Complete genome of Priestia filamentosa H146 isolated from tobacco leaves

ABSTRACT We report the complete genome of Priestia filamentosa H146 isolated from tobacco leaves. H146 contained a circular chromosome and five circular plasmids. A total of 4,669 genes were predicted, of which 4,372 genes were in the chromosome and other genes were located on plasmids. The genome sequence data provide an important basis for studying Priestia filamentosa.

P riestia is a genus of mostly Gram-positive rod-shaped bacteria in the Bacillaceae family (1)(2)(3), which has shown significant beneficial roles in various plant species that are enacted via diverse mechanisms (2)(3)(4)(5).We isolated a new strain of Priestia filamentosa H146 from leaves of cigar variety Dexue 1 from Deyang in Sichuan province in China.A single leaf from a single plant was surface sterilized by 75% ethanol and divided into many 5 × 5 mm blocks; 5 g of blocks was added to a 45-mL-sterilized phosphate solution for washing at 180 r/min for 2 h.Then, the solution was diluted to 10 −3 and spread on the starch agar plate (peptone 10 g/L, beef extract 3 g/L, sodium chloride 5 g/L, soluble starch 3g/L, agar 20g/L, pH 7.0) (6, 7), which was incubated at 37°C for 36 h-48h in aerobic condition until the isolates formed colonies.A pure colony was obtained by restreaking three times, which was then stored in the laboratory.
H146 strain was cultured in NA medium (peptone 10 g/L, beef extract 3 g/L, sodium chloride 5 g/L, pH 7.0) for shaking at 37°C with 180 rpm for 24 h.Genomic DNA was extracted from the same colony for both PacBio and Illumina sequencing by using DNeasy UltraClean/NoviPure Microbial Kits (QIAGEN, Germany).Genomic DNA was sheared into selected fragment sizes ranging from 6 kb to 20 kb by using the Covaris g-TUBE device (Woburn, USA) and size-selected by blue pippin fragment (Sage Science, USA).Libraries for single-molecule real-time sequencing were constructed, with insert size of 10 kb using the SMRT bell TM Template kit (Pacific Biosciences, USA), then sequenced using PacBio Sequel II (Pacific Biosciences, USA) in Beijing Novogene Co., Ltd. in China. 1 µg of genomic DNA was used for the construction of sequencing libraries with NEBNext Ultra DNA Library Prep Kit (NEB, USA) and sequenced by NovaSeq PE150.PacBio reads were used for assembly, and Illumina reads were only used for correction.
The Illumina read quality was controlled by Trimmomatic (version: 0.39) (8), and the reads with adapter sequences (overlap ≥15 bp), low-quality (quality value ≤38, overlap ≥40%), and a higher rate of N (>10%) were abandoned.All reads of Pacbio sequencing were used for assembly; de novo genome assembly was done by Canu software (version ： 2.0) with setting -d run1 -p H146 genome size = 4.5 m pacbio subreads.fastq.gz,which was followed by an error correction with default parameters by three rounds of Racon (version 1.4.13) with Pacbio sequencing reads and three rounds of Pilon version 1.22 with Illumina sequencing reads (9)(10)(11).Unless otherwise noted, default parameters were used for all software.If the 3′ end and 5′ end of assembled chromosome have overlapped with more than 2 k, it is considered to be looped; and the overlapping part will be removed.Resulting circular replicon sequences of chromosomes by Canu were rotated to dnaA gene.

ACKNOWLEDGMENTS
We are grateful for the funding from Anhui Wannan Tobacco Co., Ltd.(grant no.20210563004).

TABLE 1
Genome features of P. filamentosa H146 strain