Complete genome sequence of Pectobacterium brasiliense strain 21PCA_AGRO2 with antimicrobial resistance isolated from napa cabbage

ABSTRACT

We report a complete genome of Pectobacterium brasiliense strain 21PCA_AGRO2 isolated from napa cabbage, in which the genome consists of a circular chromosome comprising 4,919,671 bp with 4,399 coding DNA sequences, 22 rRNA genes, 77 tRNA genes, and 9 noncoding RNA genes.

ANNOUNCEMENT

The genus Pectobacterium is a plant pathogen that causes soft rot on a variety of economically important crops, such as potatoes, napa cabbages, and radishes (1). Long-term use of agricultural antibiotics to control the soft rot may lead to problems with antibiotic resistance (2, 3). Thus, we conducted whole-genome sequencing of P. brasiliense 21PCA_AGRO2 isolated from napa cabbage. This will provide insight into the pathogenic bacterial genome and promote further research to track the antibiotic resistance in agricultural products.

P. brasiliense 21PCA_AGRO2 was isolated from soft rot lesion in napa cabbage collected from Pyeongchang, South Korea. The infected plant tissues were sterilized with 1% hypochlorite solution for 90 s, rinsed in double distilled water, and then ground into homogenate with 1 mM MgSO₄ by stomacher (BagMixer 400 Laboratory Blender, Interscience, UK). The single colony was cultured at 28°C in LB (Luria-Bertani) media with overnight, diluted 1:100 with fresh LB broth, and then incubated 18–20 h at 28°C with shaking at 200 rpm. Colonies on plates were picked and confirmed with PCR using specific primers for pectate lyase (pel) genes (5′-TTACCGGACGCCGAGCTGTGGCGT-3′ and 5′-CAGGAAGATGTCGTTATCGCGAGT-3′) of Pectobacterium spp. and 16S rRNA (5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-GGTTACCTTGTTACGACTT-3′) sequencing. The 16S rRNA matched more than 99.5% with the reference sequences of P. brasiliense in BLAST database. Afterward, genomic DNA (gDNA) was extracted using Kit PureHelix Genomic DNA Prep Kit (solution type)-Bacteria and quantified and qualified by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, USA). The Short Read Eliminator kit was used to remove <10 kbp fragments from unsheared gDNA. The Oxford Nanopore Technologies sequencing library was prepared using the manufacturer’s ligation sequencing kit (SQK-LSK 112, UK) and sequenced on a MinION MK1b device (MIN112, R10.4) with MinKNOW software (22.05.5).

A total of 64,110 raw reads (N₅₀ 25,167 bp) were produced and then trimmed and quality filtered using Porechop v2.0.4 (https://github.com/rrwick/Porechop). The de novo assembly was conducted using flye v2.9-b1778 and confirmed genome completeness using BUSCO v5.2.2 (4, 5). Next, the genome was rotated using dnaA as the start position based on the fixstart method in Circlator v1.5.5 (6) and annotated with Prokaryotic Genome Annotation Pipeline (PGAP) v6.5 (7). As a result, the complete genome sequence comprised one circular form of 4,919,671 bp and a GC content of 51.68%, yielding 4,399 coding sequences (including 28 frameshifted genes), 22 rRNAs, 77 tRNAs, and 9 ncRNAs (Fig. 1). Subsequently, taxonomic classification was performed using GTDB-tk v1.5.1 (8) and Kraken2 v2.1.2 (9), which is identified as P. brasiliense. The average nucleotide identity (ANI) analysis was performed using FastANI v1.0 (10) to compare our genome with 31 species of published P. carotovorum and P. brasiliense genome sequences. As a consequence, our genome was closest to the P. brasiliense ZLMLSHJ5 strain (GCF_016864975.1) with an ANI value of 97.55%. Additionally, antibiotic resistance gene prediction using RGI v5.2.1 against CARD database identified 11 antibiotic resistance genes, including aminoglycoside resistance (11). Moreover, eggNOG-mapper v2.1.6 (12) was used with the eggNOG v5 (13) database to assign the Clusters of Orthologous Groups (COGs) functional categories based on biological systems. All the bioinformatics tools were used with default options unless specified otherwise.

Fig 1.

The genome map of Pectobacterium brasiliense strain 21PCA_AGRO2. Each circle indicates coding sequences (CDS) in the leading strand, CDS in the lagging strand, COG distribution, RNA, antibiotic resistance genes, and the GC contents from outer to inner. Antibiotic resistance genes are labeled.

DATA AVAILABILITY

The whole genome sequence was deposited in GenBank under the accession number CP113504https://www.ncbi.nlm.nih.gov/nuccore/CP113504.1, BioProject accession number PRJNA906323 and SRA accession number SRR22542065.