Draft Genome Sequences of Seven Chryseobacterium Type Strains.

In an honors course on "Omics Sciences," draft genome sequences of Chryseobacterium elymi KCTC 22547T, Chryseobacterium flavum KCTC 12877T, Chryseobacterium hispanicum KCTC 22104T, Chryseobacterium lathyri KCTC 22544T, "Candidatus Chryseobacterium massiliae" CCUG 51329T, Chryseobacterium piscium CCUG 51923T, and Chryseobacterium rhizosphaerae KCTC 22548T were generated to facilitate phylogenomic comparisons within the genus.

ANNOUNCEMENT

Chryseobacterium species are chemoorganotrophic, Gram-negative rods with flexirubin pigments that give the colonies a yellow-orange color. The genus contains more than 100 described species from diverse habitats, including freshwater sources, soil, marine fish, and human hosts (1–6). Our laboratories have isolated many chryseobacteria, including novel strains, from freshwater creeks and from poultry.

To determine whether a bacterium is a member if an existing species or a novel taxon, it should be compared to previously described species at the genomic level in which an average nucleotide identity (ANI) below 95% signifies that they are different species (7). Genome sequences are now required to describe new taxa, and the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project is sequencing type strain genomes for previously described species (8–11). Indeed, many Chryseobacterium type strain genomes have been sequenced as part of GEBA and also as part of the Genome Consortium for Active Teaching Next Generation Sequencing group (GCAT-SEEK) (12, 13). To provide a course-based undergraduate research experience (CURE) (14, 15) for the bioinformatics and -omics sciences honors course (BOCB 6834) at the University of the Free State (UFS) in Bloemfontein, South Africa, we selected seven unsequenced Chryseobacterium type strains from the UFS bacteria collection for analysis. Freeze-dried strains in vials were rehydrated with tryptic soy broth and streaked onto tryptic soy agar plates. After 2 days of incubation at 30°C, approximately 10 mg wet cell mass was scraped from the plates and DNA was extracted using the Zymo Quick-DNA fungal/bacterial miniprep kit according to the manufacturer’s recommendations. The purified DNA was assessed for quality by gel electrophoresis and the amount of DNA was quantified by fluorescent dye binding using a Qubit fluorometer (ThermoFisher). The source organisms’ identities were confirmed by amplification of the 16S rRNA genes from the purified DNA with universal primers 27f and 1492r (6) followed by Sanger sequencing of the amplicons with primer 27f and comparison to the type strain database on EzBioCloud (16). Nextera XT Libraries (Illumina) were prepared and 300-base paired-end sequences were generated on an Illumina MiSeq instrument at the UFS Next Generation Sequencing Unit. Reads were assembled using SPAdes v 3.10.0 with default parameters as implemented on PATRIC (17), and the assembly was assessed for quality with CheckM v 1.0.8 using the default parameters (18) on the KBase platform (19) and annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (20). The genomes ranged in size from 3.78 to 4.86 Mbp and had G+C mol% values from 33.6 to 37.1% which were all within 0.7% of values measured by HPLC or the thermal denaturation method (Table 1). Comparison of the genome-derived 16S rRNA sequences to the original sequences in GenBank revealed that while most of the originally deposited sequences were fairly accurate, the Chryseobacterium flavum CW-E 2T sequence with the GenBank accession number EF154516 had 25 differences in a small region from position 974 to 1139 relative to the genome-derived sequence and was also very different from all other Chryseobacterium 16S rRNA sequences. The authors of the C. flavum species description (3) also noted that the 16S rRNA sequence similarity to the closest relatives was below 95%. The C. flavum KCTC 12877T genome-derived 16S rRNA sequence was most similar (99.1%) to that of Chryseobacterium gleum ATCC 35910T, so the ANI was calculated using the Ortho-ANI Tool (21) and found to be 83.2%, confirming that C. flavum and C. gleum are indeed distinct species. The C. flavum KCTC 12877T 16S rRNA sequence was deposited separately into GenBank under the accession number MK116543.

TABLE 1

Genome sequence information

Organism (reference)	WGS accession no.	Genome size (bp)	G+C observed/predicted (mol%)	Coverage (×)	No. of contigs	N50 (kbp)	No. of 16S rRNA differencesa	CheckM completeness/contamination
Chryseobacterium elymiKCTC 22547T (1)	QNUH01	4,591,259	37.1/36.4	34	80	249	0	100/0.245
Chryseobacterium flavumKCTC 12877T (3)	QNUE01	4,470,829	36.7/37.2	29	40	311	25	100/0.49
Chryseobacterium hispanicumKCTC 22104T (4)	QNUG01	3,777,625	34.6/34.3	36	148	90	4	100/0.0
Chryseobacterium lathyriKCTC 22544T (1)	QNFY01	4,857,652	37.0/36.6	28	56	305	1	100/0.245
“Candidatus Chryseobacterium massiliae” CCUG 51329T (5)	QNVU01	4,409,139	36.0/NDb	29	116	79	0	100/0.49
Chryseobacterium pisciumCCUG 51923T (2)	QNVS01	4,319,594	33.6/33.6	30	158	51	0	100/0.0
Chryseobacterium rhizosphaeraeKCTC 22548T (1)	QNUF01	5,267,751	36.3/35.9	22	98	148	1	100/0.245

Differences between genome-derived 16S rRNA sequences and corresponding GenBank database entries.

ND, not determined.

Data availability.

The Chryseobacterium whole-genome shotgun (WGS) projects were deposited under the GenBank accession numbers QNUH00000000 (C. elymi KCTC 22547T), QNUE00000000 (C. flavum KCTC 12877T), QNUG00000000 (C. hispanicum KCTC 22104T), QNFY00000000 (C. lathyri KCTC 22544T), QNVU00000000 (“Ca. C. massiliae” CCUG 51329T), QNVS00000000 (C. piscium CCUG 51923T), and QNUF00000000 (C. rhizosphaerae KCTC 22544T). The versions described in this paper are the first versions, QNUH01000000, QNUE01000000, QNUG01000000, QNFY01000000, QNVU01000000, QNVS01000000, and QNUF01000000, respectively. The Sequence Read Archive (SRA) accession numbers are SRX5025940, SRX5025949, SRX5025952, SRX5026294, SRX5026665, SRX5027813, and SRX5027959, respectively.