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Sciencedirect.Com Sciencedirect [Downloaded free from http://www.ijmyco.org on Friday, February 23, 2018, IP: 194.80.229.244] International Journal of Mycobacteriology 4 (2015) 207– 216 HOSTED BY Available at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/IJMYCO The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and 5 Mycobacterium leprae Jody Phelan a,*, Arundhati Maitra b,1, Ruth McNerney a,1, Mridul Nair c, Antima Gupta b, Francesc Coll a, Arnab Pain c,2, Sanjib Bhakta b,2, Taane G. Clark a,d,2 a Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom b Mycobacteria Research Laboratory, Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, Malet Street, London WC1E 7HX, United Kingdom c Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia d Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom ARTICLE INFO ABSTRACT Article history: Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously Received 27 April 2015 been used in studies of anti-mycobacterial drugs due to its fast growth rate and low Accepted 3 May 2015 pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, Available online 4 June 2015 with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phyloge- netic analysis using whole genome alignments positioned M. aurum close to Mycobac- Keywords: terium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing Mycobacteria mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. M. aurum aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum M. tuberculosis orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis M. leprae were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyraz- Drug screening inamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two Genome homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work pro- vides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae. Ó 2015 Asian African Society for Mycobacteriology. Production and hosting by Elsevier Ltd. All rights reserved. * Corresponding author at: Pathogen Molecular Biology Department, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom. Tel.: +44 (0) 20 7636 8636. E-mail address: [email protected] (J. Phela). 1 Joint authors. 2 Joint authors. Peer review under responsibility of Asian African Society for Mycobacteriology. http://dx.doi.org/10.1016/j.ijmyco.2015.05.001 2212-5531/Ó 2015 Asian African Society for Mycobacteriology. Production and hosting by Elsevier Ltd. All rights reserved. [Downloaded free from http://www.ijmyco.org on Friday, February 23, 2018, IP: 194.80.229.244] 208 International Journal of Mycobacteriology 4 (2015) 207– 216 Introduction raw sequence data (size 0.55 Gb, 5.5 million paired reads, available from ENA ERP009288, minimum base call accuracy Mycobacterium aurum (M. aurum) is an acid-fast, gram-positive greater than 99%) underwent de novo assembly using SPAdes environmental bacteria typically found in damp conditions software [19]. The SSPACE software [20] was applied to scaf- [1,2]. It is a fast-growing mycobacterium with an in vitro dou- fold the assembly, and a combination of IMAGE [21] and bling time of 2–3 h that rarely causes infections in humans [2– GapFiller [22] routines were used to further close or reduce 6]. The M. aurum cell wall contains mycolic acids which are the length of remaining gaps. An alternative approach using analogous to those found in Mycobacterium tuberculosis [7], Velvet assembly software [23] led to a near identical assembly. and there are similarities between the antibiotic susceptibility Genomic annotation was transferred to the draft genome profiles of the two organisms [8,9]. The fast growth rate and using the Prokka pipeline [24]. The pipeline searches for genes low pathogenicity of M. aurum have encouraged its use as a present in contigs and compares them with protein and DNA surrogate for the highly pathogenic M. tuberculosis in studies databases to annotate them. The cd-hit software [25,26] was of anti-microbial activity of anti-tubercular drugs [6,10,11]. used to integrate the annotation from 8 mycobacterial species Unlike other fast-growing mycobacteria, such as to create a non-redundant blast ‘‘primary’’ database used by Mycobacterium smegmatis, M. aurum has the ability to survive the Prokka pipeline. To validate the draft assembly and anno- within macrophages [12,13] and has been used for high tation pipeline, the transferred annotation was compared throughput intracellular drug screening, allowing assessment against the kas operon sequence (GenBank: DQ268649.2). All of the ability of compounds to permeate the cell membrane 5 genes from the GenBank entry (fabD, acpM, kasA, kasB, and their stability within