Complete genome and transcriptome of Mycobacterium bovis 3488, a clinical isolate with a novel deletion at the RD1 locus

TL;DR

This paper reports the genome and transcriptome of a Mycobacterium bovis strain with a unique deletion in the RD1 locus, which is important for virulence.

Contribution

The study identifies a novel 14.2 kb deletion in the RD1 locus of a clinical Mycobacterium bovis isolate.

Findings

The isolate Mycobacterium bovis 3488 has a deletion encompassing the RD1 locus.

Genome and transcriptome data were generated for this clinical isolate.

Abstract

In members of the Mycobacterium tuberculosis complex, the RD1 locus encodes a type VII secretion system involved in virulence. Herein, we describe the genome and transcriptomic analysis of Mycobacterium bovis 3488, a clinical isolate from a cat, with a 14.2 kb deletion that encompasses the RD1 locus.

Figures

Deletions at RD1 locus across different members of MTBC. RD13488: deletion of espM′-espI′ from M. bovis3488; RD1mic: deletion from Mycobacterium microti; RD1das: deletion from “dassie bacillus”; RD1BCG: deletion of RD1 locus from M. bovis BCG; and RD1mon: deletion from Mycobacterium mungi (created with BioRender.com).

Tables

TABLE 1. Genome characteristics

Mycobacterium bovis 3488
Genome size (bp)	4,337,509
No. Illumina reads	3,915,478
No. Nanopore reads	87,050
Nanopore read N50	3,228
GC%	65.64
Coverage	41×
Contigs	1
Coding sequences	4,031
Pseudogenes	239
rRNA	3
tRNA	52
CRISPR 1	18
CRISPR 2	25

Full text

ANNOUNCEMENT

Mycobacterium bovis causes tuberculosis (TB) in animals and humans (1). The TB vaccine Mycobacterium bovis BCG (BCG) was derived by in vitro passage of M. bovis (2); deletion of the RD1 locus is a key reason for the attenuation of BCG (3, 4). All clinical M. bovis isolates described to date contain RD1 (5–7). Herein, we describe M. bovis 3488, isolated from a cat that presented to the UCD Veterinary Hospital (8), that harbors a deletion overlapping the BCG RD1 locus—a feature that complicated its original characterization (8).

Mycobacterium bovis 3488 was cultured at 37°C in Middlebrook 7H9-ADC medium with 40 mM sodium pyruvate and 0.05% Tween (9, 10). DNA extraction was performed as previously described (10) with mechanical cell lysis (Roche MagNA Lyser) followed by AMPure XP bead cleanup (Beckman Coulter). For Illumina sequencing, libraries were prepared with Nextera XT Mid output kits and on-bead tagmentation (Illumina) and sequenced on a NextSeq 500 (Illumina) to give 150 bp paired-end reads (125× depth). For Nanopore sequencing, DNA was blunt-end repaired, dA tailed, and adapter ligated using the NEBNext Ultra II DNA kit (New England Biolabs). AMPure XP beads (Beckman Coulter) were used for cleanup and size selection. Libraries were Flongle (ONT) sequenced (R9.4.1) for 24 hours; fastqs were basecalled with Guppy version 6.1.7 using SUP settings (reads 46× depth).

Short read quality was assessed with FastQC version 0.12.0 (11), filtered, and trimmed with BBduk from BBtools version 39 (12). Long reads were filtered and trimmed with Filtlong version 0.2.1 (13) and Porechop version 0.2.4 (14). The depth of sequencing was assessed with BWA (15), Minimap2 (16), and SAMtools (17). Long reads were assembled with Flye version 2.9.2 (18), corrected with Medaka version 1.7.3 (19), and polished with short reads using Polypolish version 0.5.0 (20), POLCA (from MaSuRCA version 4.1.0) (21), and Pilon version 1.24 (22). Contigs were joined and rotated to dnaA using Circlator version 1.5.5 (23). The draft sequence was annotated using Prokka version 1.14.5 (24). Assembly quality was assessed using QUAST version 4.0 (25) and with alignments to M. bovis AF2122/97 in IGV version 2.16.2 (26). Default parameters were used except where noted (https://github.com/jordysmith/Mbov-omics-annot).

The complete genome sequence (Table 1) revealed a deletion of 14,277 bp relative to M. bovis AF2122/97 (27), overlapping the BCG RD1 locus, that we designate “RD1^3488^” (Fig. 1).

Deletions at RD1 locus across different members of MTBC. RD13488: deletion of espM′-espI′ from M. bovis3488; RD1mic: deletion from Mycobacterium microti; RD1das: deletion from “dassie bacillus”; RD1BCG: deletion of RD1 locus from M. bovis BCG; and RD1mon: deletion from Mycobacterium mungi (created with BioRender.com).

RNA was sequenced commercially (Novogene). Sequence reads were aligned to the M. bovis AF2122/97 reference genome (27) with BWA-MEM (15); BAM and GTF were inputted to FeatureCounts (28) to generate gene count data. DESeq2 (29) was used for differential expression analysis. A |log_2_| fold change ≥ 2 and Benjamini–Hochberg adjusted P value ≤ 0.05 revealed 44 differentially expressed genes between M. bovis 3488 and M. bovis AF2122/97. Genes upregulated in M. bovis 3488 included the regulators whiB6 and espM and genes flanking RD1^3488^.

Beyond M. bovis BCG, deletions at the RD1 locus have been described in Mycobacterium microti (30), Mycobacterium mungi (31), Mycobacterium suricattae (32), and the dassie bacillus (33) (Fig. 1). Mycobacterium bovis 3488 is the first M. bovis clinical isolate described with a deletion encompassing the RD1 locus.