|Year : 2015 | Volume
| Issue : 3 | Page : 207-216
The draft genome of Mycobacterium aurum , a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae
Jody Phelan1, Arundhati Maitra2, Ruth McNerney1, Mridul Nair3, Antima Gupta2, Francesc Coll1, Arnab Pain3, Sanjib Bhakta2, Taane G Clark4
1 Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom
2 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
3 Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia
4 Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine; Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom
|Date of Web Publication||23-Feb-2017|
Pathogen Molecular Biology Department, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT
Source of Support: None, Conflict of Interest: None
Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two emb B homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum , this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.
Keywords: Mycobacteria, M. aurum, M. tuberculosis, M. leprae, Drug screening, Genome
|How to cite this article:|
Phelan J, Maitra A, McNerney R, Nair M, Gupta A, Coll F, Pain A, Bhakta S, Clark TG. The draft genome of Mycobacterium aurum , a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae. Int J Mycobacteriol 2015;4:207-16
|How to cite this URL:|
Phelan J, Maitra A, McNerney R, Nair M, Gupta A, Coll F, Pain A, Bhakta S, Clark TG. The draft genome of Mycobacterium aurum , a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae. Int J Mycobacteriol [serial online] 2015 [cited 2019 Sep 18];4:207-16. Available from: http://www.ijmyco.org/text.asp?2015/4/3/207/200824
| Introduction|| |
Mycobacterium aurum (M. aurum) is an acid-fast, gram-positive environmental bacteria typically found in damp conditions ,. It is a fast-growing mycobacterium with an in vitro doubling time of 2–3 h that rarely causes infections in humans ,,,,. The M. aurum cell wall contains mycolic acids which are analogous to those found in Mycobacterium tuberculosis  , and there are similarities between the antibiotic susceptibility profiles of the two organisms ,. The fast growth rate and low pathogenicity of M. aurum have encouraged its use as a surrogate for the highly pathogenic M. tuberculosis in studies of anti-microbial activity of anti-tubercular drugs ,,. Unlike other fast-growing mycobacteria, such as Mycobacterium smegmatis, M. aurum has the ability to survive within macrophages , and has been used for high throughput intracellular drug screening, allowing assessment of the ability of compounds to permeate the cell membrane and their stability within the cell ,. The emergence of strains of M. tuberculosis resistant to multiple first- and second-line drugs threatens efforts to control tuberculosis (TB) and has renewed interest in the search for new anti-tubercular agents . Rapid-growing models for screening putative anti-tubercular compounds are needed to accelerate drug discovery studies. Similarly, surrogate bacteria are needed to enable studies on drugs that may improve treatment for infection with non-culturable Mycobacterium leprae. Knowledge of the bacterial genome could enhance understanding of the molecular basis for drug resistance, and to this end, the genome of M. aurum has been sequenced and annotated. The genome was placed in a mycobacterium phylogeny, and comparisons with M. tuberculosis, M. leprae and M. smegmatis genomes were made in relation to susceptibility towards anti-tubercular drugs.
| Materials and methods|| |
M. aurum sample and DNA extraction
The M. aurum (NCTC 10437) was grown in 7H9 Middlebrook broth (Becton Dickinson, USA) supplemented with 10% albumin–dextrose–catalase (ADC) at 35 °C. DNA was extracted using the Bilthoven RFLP protocol . In brief, log phase growth bacteria were treated with lysozyme, sodium dodecyl sulphate, proteinase K, N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB) and chloroform-isoamyl alcohol prior to precipitation with isopropanol. Minimum inhibitory concentration (MIC) values for ethambutol, isoniazid, pyrazinamide and rifampicin drugs for the same M. aurum strain are available . Duplications in M. aurum of embB and katG loci were confirmed by Sanger sequencing. For details of primers used, see Supplementary [Table 1].
