Journal List > J Vet Sci > v.18(S1) > 1148330

TOOLS
Kim, Jang, Kim, Ryoo, Kwon, Kim, Kang, Byeon, Lee, Lim, and Kim: Molecular and genomic features of Mycobacterium bovis strain 1595 isolated from Korean cattle
Original Article

J Vet Sci 2017;18(S1):333-341.

Published online: 4 December 2017

DOI: https://doi.org/10.4142/jvs.2017.18.S1.333

Molecular and genomic features of Mycobacterium bovis strain 1595 isolated from Korean cattle

Narae Kim1, 2, Yunho Jang1, Jin Kyoung Kim1, Soyoon Ryoo1, Ka Hee Kwon1, Miso Kim1, Shin Seok Kang4, Hyeon Seop Byeon4, Hee Soo Lee1, Young-Hee Lim3, Jae-Myung Kim1, *

1Bacterial Disease Division, Animal and Plant Quarantine Agency, Anyang 14089, Korea

2Department of Integrated Biomedical and Life Science, College of Health Science, Korea University, Seoul 02841, Korea

3Department of Public Health Science (Brain Korea 21 PLUS Program), Graduate School, Korea University, Seoul 02841, Korea

4Chungcheongbukdo Veterinary Service, Chungju 27492, Korea

*Corresponding authors: Tel: +82-31-467-1823; Fax: +82-31-467-1778; E-mails: kimjm88@korea.kr (JM Kim), yhlim@korea.ac.kr (YH Lim)

Received 11 July 2016       Revised 11 October 2016       Accepted 2 January 2017

Copyright © 2017 The Korean Society of Veterinary Science

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The aim of this study was to investigate the molecular characteristics and to conduct a comparative genomic analysis of Mycobacterium (M.) bovis strain 1595 isolated from a native Korean cow. Molecular typing showed that M. bovis 1595 has spoligotype SB0140 with mycobacterial interspersed repetitive units– variable number of tandem repeats typing of 4-2-5-3-2-7-5-5-4-3-4-3-4-3, representing the most common type of M. bovis in Korea. The complete genome sequence of strain 1595 was determined by single-molecule real-time technology, which showed a genome of 4351712 bp in size with a 65.64% G + C content and 4358 protein-coding genes. Comparative genomic analysis with the genomes of Mycobacterium tuberculosis complex strains revealed that all genomes are similar in size and G + C content. Phylogenetic analysis revealed all strains were within a 0.1% average nucleotide identity value, and MUMmer analysis illustrated that all genomes showed positive collinearity with strain 1595. A sequence comparison based on BLASTP analysis showed that M. bovis AF2122/97 was the strain with the greatest number of completely matched proteins to M. bovis 1595. This genome sequence analysis will serve as a valuable reference for improving understanding of the virulence and epidemiologic traits among M. bovis isolates in Korea.

