Draft Genome Sequence of a Rare Pigmented Mycobacterium avium subsp. paratuberculosis Type C Strain

ABSTRACT Mycobacterium avium subsp. paratuberculosis is the causative agent of paratuberculosis. We report here the draft genome sequence of a rare pigmented M. avium subsp. paratuberculosis type C strain, comprising 58 contigs and having a genome size of 4,851,414 bp. The genome will assist in the execution of pigmentation and virulence studies on this mycobacterium.

M ycobacterium avium subsp. paratuberculosis infects a wide range of animals, including humans, and is the causative agent of paratuberculosis, which affects ruminants, camelids, rabbits, and hares (1)(2)(3). M. avium subsp. paratuberculosis strains are classified into two major groups: types C and S. Type C strains are isolated from several hosts and are the predominant strain type infecting cattle, while type S strains have a host species preference for sheep and goats (4)(5)(6). The two types can be distinguished by their growth characteristics in culture medium (growth of type S strains is considerably slower) and genotyping assays. While yellow pigmentation in type S strains has been observed, to date only a single report of a putative pigmented type C strain has been published (7), but the strain is unavailable. Recently, a yellow pigmented strain was isolated from a goat fecal sample from Azores, Portugal. This strain was confirmed to be type C, based on its growth characteristics and single nucleotide polymorphism (SNP) analysis (8) results. Here, we announce the complete genome sequence of this rare pigmented type C Portuguese strain.
DNA was extracted from a single colony using a QIAamp DNA minikit (Qiagen). High-throughput sequence data were generated with Ion Torrent PGM (Thermo Fisher Scientific, Inc., USA), using a 400-bp sequencing kit and a 316 V1 chip. A total of 3,009,190 reads were produced, with a mean length of 299 bp. The genome assembly was created with MIRA (9) using the raw reads with preprocessing modules included in the software. The assembly contains 58 contigs, which encompass 4,851,414 bp. The largest contig obtained was 521,843 bp long and the N 50 value was 188,875 bp, while the GC content was 69.3%.
Structural genome annotation was performed with Prokka (10), through Prodigal (11), yielding 4,666 protein-coding genes, 3 rRNAs, 58 tRNAs, and 1 transfer-messenger RNA. Functional annotation was executed with HMMER (12) against the eggNOG version 4.5 (13) database. The search space was reduced by selecting only hidden Markov models from actinobacteria. A total of 4,178 HMMER hits (90.8%) were detected, and 3,425 unique nonsupervised orthologous groups were found. The most common orthologs found in the genome were as follows: members of the PPE family, specific to mycobacteria and possibly playing a role in infection and virulence (14); S-adenosyl-L-methioninedependent methyltransferase activity; cytochrome P450; acyl-CoA synthetase; synthase; transport proteins; and virulence factor mammalian cell entry family proteins.
A relatedness analysis was performed using 10,515 identified variants. The pigmented strain and 47 other M. avium subsp. paratuberculosis (15) strains were compared by mapping the reads to the K10 M. avium subsp. paratuberculosis reference genome (14) using BWA-MEM (16) and calling variation with GATK Haplotype Caller (17). This resulted in a dendrogram, built with SNPRelate (18), with three clearly differentiated groups for the type C, type S pigmented, and type S nonpigmented strains. The pigmented M. avium subsp. paratuberculosis isolate was unequivocally placed among the type C clade, confirming it to be a type C M. avium subsp. paratuberculosis strain. The first genome sequence of a pigmented type C M. avium subsp. paratuberculosis strain will assist future research focusing on pigmentation and virulence mechanisms of this bacterium.
Accession number(s). This whole-genome shotgun project has been deposited in GenBank under accession number MWPB00000000.

ACKNOWLEDGMENTS
Financial support for P.B., A.U., and A.M.R. was provided by Investigador FCT (Fundação para a Ciência e a Tecnologia) project IF/01015/2013/CP1209/CT0001, "Genetic characterization of national animal and plant resources using next-generation sequencing." Funding was also provided by FCT projects PTDC/CVT/111634/2009, "Novas abordagens moleculares para a detecção e discriminação de membros do Complexo Mycobacterium tuberculosis associados a tuberculose animal e avaliação do potencial zoonótico destas espécies em Portugal," and UID/AGR/00115/2013. K.S. was funded by the Rural and Environmental Science and Analytical Services Division of the Scottish Government.