islands	5	0	1	1
No. of CDS in genetic islands	165 [9]	0	22 [1]	48 [1]
CRISPR/Cas loci	Type III	0	Type I-B	0
No. of insertion sequence elements	6; 3	0	[1]	0
Gene content/pathways
Signal transduction
No. of Che proteins	6	7	9	7
No. of methyl-accepting chemotaxis proteins	10 [5]; 0	11 [2]	10 [5]	13 [1]; 0
No. of RRs	13; 0	9	15	9; 0
No. of HKs	9 [1]; 0	6	12 [2]	6; 0
No. of RR/HK fusions	1; 0	0	0	0
No. of diguanylate cyclases/phosphodiesterases	9; 0	2	7	2; 0
No. of other signal transduction genes	2; 0	2	3 [1]	2; 0
Motility
Flagellin genes	flaAB	flaAB	fla1-2	flaAB
Restriction/modification systems
No. of type I systems (hsd)	1; 0	1	1	[1]; 0
No. of type II systems	1; 0	2	3	3; 0
No. of type III systems	0	0	0	0
Transcription/translation
No. of transcriptional regulatory proteins	25 [2]; 0	22 [1]	24 [1]	18; 0
Sigma factors	σ28, σ54, σ70	σ28, σ54, σ70	σ28, σ54, σ70	σ28, σ54, σ70
No. of tRNAs, ribosomal loci	44, 3	46, 3	47, 3	46, 3
Catalase gene (katA)	Yes	Yes	No	Yes
Cytolethal distending toxin genes (cdtABC)	Yes (3×)	Yes	No	Yes
Histidine biosynthesis genese	I	II	I	II
Nuo clusterf	I	II	III	II
Tryptophan biosynthesis [trpABC(DG)EF]	Yes	No	Yes	No

RRs, response regulators; HKs, histidine kinases.

Numbers in brackets indicate pseudogenes or fragments. Values following a semicolon are plasmid borne. NA, not applicable.

Size listed in kilobase pairs due to length variation at the hypervariable GC tracts.

Numbers do not include pseudogenes. CDS, coding sequences.

I represents genes hisA, hisB, hisC, hisD, hisF, hisG(S), hisH, hisIE, hisJ, and hisZ; II represents genes hisA, hisBJ, hisC, hisD, hisF, hisG(L), hisH, and hisIE.

I represents nuoA-nuoB-nuoCD-nuoE-nuoF-nuoG-nuoH-nuoI-nuoJ-nuoK-nuoL-nuoM-nuoN; II represents genes nuoA-nuoB-nuoC-nuoD-ORF-ORF-nuoG-nuoH-nuoI-nuoJ-nuoK-nuoL-nuoM-nuoN; and III represents genes nuoA-nuoB-nuoC-nuoD-nuoE-nuoF-nuoG-nuoH-nuoI-nuoJ-nuoK-nuoL-nuoM-nuoN.

The genomic data are presented in Table 1. Protein-, rRNA-, and tRNA-encoding genes were identified using GeneMark, RNAmmer, and ARAGORN (13–15), respectively, and annotated as described previously (16). The genome sizes, G+C content values, and gene contents of these four strains are similar to those reported for other strains of the same taxa (7, 9, 12). With these genome sequences, genomic data are now available for type strains representing all current taxa in the genus Campylobacter.

Data availability.

The complete genome sequences of the four Campylobacter type strains have been deposited in GenBank, and all MiSeq and PacBio sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA). The accession numbers are provided in Table 1.