Skip to content
1887

Microbiology Society logo Microbiology

Volume 170, Issue 1

A toolbox for manipulating the genome of the major goat pathogen, subsp. Open Access

Abstract

subspecies () is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to . CReasPy-Cloning was used to simultaneously clone and engineer the genome in yeast, prior to whole-genome transplantation into subsp. recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific mutants. The Cre–lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of and the rational design of novel, improved vaccines for the control of CCPP.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Cre–lox recombination system , in-yeast genome cloning/engineering , Mycoplasma capricolum subsp. capripneumoniae , oriC plasmid , S41 serine protease and whole-genome transplantation
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001423
2024-01-09
2025-03-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/170/1/mic001423.html?itemId=/content/journal/micro/10.1099/mic.0.001423&mimeType=html&fmt=ahah

References

  1. Manso-Silván L, Thiaucourt F. Contagious caprine pleuropneumonia. In Transboundary Animal Diseases in Sahelian Africa and Connected Regions 2019 pp 439–458 [View Article]
     [Google Scholar]
  2. Renault V, Hambe HA, Van Vlaenderen G, Timmermans E, Mohamed AM et al. Economic impact of contagious caprine pleuropneumonia and cost-benefit analysis of the vaccination programmes based on a one-year continuous monitoring of flocks in the arid and semi-arid lands of Kenya. Transbound Emerg Dis 2019; 66:2523–2536 [View Article] [PubMed]
     [Google Scholar]
  3. WOAH Contagious caprine pleuropneumoniae. In OIE Terrestrial Manual - Chapter 3-7-4 pp 1441–1455
     [Google Scholar]
  4. Ozdemir U, Ozdemir E, March JB, Churchward C, Nicholas RAJ. Contagious caprine pleuropneumonia in the Thrace region of Turkey. Vet Rec 2005; 156:286–287 [View Article] [PubMed]
     [Google Scholar]
  5. Srivastava AK, Meenowa D, Barden G, Salguero FJ, Churchward C et al. Contagious caprine pleuropneumonia in Mauritius. Vet Rec 2010; 167:304–305 [View Article] [PubMed]
     [Google Scholar]
  6. Arif A, Schulz J, Thiaucourt F, Taha A, Hammer S. Contagious caprine pleuropneumonia outbreak in captive wild ungulates at Al Wabra Wildlife Preservation, state of Qatar. J Zoo Wildl Med 2007; 38:93–96 [View Article] [PubMed]
     [Google Scholar]
  7. Chaber AL, Lignereux L, Al Qassimi M, Saegerman C, Manso-Silván L et al. Fatal transmission of contagious caprine pleuropneumonia to an Arabian oryx (Oryx leucoryx). Vet Microbiol 2014; 173:156–159 [View Article] [PubMed]
     [Google Scholar]
  8. Lignereux L, Chaber A-L, Saegerman C, Manso-Silván L, Peyraud A et al. Unexpected field observations and transmission dynamics of contagious caprine pleuropneumonia in a sand gazelle herd. Prev Vet Med 2018; 157:70–77 [View Article] [PubMed]
     [Google Scholar]
  9. Yu Z, Wang T, Sun H, Xia Z, Zhang K et al. Contagious caprine pleuropneumonia in endangered Tibetan antelope, China, 2012. Emerg Infect Dis 2013; 19:2051–2053 [View Article] [PubMed]
     [Google Scholar]
  10. Thiaucourt F. Contagious caprine pleuropneumonia. In Coetze JAW, Tustin RC. eds Infection Diseases of Livestock Oxford, UK: Oxford University Press; 2018 pp 2060–2065
     [Google Scholar]
  11. Ozdemir U, Loria GR, Godinho KS, Samson R, Rowan TG et al. Effect of danofloxacin (Advocin A180) on goats affected with contagious caprine pleuropneumonia. Trop Anim Health Prod 2006; 38:533–540 [View Article] [PubMed]
     [Google Scholar]
  12. Murray CJL, Ikuta KS, Sharara F, Swetschinski L, Robles Aguilar G et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet 2022; 399:629–655 [View Article]