|Table 1: Genomic characteristics of M. aurum in the context of related species.|
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DNA sequencing and genome assembly
The M. aurum genomic DNA was sequenced using a 101 bp paired-end library on the Illumina HiSeq2000 platform. The raw sequence data (size 0.55 Gb, ˜5.5 million paired reads, available from ENA ERP009288, minimum base call accuracy greater than 99%) underwent de novo assembly using SPAdes software . The SSPACE software  was applied to scaffold the assembly, and a combination of IMAGE  and GapFiller  routines were used to further close or reduce the length of remaining gaps. An alternative approach using Velvet assembly software  led to a near identical assembly. Genomic annotation was transferred to the draft genome using the Prokka pipeline . The pipeline searches for genes present in contigs and compares them with protein and DNA databases to annotate them. The cd-hit software , was used to integrate the annotation from 8 mycobacterial species to create a non-redundant blast “primary” database used by the Prokka pipeline. To validate the draft assembly and annotation pipeline, the transferred annotation was compared against the kas operon sequence (GenBank: DQ268649.2). All 5 genes from the GenBank entry (fabD, acpM, kasA, kasB, accD6) were annotated in the correct order and orientation in the assembly.
Genomes from 27 species used in whole genome comparisons were downloaded from ensembl (bacteria.ensembl.org), and the Uniprot taxon identification numbers are listed in [Table 1]. Gene multiple alignments were constructed using clustalw2  for 16S rRNA and MACSE software  for rpoB sequences. Raxml software  was used to construct the best scoring maximum likelihood tree, which was rooted using the Corynebacterium glutamicum (strain: ATCC 13032) reference sequence, an organism closely related to the mycobacterium genus . Pairwise gene alignments were constructed using MACSE software, which uses the translated amino acid sequence and accounts for frame shifts and premature stop codons. Sequence identities were calculated using the SIAS webserver. Gaps were not used in the calculation of the percent identity. Whole genome alignments were constructed using mercator and mavid programs  , and the resulting homology map was inspected and drawn using CIRCOS . Orthologue clusters were created using OrthoMCL . To identify any protein coding genes under selective pressure across M. aurum , M. tuberculosis, Mycobacterium bovis – BCG, M. smegmatis, and M. leprae , the Ka/Ks ratio was calculated, where Ka is the number of non-synonymous substitutions per non-synonymous site, and Ks is the number of synonymous substitutions per synonymous site. Ratio values less than one imply stabilizing or purifying selection, whilst values greater than one imply positive selection. To measure the degree of polymorphism across the genes, the nucleotide diversity (π) was also calculated using the same mycobacterial sample alignments. The Ka/Ks and π metrics were calculated using variscan (http://www.ub.edu/softevol/variscan) and PAML (http://abacus.gene.ucl.ac.uk/software/paml.html) software, respectively.
| Results|| |
The M. aurum genome
A total of ˜5.5 million high quality paired end (101 bp) reads were used to assemble the M. aurum genome. The final M. aurum assembly consisted of 46 contigs, 43 of which were over 500 bp in length. The median contig length (N50) was 265 Kb (minimum 315 bp, maximum 742,983 bp). The total genome length (˜6.02 Mb, G + C content 67.52%) is longer than M. tuberculosis (4.4 Mbp) and Mycobacterium canetti (4.5 Mbp), but shorter than Mycobacterium marinum (6.6 Mbp) and M. smegmatis (7.0 Mbp) ([Table 1]). A total of 5684 coding sequences, 1 tmRNA, 4 rRNA and 51 tRNA features were annotated, and of these 4306 (75%) were assigned a function ([Figure 1]). The final contigs and annotation are available for download (pathogenseq.lshtm.ac.uk/m_aurum).
|Figure 1: An annotated circular view of the M. aurum genome (length ˜6.02 Mb). Innermost track: G + C% content; middle track: the 46 contigs, alternating between brown and orange with green and grey lines representing tRNA and rRNA, respectively; outer track: the 5684 forward and reverse genes.|
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M. aurum and the mycobacteria phylogeny
A phylogenetic analysis using 27 mycobacterial whole genome sequences revealed that M. aurum clustered with Mycobacterium vaccae and Mycobacterium vanbaalenii within a clade related to fast-growing mycobacteria ([Figure 2]). Slow-growing bacteria, including M. tuberculosis , clustered within a distinct clade. However, Mycobacterium indicus pranii , a fast-growing mycobacterium and immunotherapy and vaccine candidate for leprosy and tuberculosis  , clustered within the slow-growing clade. The very high bootstrap support values for the phylogenetic tree (median 100%, range 77–100%) indicates the high precision afforded when using whole genome data. Previously, hsp65, sodA, recA, rpoB and 16S rRNA gene sequence data were used to barcode bacteria, with the latter approach being adopted widely . The assembled 16S rRNA sequence for M. aurum had the highest identity with M. vanbaalenii (99%), Mycobacterium rhodesiae (99%), and Mycobacterium austroafricanum (99%), in concordance with previous reports ,. The phylogenetic tree constructed using 16S rRNA sequences was broadly similar to that from whole genome data (Supplementary [Figure 1]). However, M. aurum and M. vanbaalenii clustered closer to Mycobacterium abscessus rather than Mycobacterium gilvum , and the topology was less robust with lower bootstrap support values.