the cell [14,15]. The emergence of accD6) were annotated in the correct order and orientation strains of M. tuberculosis resistant to multiple first- and in the assembly. second-line drugs threatens efforts to control tuberculosis (TB) and has renewed interest in the search for new anti- tubercular agents [16]. Rapid-growing models for screening Comparative genomics putative anti-tubercular compounds are needed to accelerate drug discovery studies. Similarly, surrogate bacteria are Genomes from 27 species used in whole genome compar- needed to enable studies on drugs that may improve treat- isons were downloaded from ensembl (bacteria.ensembl.org), ment for infection with non-culturable Mycobacterium leprae. and the Uniprot taxon identification numbers are listed in Knowledge of the bacterial genome could enhance under- Table 1. Gene multiple alignments were constructed using standing of the molecular basis for drug resistance, and to clustalw2 [27] for 16S rRNA and MACSE software [28] for this end, the genome of M. aurum has been sequenced and rpoB sequences. Raxml software [29] was used to construct annotated. The genome was placed in a mycobacterium phy- the best scoring maximum likelihood tree, which was rooted logeny, and comparisons with M. tuberculosis, M. leprae and M. using the Corynebacterium glutamicum (strain: ATCC 13032) smegmatis genomes were made in relation to susceptibility reference sequence, an organism closely related to the towards anti-tubercular drugs. mycobacterium genus [30]. Pairwise gene alignments were constructed using MACSE software, which uses the trans- Materials and methods lated amino acid sequence and accounts for frame shifts and premature stop codons. Sequence identities were calcu- M. aurum sample and DNA extraction lated using the SIAS webserver. Gaps were not used in the calculation of the percent identity. Whole genome align- The M. aurum (NCTC 10437) was grown in 7H9 Middlebrook ments were constructed using mercator and mavid programs broth (Becton Dickinson, USA) supplemented with 10% albu [31], and the resulting homology map was inspected and min–dextrose–catalase (ADC) at 35 °C. DNA was extracted drawn using CIRCOS [32]. Orthologue clusters were created using the Bilthoven RFLP protocol [17]. In brief, log phase using OrthoMCL [33]. To identify any protein coding genes growth bacteria were treated with lysozyme, sodium dodecyl under selective pressure across M. aurum, M. tuberculosis, sulphate, proteinase K, N-cetyl-N,N,N-trimethyl ammonium Mycobacterium bovis – BCG, M. smegmatis, and M. leprae, the bromide (CTAB) and chloroform-isoamyl alcohol prior to pre- Ka/Ks ratio was calculated, where Ka is the number of non- cipitation with isopropanol. Minimum inhibitory concentra- synonymous substitutions per non-synonymous site, and tion (MIC) values for ethambutol, isoniazid, pyrazinamide Ks is the number of synonymous substitutions per synony- and rifampicin drugs for the same M. aurum strain are avail- mous site. Ratio values less than one imply stabilizing or able [18]. Duplications in M. aurum of embB and katG loci were purifying selection, whilst values greater than one imply confirmed by Sanger sequencing. For details of primers used, positive selection. To measure the degree of polymorphism p see Supplementary Table 1. across the genes, the nucleotide diversity ( ) was also calcu- lated using the same mycobacterial sample alignments. The DNA sequencing and genome assembly Ka/Ks and p metrics were calculated using variscan (http:// www.ub.edu/softevol/variscan) and PAML (http://abacus. The M. aurum genomic DNA was sequenced using a 101 bp gene.ucl.ac.uk/software/paml.html) software, respectively. paired-end library on the Illumina HiSeq2000 platform. The [Downloaded freefromhttp://www.ijmyco.orgonFriday,February23,2018,IP:194.80.229.244] Table 1 – Genomic characteristics of M. aurum in the context of related species. Organism Chromosome Uniprot Strain Assembled G + C content No. genes Relative in vitro ACDP risk classa accession number taxon id genome (bp) growth rate International Journal of Mycobacteriology M. leprae AL450380.1 272631 3268203 57.80 1605 Unculturable 3 C. glutamicum HE802067.1 1204414 3309401 53.81 3099 Rapid 1 M. bovis BX248333 233413 4345492 65.63 3952 Slow 3 M. tuberculosis AL123456.3 83332 4411532 65.61 4047 Slow 3 M. xenopi AJFI01000000 1150591 4434836 66.11 4281 Slow 2 M. canetti HE572590.1 1048245 4482059 65.62 3981 Slow 3 M. thermoresistibile AGVE01000000 1078020 4870742 69.02 4614 Rapid 1 M. hassiacum AMRA01000000 1122247 5000164 69.46 4959 Rapid 1 M. abscessus CU458896.1 36809 5067172 64.15 4942 Rapid 1 M.
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