     [Google Scholar]
  13. Rurangirwa FR, McGuire TC, Kibor A, Chema S. An inactivated vaccine for contagious caprine pleuropneumonia. Vet Rec 1987; 121:397–400 [View Article] [PubMed]
     [Google Scholar]
  14. Thiaucourt F, Pible O, Miotello G, Nwankpa N, Armengaud J. Improving quality control of contagious caprine pleuropneumonia vaccine with tandem mass spectrometry. Proteomics 2018; 18:e1800088 [View Article] [PubMed]
     [Google Scholar]
  15. Jores J, Baldwin C, Blanchard A, Browning GF, Colston A et al. Contagious bovine and caprine pleuropneumonia: a research community’s recommendations for the development of better vaccines. NPJ Vaccines 2020; 5:1–9 [View Article] [PubMed]
     [Google Scholar]
  16. Janis C, Lartigue C, Frey J, Wróblewski H, Thiaucourt F et al. Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum. Appl Environ Microbiol 2005; 71:2888–2893 [View Article] [PubMed]
     [Google Scholar]
  17. Ishag HZA, Xiong Q, Liu M, Feng Z, Shao G. Development of oriC-plasmids for use in Mycoplasma hyorhinis. Sci Rep 2017; 7:10596 [View Article] [PubMed]
     [Google Scholar]
  18. Lee S-W, Browning GF, Markham PF. Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum. Microbiology 2008; 154:2571–2580 [View Article] [PubMed]
     [Google Scholar]
  19. Lartigue C, Lebaudy A, Blanchard A, El Yacoubi B, Rose S et al. The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA. Nucleic Acids Res 2014; 42:8073–8082 [View Article] [PubMed]
     [Google Scholar]
  20. Blötz C, Lartigue C, Valverde Timana Y, Ruiz E, Paetzold B et al. Development of a replicating plasmid based on the native oriC in Mycoplasma pneumoniae. Microbiology 2018; 164:1372–1382 [View Article] [PubMed]
     [Google Scholar]
  21. Vashee S, Arfi Y, Lartigue C. Budding yeast as a factory to engineer partial and complete microbial genomes. Curr Opin Syst Biol 2020; 24:1–8 [View Article] [PubMed]
     [Google Scholar]
  22. Jores J, Ma L, Ssajjakambwe P, Schieck E, Liljander A et al. Removal of a subset of non-essential genes fully attenuates a highly virulent Mycoplasma strain. Front Microbiol 2019; 10:664 [View Article] [PubMed]
     [Google Scholar]
  23. Lartigue C, Valverde Timana Y, Labroussaa F, Schieck E, Liljander A et al. Attenuation of a pathogenic Mycoplasma strain by modification of the obg gene by using synthetic biology approaches. mSphere 2019; 4:e00030-19 [View Article] [PubMed]
     [Google Scholar]
  24. Talenton V, Baby V, Gourgues G, Mouden C, Claverol S et al. Genome engineering of the fast-growing Mycoplasma feriruminatoris toward a live vaccine chassis. ACS Synth Biol 2022; 11:1919–1930 [View Article] [PubMed]
     [Google Scholar]
  25. Tsarmpopoulos I, Gourgues G, Blanchard A, Vashee S, Jores J et al. In-yeast engineering of a bacterial genome using CRISPR/Cas9. ACS Synth Biol 2016; 5:104–109 [View Article] [PubMed]
     [Google Scholar]
  26. Kannan K, Tsvetanova B, Chuang R-Y, Noskov VN, Assad-Garcia N et al. One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9. Sci Rep 2016; 6:30714 [View Article] [PubMed]
     [Google Scholar]
  27. Lartigue C, Vashee S, Algire MA, Chuang R-Y, Benders GA et al. Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 2009; 325:1693–1696 [View Article] [PubMed]
     [Google Scholar]
  28. Labroussaa F, Lebaudy A, Baby V, Gourgues G, Matteau D et al. Impact of donor-recipient phylogenetic distance on bacterial genome transplantation. Nucleic Acids Res 2016; 44:8501–8511 [View Article] [PubMed]
     [Google Scholar]
  29. Baby V, Labroussaa F, Brodeur J, Matteau D, Gourgues G et al. Cloning and transplantation of the Mesoplasma florum genome. ACS Synth Biol 2018; 7:209–217 [View Article] [PubMed]