|Figure 2: M. aurum and the mycobacterium phylogeny* constructed using 27 whole genome reference sequences. *Constructed using RaXML and statistic support for lineages was based on 100 bootstrap samples. 27 reference sequences used are described in [Table 1].|
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Comparison to the M. tuberculosis and M. leprae genomes
The M. aurum assembled contigs were ordered according to the M. tuberculosis H37Rv reference genome (AL123456.3), leading to 10 gapped scaffolds. Most of the M. tuberculosis genome (86%) had regions with synteny in M. aurum. The map of homology between the 10 M. aurum scaffolds and the M. tuberculosis genome consisted of 67 regions of synteny (Supplementary [Table 2] and [Figure 3]a). Although there was high similarity between M. aurum and M. tuberculosis , there was evidence for large-scale rearrangements ([Figure 3] a). Twenty-eight genes required for survival within macrophages were observed, but a further two (lpqY and eccA 1) could not be found  (Supplementary [Table 3]). The putative proteome for M. aurum suggests it lacks 1002 proteins present in M. tuberculosis , but has an additional 2090 proteins not seen in M. tuberculosis (see [Table 2]).
|Table 2: Drug minimum inhibitory concentrations (MICs) and candidate resistance gene identity between M. aurum and M. tuberculosis at drug resistance loci.|
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|Table 3: A comparison across M. aurum, M. tuberculosis, M. bovis – BCG, M. smegmatis, and M. leprae alignments at drug targets or other important loci.|
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|Figure 3: Homology between M. aurum and M. tuberculosis and M. leprae. (a) M. aurum (green) and M. tuberculosis H37Rv (blue). The ten contigs provide 67 segments of synteny with M. tuberculosis H37Rv. The segments range from 2,266 bp to 391,674 bp in length. (b) M. aurum (green) and M. leprae (blue). The ten contigs provide 73 segments of synteny with M. leprae. The segments range from 2,495 bp to 193,922 bp in length.|
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The map of homology between the 10 M. aurum scaffolds and the M. leprae genome consisted of 73 segments of synteny (Supplementary [Table 2] and [Figure 3] b). For M. aurum and M. leprae there were 2047 and 222 unique proteins, respectively, which had no orthologue in the other mycobacteria (see Supplementary [Table 2]). M. smegmatis is often used as a fast-growing model of M. tuberculosis. A similar analysis carried out between M. tuberculosis and M. smegmatis revealed 979 and 2314 unique proteins for each, respectively, which had no orthologue in the other mycobacteria. When compared with the M. aurum – M. tuberculosis analysis, the number of apparently unique proteins in M. smegmatis was higher by 224 proteins.
Drug resistance candidate genes
Pairwise alignments were constructed for the known drug target genes to establish the degree of homology between M. aurum and M. tuberculosis ([Table 2]). The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). The percentage of amino acid identity was higher than the sequence identity, being high among all drug resistance candidate genes analysed (range 90.6–99.2%). Interestingly, two genes at different locations were annotated as katG in the M. aurum genome, and denoted as katG 1 and katG 2. The percent identity between the two genes and their M. tuberculosis homologue at the DNA level are 73.6% and 68.8% (Supplementary [Figure 2]) The putative M. aurum katG 1 found in contig 20 (aurum 03417) demonstrated the highest homology to the M. tuberculosis katG gene (Rv1908c) and M. smegmatis MSMEG_ 6384. The second M. aurum katG 2 (aurum 02416) located in contig 2 (katG 2) was most homologous with M. smegmatis MSMEG_ 3461. A third M. smegmatis gene, MSMEG_3729 , showed weak homology to each of the katG genes in M. aurum and M. tuberculosis. Two copies of embB , a gene associated with ethambutol in M. tuberculosis , were also found in different locations in M. aurum (72.3% and 47.7% identity). The semi-identical duplications for each of katG and embB were confirmed by PCR and Sanger sequencing (Supplementary Table 4).
