Complete genome sequences of eight Caryophanales strains isolated from the rhizosphere of strawberry in Canada

ABSTRACT

Here, we present the complete genome sequences of eight bacterial strains isolated from the rhizosphere of strawberry plants grown in an agricultural field located in Bouctouche, New Brunswick, Canada. The strains belong to four different genera (Bacillus, Lysinibacillus, Psychrobacillus, and Priestia) within the order Caryophanales.

ANNOUNCEMENT

The genus Bacillus (order Caryophanales) is an important genus of endospore-forming rod-shaped bacteria that includes both human pathogens and plant-beneficial strains (1, 2). Over the years, several genera were created from species previously included in this genus, such as Lysinibacillus, Psychrobacillus, and Priestia (3–5). Many of these genera also include strains with plant growth promotion activity (6, 7). Genome sequencing of plant-beneficial strains belonging to the genus Bacillus and its related genera will promote the development of microbial biofertilizers and contribute to a better understanding of the plant growth promotion molecular mechanisms involved. Here, we report the complete genome sequences of eight Caryophanales strains.

The strains were isolated in 2004 from the rhizosphere of strawberry plants (Fragaria x ananassa Duch.) grown in an agricultural field located in Bouctouche, New Brunswick, Canada (46.432232,-64.769877) using a protocol described previously (8). Briefly, rhizosphere soil samples were collected and kept at 4°C. One gram of soil was suspended in 100 mL of a 0.9% NaCl solution and agitated for 5 min at 250 rpm. The suspension was serially diluted and plated onto tryptic soy agar plates (BD Difco), which were incubated at 25°C for 48 h. Isolated colonies were subsequently purified on the same medium. Bacterial strains were cryopreserved at −80°C in sterile distilled water supplemented with 25% (v/v) glycerol.

Bacterial strains were grown directly from frozen stocks in tryptic soy broth (BD Difco) for 24 h at 25°C, and genomic DNA was extracted using the DNeasy UltraClean Microbial kit (Qiagen) following the manufacturer’s instructions. Library preparation and genome sequencing were performed at the Integrated Microbiome Resource (Halifax, NS, Canada). Genomic DNA was mechanically sheared and size-selected using g-TUBE devices (Covaris). The resulting DNA fragments (~8-10 kb) were converted into a WGS library using the SMRTbell Prep Kit 3.0 (Pacific Biosciences) as per the manufacturer’s instructions. Genome sequencing was performed on a Sequel II sequencer (Pacific Biosciences) loaded with a SMRT Cell 8M (v2.0 chemistry). FastQC v0.11.9 (9) was used to check the quality of the raw reads. Genome assembly was performed with Flye v2.9-b1768 using the command “--pacbio-hifi” (HiFi reads) or “--pacbio-raw” (CLR reads). Chromosome and plasmid sequences were automatically circularized by the Flye algorithm and rotated using Geneious Prime v2025.1.2 (Biomatters). The origin of the chromosome sequences was set at the start of the dnaA gene. The genomes were annotated by the NCBI Prokaryotic Genome Annotation Pipeline v6.10 (10). Default parameters were used for all software, unless otherwise specified. Sequencing, assembly metrics, and genome features are presented in Table 1.

TABLE 1.