Keywords: Korea, Mycobacterium bovis, cattle, genomics

References

1. Achtman M. Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. Annu Rev Microbiol. 2008; 62:53–70.
crossref
2. Allix C, Walravens K, Saegerman C, Godfroid J, Supply P, Fauville-Dufaux M. Evaluation of the epidemiological relevance of variable-number tandem-repeat genotyping of Mycobacterium bovis and comparison of the method with IS6110 restriction fragment length polymorphism analysis and spoligotyping. J Clin Microbiol. 2006; 44:1951–1962.
3. Bailo R, Bhatt A, Aínsa JA. Lipid transport in Mycobacterium tuberculosis and its implications in virulence and drug development. Biochem Pharmacol. 2015; 96:159–167.
4. Belisle JT, Mahaffey SB, Hill PJ. Isolation of Mycobacterium species genomic DNA. Methods Mol Biol. 2009; 465:1–12.
5. Bouklata N, Supply P, Jaouhari S, Charof R, Seghrouchni F, Sadki K, El Achhab Y, Nejjari C, Filali-Maltouf A, Lahlou O, El Aouad R. Molecular typing of Mycobacterium tuberculosis complex by 24-locus based MIRU-VNTR typing in conjunction with spoligotyping to assess genetic diversity of strains circulating in Morocco. PLoS One. 2015; 10:e0135695.
6. Carver T, Berriman M, Tivey A, Patel C, Böhme U, Barrell BG, Parkhill J, Rajandream MA. Artemis and ACT: viewing, annotating and comparing sequences stored in a relational database. Bioinformatics. 2008; 24:2672–2676.
crossref
7. Cho YJ, Yi H, Chun J, Cho SN, Daley CL, Koh WJ, Shin SJ. The genome sequence of ‘Mycobacterium massiliense' strain CIP 108297 suggests the independent taxonomic status of the Mycobacterium abscessus complex at the subspecies level. PLoS One. 2013; 8:e81560.
8. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 1999; 27:4636–4641.
crossref
9. Forrellad MA, Klepp LI, Gioffré A, Sabio y García J, Morbidoni HR, de la Paz Santangelo M, Cataldi AA, Bigi F. Virulence factors of the Mycobacterium tuberculosis complex. Virulence. 2013; 4:3–66.
10. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007; 57:81–91.
crossref
11. Hotter GS, Collins DM. Mycobacterium bovis lipids: virulence and vaccines. Vet Microbiol. 2011; 151:91–98.
12. Humblet MF, Boschiroli ML, Saegerman C. Classification of worldwide bovine tuberculosis risk factors in cattle: a stratified approach. Vet Res. 2009; 40:50.
crossref
13. Jackson M, Stadthagen G, Gicquel B. Long-chain multiple methyl-branched fatty acid-containing lipids of Mycobacterium tuberculosis: biosynthesis, transport, regulation and biological activities. Tuberculosis (Edinb). 2007; 87:78–86.
14. Je S, Ku BK, Jeon BY, Kim JM, Jung SC, Cho SN. Extent of Mycobacterium bovis transmission among animals of dairy and beef cattle and deer farms in South Korea determined by variable-number tandem repeats typing. Vet Microbiol. 2015; 176:274–281.
15. Jensen LJ, Julien P, Kuhn M, von Mering C, Muller J, Doerks T, Bork P. eggNOG: automated construction and annotation of orthologous groups of genes. Nucleic Acids Res. 2008; 36:D250–254.
crossref
16. Jeon B, Je S, Park J, Kim Y, Lee EG, Lee H, Seo S, Cho SN. Variable number tandem repeat analysis of Mycobacterium bovis isolates from Gyeonggi-do, Korea. J Vet Sci. 2008; 9:145–153.
17. Jeon BY, Kim SC, Je S, Kwak J, Cho JE, Woo JT, Seo S, Shim HS, Park BO, Lee SS, Cho SN. Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea. Res Vet Sci. 2010; 88:390–393.
crossref
18. Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, van Embden J. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 1997; 35:907–914.
19. Käser M, Ruf MT, Hauser J, Pluschke G. Optimized DNA preparation from mycobacteria. Cold Spring Harb Protoc. 2010; 2010:pdb. prot5408.
crossref
20. Kim J, Jang Y. [Investigation of tuberculosis in wildlife animals and dogs and cats residing on infected cattle and deer farms, and molecular characterization of Mycobacterium bovis isolated from animals]. Animal and Plant Quarantine Agency. (ed.).Annual Research Report. pp.p. 135–136. Animal and Plant Quarantine Agency;Gimcheon: 2014. Korean.
21. Kim N, Jang Y, Kim JK, Ryoo S, Kwon KH, Kang SS, Byeon HS, Lee HS, Lim YH, Kim JM. Complete genome sequence of Mycobacterium bovis clinical strain 1595, isolated from the laryngopharyngeal lymph node of South Korean cattle. Genome Announc. 2015; 3:e01124–15.
crossref
22. Kim N, Jang Y, Park SY, Song WS, Kim JT, Lee HS, Lim YH, Kim JM. Whole-genome squence of Mycobacterium bovis W-1171, isolated from the laryngopharyngeal lymph node of a wild boar in South Korea. Genome Announc. 2015; 3:e01464–15.
crossref
23. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol. 2012; 62:716–721.
crossref
24. Koren S, Schatz MC, Walenz BP, Martin J, Howard JT, Ganapathy G, Wang Z, Rasko DA, McCombie WR, Jarvis ED, Adam M. Phillippy. Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat Biotechnol. 2012; 30:693–700.
25. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. Versatile and open software for comparing large genomes. Genome Biol. 2004; 5:R12.
26. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007; 35:3100–3108.
crossref
27. Le Flèche P, Fabre M, Denoeud F, Koeck JL, Vergnaud G. High resolution, online identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing. BMC Microbiol. 2002; 2:37.
28. Lee SH, Lee WC. Epidemiological patterns and testing policies for bovine tuberculosis in the domestic cattle in Korea from 1961 to 2010. Japan J Vet Res. 2013; 61:19–23.
29. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955–964.
crossref
30. Ministry of Food and Drug Safety (KR). Livestock Product Sanitary Control. Act 12672. May 21, 2014.
31. Pirson C, Jones GJ, Steinbach S, Besra GS, Vordermeier HM. Differential effects of Mycobacterium bovis‒derived polar and apolar lipid fractions on bovine innate immune cells. Vet Res. 2012; 43:54.
32. Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A. 2009; 106:19126–19131.
crossref
33. Ruettger A, Nieter J, Skrypnyk A, Engelmann I, Ziegler A, Moser I, Monecke S, Ehricht R, Sachse K. Rapid spoligotyping of Mycobacterium tuberculosis complex bacteria by use of a microarray system with automatic data processing and assignment. J Clin Microbiol. 2012; 50:2492–2495.
34. Tatusova T, Ciufo S, Fedorov B, O'Neill K, Tolstoy I. RefSeq microbial genomes database: new representation and annotation strategy. Nucleic Acids Res. 2014; 42:D553–559.
crossref
35. Wiker HG, Harboe M. The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol Rev. 1992; 56:648–661.