Across a range of therapeutic agents, potential differences in minimum inhibitory concentration (MIC) levels between M. tuberculosis (H37Rv) and M. aurum for isoniazid, ethambutol and ofloxacin ([Table 2]) are available , , with the biggest difference for isoniazid. The MIC values for isoniazid were greatest in M. smegmatis (2 mg/L), followed by M. aurum (0.4) and M. tuberculosis (0.02–0.2). No known M. tuberculosis mutations were identified in the katG, inhA (isoniazid), ethA, ethR (ethambutol), and gyrA/B (ofloxacin) orthologues in M. aurum. Homologues of ahpC and embR genes, associated with isoniazid and ethambutol drug resistance respectively, were not observed in the M. aurum genome.
The alignments were compared across M. aurum , M. tuberculosis, M. bovis – BCG, M. smegmatis, and M. leprae at the loci considered drug targets or those loci considered to have important functional roles ([Table 3]). All loci had a high percentage (˜90%) of their nucleotides analyzable across the mycobacteria, except fas and gyrA where there were large insertions in M. aurum and M. leprae , respectively. Only three loci did not have alignment gaps: inhA (isoniazid drug-related); rpsL (streptomycin); and kasA (thiolactomycin). The ddn (delamanid) , fpol1 (para-aminosalicylic acid) , murC/D/E/F family (isoquinolines), and nat (cholesterol metabolism) loci were the most polymorphic (>40% sites segregating, nucleotide diversity π > 0.2). In contrast, the rrs gene associated with streptomycin drug resistance was the most conserved (2.9% segregating sites, pairwise diversity π = 0.029). In general, there was a modest degree of conservation in most genes (all with >50% of sequence conserved), which would be expected given the known synergistic drug effects across mycobacteria. All candidate genes reported Ka/Ks values much lower than 1, consistent with the selective removal of alleles that are deleterious (purifying selection). The highest Ka/Ks value was observed for nat (Rv3566c) , a gene encoding arylamine acetylase that is associated with resistance to isoniazid .
| Discussion|| |
The draft genome sequence of M. aurum (length ˜6.02 Mb, G + C content 67.52%) has been assembled. The genome assembly consists of 46 contigs and provides the first insight into the genetic code of M. aurum. Lack of alternative sequence data for this bacterium, particularly from technologies with longer reads, prevents closure of the gaps at this time. Using whole genome alignments, the placement of M. aurum within the mycobacterial phylogeny, close to M. vaccae and M. vanbaalenii , was confirmed. The analysis of loci involved in drug resistance demonstrated homology with M. tuberculosis and M. leprae. This insight corroborates earlier investigations of inhA gene mutants of M. aurum that showed similarity in drug resistance mechanisms against isoniazid and ethionamide between M. aurum and M. tuberculosis ,. The draft M. aurum genome is larger than that of M. tuberculosis with an additional 2090 genes not observed in M. tuberculosis ; it is also lacking 1002 of the genes found in M. tuberculosis. Multiple copies of some homologous genes were observed. Of particular interest are two putative copies of embB , a gene involved in the biosynthesis of the mycobacterial cell wall component arabinan and that is associated with resistance to ethambutol in M. tuberculosis. Similarly, two annotated catalase-peroxidase (katG) genes that may be involved in the activation of the anti-tuberculosis pro-drug isoniazid were identified and confirmed. Multiple katG genes have been reported in other mycobacteria, for example in Mycobacterium fortuitum . It could be hypothesized that the duplications of katG in M. aurum and M. smegmatis could have an effect on the MIC values. Further laboratory work is underway to elucidate the endogenous function of the observed duplications.
In summary, these genomic analyses support the use of M. aurum as a potential model organism for providing insights into M. tuberculosis biology, particularly for new drug development, with the possibility of leading to new control measures for tuberculosis disease. Further insight may be gained from the genome sequence of additional strains and related mycobacteria.
| Conflicts of interest|| |
The authors declare no conflict of interests.
| Acknowledgements|| |
T.G.C. is funded by the Medical Research Council UK (Grant No. MR/K000551/1). J.P. is supported by a BBSRC UK Ph.D. studentship. The project was supported by the KAUST faculty baseline research fund to A.P.
| Appendix A. Supplementary data|| |
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmyco.2015.05.001.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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