Genome sequencing/assembly metrics and genome features

Metrics	Strains
	LBUM116	LBUM136	LBUM169	LBUM342	LBUM349	LBUM447	LBUM564	LBUM632
No. of CLR reads	3,921,600	2,573,417	25,834,315	1,750,168	15,855,285	4,072,462	1,794,769	2,497,778
CLR read N50 (bp)	4,877	5,896	4,200	5,046	2,408	4,848	5,932	6,045
No. of HiFi reads	96,083	70,512	544,216	42,597	232,162	94,911	49,220	72,518
HiFi read N50 (bp)	6,693	7,977	6,829	7,193	4,065	6,804	8,364	8,196
Reads used for assembly	HiFi	HiFi	HiFi	CLR	CLR	HiFi	HiFi	CLR
Coverage (×)	103	70	461	1,107	4,517	80	76	1,708
Genome size (bp)	4,731,136	5,911,531	5,544,785	5,416,514	6,123,168	5,968,817	4,009,808	6,065,531
G + C content (%)	37.6	35.3	35.6	36.8	37.6	35.3	36.2	37.6
No. of chromosomes	1	1	1	1	1	1	1	1
No. of plasmids	0	2	7	1	8	5	0	10
No. of coding DNA sequences	4,539	5,952	5,528	5,158	6,190	6,035	3,884	6,085
No. of pseudogenes	59	239	405	118	95	288	45	102
No. of rRNAs	34	42	39	43	47	42	30	47
No. of tRNAs	107	107	107	110	144	107	74	152
Closest type strain (accession no.)	Lysinibacillus fusiformis ATCC 7055T (NZ_PYWL00000000)	Bacillus mycoides DSM 2048T (NZ_CM000742)	Bacillus pseudomycoides DSM 12442T (NZ_CM000745)	Lysinibacillus xylanilyticus DSM 23493T (NZ_LFXJ00000000)	Priestia megaterium ATCC 14581T (NZ_CP069288)	Bacillus mycoides DSM 2048T (NZ_CM000742)	Psychrobacillus psychrodurans DSM 11713T (NZ_JAMKBK000000000NZ_JAMKBK000000000)	Priestia megaterium ATCC 14581T (NZ_CP069288)
Digital DNA-DNA hybridization value (%)	97.1	70.8	68.5	69.3	72.7	93	79.6	71.2
Average nucleotide identity (%)	99.4	95.9	95.6	96	95.6	98.7	97.5	95.5
Identification	Lysinibacillus fusiformis	Bacillus mycoides	Bacillus pseudomycoides	Lysinibacillus xylanilyticus	Priestia megaterium	Bacillus mycoides	Psychrobacillus psychrodurans	Priestia megaterium
GenBank accession no.	CP189820	CP189817 CP189818 CP189819	CP189809 CP189810 CP189811 CP189812 CP189813 CP189814 CP189815 CP189816	CP189807 CP189808	CP189798 CP189799 CP189800 CP189801 CP189802 CP189803 CP189804 CP189805 CP189806	CP189792 CP189793 CP189794 CP189795 CP189796 CP189797	CP189791	CP189780 CP189781 CP189782 CP189783 CP189784 CP189785 CP189786 CP189787 CP189788 CP189789 CP189790
SRA accession no.	SRR33306138	SRR33306137	SRR33306136	SRR33306135	SRR33306134	SRR33306133	SRR33306132	SRR33306131

Genome size ranges from 4.01 to 6.12 Mb, and the G + C content fluctuates between 35.3% and 37.6%. The Type (Strain) Genome Server (11) and the JSpeciesWS web server (12) were used to identify the strains to the species level (Table 1). The strains belong to four different genera (Bacillus, Lysinibacillus, Psychrobacillus, and Priestia) within the order Caryophanales. To visualize the phylogenetic relationships between the eight strains and closely related type strains, we performed a multilocus sequence analysis (Fig. 1).

Fig 1.

Phylogenetic relationships between eight Caryophanales strains and closely related type strains. The genomes of type strains belonging to the B. cereus group and the Priestia/Psychrobacillus/Lysinibacillus genera were downloaded from GenBank. The complete nucleotide sequences of four housekeeping genes (gyrA, gyrB, rpoB, and rpoC) were extracted from each genome, concatenated, and subsequently aligned using Clustal Omega v1.2.2 (13). The resulting alignment was used to generate a phylogenetic tree using FastTree v2.1 (14) and the generalized time reversible model. The tree topology was verified by computing Shimodaira Hasegawa support values (15). Only support values above 0.5 are displayed at the nodes. Lactobacillus delbrueckii DSM 20074^T was used as an outgroup. The eight strains under study are highlighted in bold. The scale bar indicates sequence divergence.

DATA AVAILABILITY

The complete genome sequences of the eight bacterial strains (BioProject PRJNA1255136) have been deposited at GenBank. The versions described in this paper are the first versions. The raw sequencing data have been deposited to the Sequence Read Archive (SRA). Accession numbers are provided in Table 1.