Complete genome sequence of Burkholderia contaminans PCR-C_Y9 isolated from Pericarpium citri reticulatae 'Chachiensis'

TL;DR

This paper reports the full genome sequence of a bacterium that degrades lipids in aged citrus pericarps.

Contribution

The study provides the first complete genome sequence of Burkholderia contaminans PCR-C_Y9 and its potential role in pericarp aging.

Findings

The genome size is 8.70 Mbp with a GC content of 66.23%.

The bacterium can degrade lipids in Pericarpium citri reticulatae.

Genome annotation helps understand its properties and aging contribution.

Abstract

Burkholderia contaminans PCR-C_Y9 was isolated from Pericarpium citri reticulatae ‘Chachiensis’ (PCR-C), the natural aged pericarps of Citrus reticulata Blanco ‘Chachiensis.’ It could degrade PCR-C lipids. Its genome was sequenced as 8.70 Mbp with a guanine–cytosine content 66.23%. It was annotated to understand properties and possible contribution to aging of PCR-C.

Tables

TABLE 1. Information on sequencing data statistics

Illumina NovaSeq		Nanopore PromethION
Statistical item	Value	Statistical item	Value
Raw paired reads	4,839,150	Raw reads	279,342
Raw base (Mbp)	1,452	Raw base (Mbp)	1,556
Insert size (bp)	350	Clean reads	267,737
Clean reads length (bp)	150:150	Clean base (Mbp)	1,483
Clean data (Mb)	1,341	Mean read length (bp)	5,541
Scaffolds total depth	131	N50 read length (bp)	8,868

Full text

ANNOUNCEMENT

Pericarpium citri reticulatae (PCR) is the dried and aged pericarps of Citrus reticulata Blanco and its cultivars, while PCR-Chachiensis (PCR-C) is the most premium one (1). They have been used as medicine and food additive for hundreds of years (1), and we have revealed the central bacterial community and identified probiotic bacterial strains from PCR-Chachiensis (2, 3). Screening the lipid-degrading microbe in PCR-C is an interesting topic for further characterization. Burkholderia contaminans PCR-C_Y9 was isolated from PCR-C collected from an orchard in Xinhui District in Guangdong, China through screening on basic salt culture medium [(NH₄)₂SO₄ 2.38 g/L, KH₂PO₄ 1.36 g/L, CaCl₂·2H₂O 6.63 mg/L, MgSO₄·7H₂O 0.25 g/L, Na₂HPO₄ 1.42 g/L, FeSO₄·7H₂O 2.8 mg/L, 2.5 ‰ Triton X-100 (v/v), 1.5% agar (m/v)] supplemented with 1% lipid (m/v), which was extracted from PCR-C through Soxhlet methods with petroleum ether (4). Clear and transparent hydrolysis cycle around PCR-C_Y9 colony indicated degradation of PCR-C lipid. PCR-C_Y9 was identified as Burkholderia contaminans through 16S rRNA gene sequencing (5).

PCR-C_Y9 DNA was extracted from an overnight lysogeny broth shaking culture through DNA extraction following an initial washing step with STE buffer (0.25 M sucrose, 0.03 M Tris, 0.05 M EDTA) (6). Whole-genome sequencing was performed by Nanopore PromethION and Illumina NovaSeq. To construct 10 kb library for Nanopore, large fragments of DNA were recovered by using a BluePippin automatic nucleic acid fragment recovery system, and barcode was added by using an EXP-NBD104 Kit (Oxford Nanopore, UK). Afterwards, the SQK-LSK109 Kit was used to link the adapter. Library for Illumina NovaSeq was generated using NEBNext Ultra DNA Library Preparation Kit (NEB, USA) after sonification of DNA to a size of 350 bp. Raw nanopore data attained from Nanopore flow cell R10.4.1 was filtered with NanoPlot v1.29.1 (threshold Q > 7), and Illumina data were filtered with Trimmomatic v0.40 (Table 1). Default parameters were used for all software unless otherwise specified.

Unicycler v0.4.8 (7) was used to combine Illumina and Nanopore data for genome assembly. The assembled 8.70 Mbp PCR-C_Y9 genome comprised three circular chromosomes and two circular plasmids. The sizes for chromosomes 1, 2, and 3 and plasmids 1 and 2 were 1.53, 3.25, and 3.61 Mbp and 0.24 and 0.07 Mbp, respectively. Their guanine–cytosine content was 65.78, 66.71, and 66.42% and 61.99 and 58.96%, respectively. NCBI Prokaryotic Genome Annotation Pipeline v6.8 (8) was applied to annotate the genome as 7,723 protein-coding sequences, 18 rRNA, and 69 tRNA genes. SignalP v6.0 (9) and DeepTMHMM v1.0.42 was used to predict secretory protein. Among the 545 secretory proteins predicted, four phospholipase, two glycerophosphodiester phosphodiesterase, and two lipase were annotated. Neighboring to the secreted triacylglycerol lipase gene is the gene predicted to encode lipase secretion chaperone. Meanwhile, six secreted alpha/beta fold hydrolases might contribute to the lipid-degrading capacity of PCR-C_Y9 since they contain amino acid sequences conserved in esterase as the catalytic site in a three-dimensional structure predicted by AlphaFold 3 (10, 11). Availability of the Burkholderia contaminans PCR-C_Y9 genome sequence will allow further investigation into the lipid degrading properties of this bacterium and its contribution to the aging process of high-quality PCR-C.