A complete genome sequence of Leifsonia sp. McL0608, a soil bacterium from Pinus radiata forest

TL;DR

This paper presents the full genome sequence of a soil bacterium from a New Zealand pine forest, revealing its potential to produce alkanes, terpenes, and auxins.

Contribution

The study provides the first complete genome sequence of Leifsonia sp. McL0608, including biosynthetic pathway predictions.

Findings

The genome is 4.2 Mb in size and contains 3,942 coding sequences.

Biosynthetic pathways for alkanes, terpenes, and auxins were identified.

The bacterium was isolated from Pinus radiata forest soil in New Zealand.

Abstract

Leifsonia sp. McL0608, isolated from Pinus radiata forest soil in New Zealand, has a complete 4.2 Mb circular genome assembled from Nanopore reads. Genome annotations identified 3,942 coding sequences and predicted biosynthetic pathways for alkanes, terpenes, and auxins.

Full text

ANNOUNCEMENT

Pinus radiata D Don (Monterey pine) is one of the most widely planted tree species globally (1). Soils beneath P. radiata plantations support essential ecosystem services, such as nutrient cycling, carbon storage, and biodiversity (2). Actinobacteria comprise ~20% of the bacterial community associated with P. radiata roots and soils (3). Despite being widespread, most Actinobacteria are poorly described in terms of function and ecological roles (4). Incorporating annotated genomes of cultured isolates into reference databases is essential for improving functional interpretations of sequencing data.

Leifsonia sp. McL0608 was isolated from topsoil collected at 0–10 cm depth in a second rotation P. radiata plantation in New Zealand (43.4656 S 172.3992 E) in autumn 2022. The soil is classified as fluvial recent soil from alluvial floodplains; it is stony, well-draining, and nutrient poor (5). A 1:100 soil:water mix was shaken for 4 h then serially diluted to 10^−6^ g soil mL^−1^. Aliquots of 100 µL were spread on Reasoner’s 2A agar and incubated at room temperature under dark conditions until a single colony formed. After 1 week, a circular creamy colony was putatively identified via full-length 16S rRNA gene sequencing using 785F/907R primers (Macrogen; South Korea), showing similarity to Diaminobutyricibacter tongyongensis (GenBank accession NR_134019.1) after performing a BLASTn version 2.16.0 + search against the NCBI RefSeq 16S rRNA database, release number 229 (6).

DNA was extracted using the Wizard Genomic DNA Kit (Promega, USA) following the manufacturer’s gram-positive bacteria protocol. Genomic DNA was sequenced using long-read Oxford Nanopore Technology (ONT; UK) using a FLO-MIN114 R10.4.1 flow cell on a PromethION platform. DNA was prepared for sequencing using the Rapid Barcoding Kit V14 (SQK-RBK114.96) following the ONT DNA V14 protocol; no shearing or size selection was performed. “Super-accurate” base calling was performed using Guppy version 6.3.9 (7).

Sequencing ran for 68 h and generated 28,663 reads (average length 3,363 bp; max length 77,981 bp; N_50_ 8,698 bp). Quality control and trimming were performed using chopper version 0.8.0 (8) with a Q-score of 10; 57% of reads passed quality filtering. The genome was assembled with raven-assembler version 1.8.3 (9) and polished with medaka version 1.12.1 (10).

The genome consists of a single contig, with an N_50_ and a total size of 4,196,112 bp, GC content of 66.74%, and 21× coverage/depth. The circular nature of the genome was confirmed by Bandage version 0.8.1 (11), which showed a closed circular contig without dead ends or breaks. The genome was not rotated.

Genome annotation was performed using NCBI Prokaryotic Genome Annotation Pipeline version 6.7 (12) resulting in 3,942 CDSs (protein-coding sequence), 3 rRNA, 46 tRNA, and 1 tmRNA. Secondary metabolism was predicted using antiSMASH version 8.0 (13) and RAST (14), which includes genes for non-alpha poly-amino acid, ribosomally synthesized and post-translationally modified peptide products, type III polyketide synthase, terpene synthesis, auxin synthesis, and alkane biosynthesis.

Taxonomic placement of McL0608 was initially assigned to the genus Diaminobutyricibacter based on genome phylogeny using Genome Taxonomy Database version 10-RS226 (15). However, this genus has since been reclassified as a later heterotypic synonym of Leifsonia (16).