Fig. 1.
The entire genomic sequence of Mycobacterium bovis strain 1595. The scale is shown in megabases on the outer black circle. From the outside to the center: RNA features (ribosomal RNAs are shown in blue, and transfer RNAs are shown in red), genes on the forward strand, and genes on the reverse strand (colored according to the clusters of orthologous groups categories). The inner two circles show the GC ratio and GC skew. The GC ratio and GC skew shown in orange and red indicate positive values, respectively, and those shown in blue and green indicate negative values, respectively.
jvs-18-333f1.tif
Fig. 2.
Genome tree based on the average nucleotide identity (ANI) values showing the relationships among Mycobacterium (M.) tuberculosis complex strains including M. bovis 1595. To convert the ANI value into a genetic distance, its complement to 1 was taken. From this pairwise distance matrix, an ANI tree was constructed using the unweighted pair group method and the arithmetic mean clustering method.
jvs-18-333f2.tif
Fig. 3.
Nucleotide-based alignments with NUCmer. X-axis: Mycobacterium bovis strain 1595. Y-axis: (A) Mycobacterium bovis W-1171, (B) AF2122/97, (C) BCG Pasteur 1173P2, and (D) Mycobacterium tuberculosis H37Rv. Aligned segments are presented as dots or lines in the NUCmer alignment and were generated by the MUMmer plot script.
jvs-18-333f3.tif
Table 1.
Clusters of orthologous groups (COGs) of the genome of Mycobacterium bovis strain 1595
COG Description Number of genes (n = 3,663)
J Translation, ribosomal structure, and biogenesis 152 (4.15)
K Transcription 232 (6.33)
L Replication, recombination, and repair 214 (5.84)
D Cell cycle control, cell division, chromosome partitioning 47 (1.28)
O Posttranslational modification, protein turnover, chaperones 102 (2.78)
M Cell wall/membrane/envelope biogenesis 118 (3.22)
N Cell motility 20 (0.55)
P Inorganic ion transport and metabolism 144 (3.93)
T Signal transduction mechanisms 114 (3.11)
C Energy production and conversion 214 (5.84)
G Carbohydrate transport and metabolism 163 (4.45)
E Amino acid transport and metabolism 204 (5.57)
F Nucleotide transport and metabolism 72 (1.97)
H Coenzyme transport and metabolism 123 (3.36)
I Lipid transport and metabolism 219 (5.98)
Q Secondary metabolites biosynthesis, transport, and catabolism 152 (4.15)
R General function prediction only 403 (11.00)
S Function unknown 970 (26.48)

Data are presented as number (%).

Table 2.
Genomic characteristics of strains used in this study
Characteristic Mycobacterium (M.) bovis 1595 M. bovis W-1171 M. bovis AF2122/97 M. bovis BCG Pasteur1173P2 M. tuberculosis H37Rv
Genome size (bp) 4351712 4304865 4345492 4374522 4411532
%G + C 65.64 65.57 65.63 65.64 65.61
Status Complete Assembly Complete Complete Complete
Contigs 1 50 1 1 1
CDSs 4,358 3,964 3,918 3,992 3,906
rRNA 3 3 3 3 3
tRNA 45 46 45 47 45

Contigs, contiguous; CDSs, coding sequences; rRNA, ribosomal RNA; tRNA, transfer RNA.

Table 3.
Similarities of open reading frames (ORFs) of various Mycobacterium (M.) tuberculosis complex strains compared to M. bovis 1595 (n = 4,358)
Match M. bovis W-1171 M. bovis AF2122/97 M. bovis BCG Pasteur 1173P2 M. tuberculosis H37Rv
100% 3,515 (80.66) 3,657 (83.91) 3,396 (77.93) 3,237 (74.28)
99% 82 (1.88) 75 (1.72) 148 (3.40) 309 (7.09)
95% 73 (1.68) 45 (1.03) 96 (2.20) 89 (2.04)
90% 93 (2.13) 39 (0.89) 93 (2.13) 50 (1.15)
80% 24 (0.55) 37 (0.85) 30 (0.69) 49 (1.12)
70% 64 (1.47) 8 (0.18) 68 (1.56) 15 (0.34)
10% 45 (1.03) 17 (0.39) 40 (0.92) 38 (0.87)
Paralog 92 (2.11) 62 (1.42) 120 (2.75) 172 (3.95)
Missing 0 (0) 1 (0.02) 1 (0.02) 2 (0.05)
No match 370 (8.49) 417 (9.57) 366 (8.40) 397 (9.11)

Data present number (%) of ORFs.

TOOLS
Similar articles