Whole-Genome Sequences of Propionibacterium australiense NML (LCDC) 98A072T and NML (LCDC) 98A078, Associated with Granulomatous Bovine Lesions.

Draft genome sequences of Propionibacterium australiense isolates NML 98A072T and NML 98A078, derived from granulomatous lesions of infected bovines, were assembled and studied. Respectively, the genome sizes were 2.99 and 3.01 Mb, with G+C contents of 68.4% and 68.5%.

ANNOUNCEMENT

In 2000, Forbes-Faulkner et al. described a novel granulomatous infection in cattle that was associated with an unidentifiable Propionibacterium-like organism (1). Propionibacterium australiense sp. nov. was subsequently described as this novel bovine pathogen (2). Here, we have characterized draft genome sequences for two P. australiense strains, NML/LCDC 98A072T (= ATCC BAA-264T = CCUG 46075T) and NML/LCDC 98A078 (= ATCC BAA-263 = CCUG 46174). Identifiers using NML (National Microbiology Laboratory) or the older acronym LCDC (Laboratory Centre for Disease Control) are to be considered synonyms. Bacteria were subcultured after storage at −80°C in Microbank vials (Pro-Lab) from NML stocks and passed twice at 35°C on Brucella blood agar plates (BBA; Thermo Fisher) for 48 h under anaerobic conditions in a jar containing a GasPak (BD).

A loopful of plate culture was passed and grown anaerobically in prereduced peptone-yeast extract broth (Anaerobe Systems) for 18 h at 35°C. DNA was extracted using a DNA minikit (Qiagen), and paired-end whole-genome shotgun libraries were constructed using a Nextera XT library preparation kit. Samples were run separately for sequencing on the MiSeq 600-cycle kit (version 3) on a MiSeq sequencer (Illumina). Read quality was assessed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and assembled using default settings of SPAdes (version 3.9.0 [3]) after merging short paired-end reads with Fast-Length Adjustment of Short Reads (FLASH) with default settings (4).

The genomes were compared to each other using JSpeciesWS to calculate the average nucleotide identity values using BLAST+ (ANIb) (5). With that approach, 98A072T and 98A078 had ANIb scores more than 99.6% similar to each other but only 73.56% to 84.11% similar to genomes from Propionibacterium freudenreichii subsp. freudenreichii DSM 20271T (GenBank accession number CP010341) and Propionibacterium acidifaciens DSM 21887T (GenBank accession number AUFR00000000), species selected from the genus Propionibacterium after emendation in 2016 (6). The genome-to-genome distance calculator (7) was used to estimate in silico DNA-DNA hybridization values between strains. The two NML strains were found to have 97.6% similarity to each other using the recommended formula 2 for draft genomes, but values were low (∼20%) when compared to those of P. freudenreichii subsp. freudenreichii DSM 20271T.

The sequencing run for NML 98A072 produced 462,927 sequences of 35 to 301 bases in length, with a total of 136,192,279 bases. The draft genome of NML98A072T was comprised of 2,996,433 bp, which assembled into 63 contigs with 82× coverage, a G+C content of 68.4%, and an N50 contig length of 138,977. The genome, annotated by Prokka (version 1.13) (8), coded for 2,578 proteins, of which 85% were assigned to Clusters of Orthologous Groups (COG) categories using eggNOG-mapper (9). This genome encoded 3 rRNA genes, 51 tRNAs, and 9 repeat CRISPR elements. The sequencing run for NML 98A078 produced 408,707 sequencing reads (lengths of 35 to 301 bases), with a total of 113,773,758 bases. The draft genome of NML 98A078 consisted of 3,016,395 bp, assembled into 63 contigs with 64× coverage, a G+C content of 68.5%, and an N50 contig length of 129,689. eggNOG-mapper assigned 83.5% of the 2,644 coding regions to COG categories, and the draft genome encoded 3 rRNA genes, 51 tRNAs, and 4 CRISPR repeats. Neither genome harbored intact phages, as evaluated using PHASTER (10). The G+C contents of the draft genomes were consistent with members of the emended genus Propionibacterium (6, 11).

Data availability.

Raw reads for these projects were assigned accession numbers SRX4875582 and SRX4875583 in the NCBI Sequence Read Archive. Draft genome sequences of Propionibacterium australiense 98A072T and 98A078 were deposited in DDBJ/ENA/GenBank under the accession numbers RCIV00000000 and RCIW00000000, and the versions described in this paper are RCIV01000000 and RCIW01000000.