     [Google Scholar]
  30. Zhao G, Lu D, Li M, Wang Y. Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation. Front Microbiol 2023; 14:1191812 [View Article] [PubMed]
     [Google Scholar]
  31. Lyon BR, May JW, Skurray RA. Tn4001: a gentamicin and kanamycin resistance transposon in Staphylococcus aureus. Mol Gen Genet 1984; 193:554–556 [View Article] [PubMed]
     [Google Scholar]
  32. Clewell DB, Gawron-Burke C. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu Rev Microbiol 1986; 40:635–659 [View Article] [PubMed]
     [Google Scholar]
  33. Hutchison CA, Peterson SN, Gill SR, Cline RT, White O et al. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 1999; 286:2165–2169 [View Article] [PubMed]
     [Google Scholar]
  34. Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR et al. Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A 2006; 103:425–430 [View Article] [PubMed]
     [Google Scholar]
  35. Dybvig K, French CT, Voelker LL. Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis. J Bacteriol 2000; 182:4343–4347 [View Article] [PubMed]
     [Google Scholar]
  36. Pour-El I, Adams C, Minion FC. Construction of mini-Tn4001tet and its use in Mycoplasma gallisepticum. Plasmid 2002; 47:129–137 [View Article] [PubMed]
     [Google Scholar]
  37. Zimmerman C-U, Herrmann R. Synthesis of a small, cysteine-rich, 29 amino acids long peptide in Mycoplasma pneumoniae. FEMS Microbiol Lett 2005; 253:315–321 [View Article] [PubMed]
     [Google Scholar]
  38. Piñero-Lambea C, Garcia-Ramallo E, Martinez S, Delgado J, Serrano L et al. Mycoplasma pneumoniae genome editing based on oligo recombineering and Cas9-mediated counterselection. ACS Synth Biol 2020; 9:1693–1704 [View Article] [PubMed]
     [Google Scholar]
  39. Ipoutcha T, Gourgues G, Lartigue C, Blanchard A, Sirand-Pugnet P. Genome engineering in Mycoplasma gallisepticum using exogenous recombination systems. ACS Synth Biol 2022; 11:1060–1067 [View Article] [PubMed]
     [Google Scholar]
  40. Piñero-Lambea C, Garcia-Ramallo E, Miravet-Verde S, Burgos R, Scarpa M et al. SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome. Nucleic Acids Res 2022; 50:e127 [View Article] [PubMed]
     [Google Scholar]
  41. Mahdizadeh S, Sansom FM, Lee SW, Browning GF, Marenda MS. Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system. Vet Microbiol 2020; 250:108868 [View Article] [PubMed]
     [Google Scholar]
  42. Klose SM, Wawegama N, Sansom FM, Marenda MS, Browning GF. Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum. Vet Microbiol 2022; 269:109436 [View Article] [PubMed]
     [Google Scholar]
  43. Ipoutcha T, Rideau F, Gourgues G, Arfi Y, Lartigue C et al. Genome editing of veterinary relevant Mycoplasmas using a CRISPR-cas base editor system. Appl Environ Microbiol 2022; 88:e0099622 [View Article] [PubMed]
     [Google Scholar]
  44. Mariscal AM, Kakizawa S, Hsu JY, Tanaka K, González-González L et al. Tuning gene activity by inducible and targeted regulation of gene expression in minimal bacterial cells. ACS Synth Biol 2018; 7:1538–1552 [View Article] [PubMed]
     [Google Scholar]
  45. Evsyutina DV, Fisunov GY, Pobeguts OV, Kovalchuk SI, Govorun VM. Gene silencing through CRISPR interference in Mycoplasmas. Microorganisms 2022; 10:1159 [View Article] [PubMed]
     [Google Scholar]
  46. Mariscal AM, González-González L, Querol E, Piñol J. All-in-one construct for genome engineering using Cre-lox technology. DNA Research : An International Journal for Rapid Publication of Reports on Genes and Genomes 2016; 23263–270 [View Article] [PubMed]
     [Google Scholar]
  47. Garcia-Morales L, Ruiz E, Gourgues G, Rideau F, Piñero-Lambea C et al. A RAGE based strategy for the genome engineering of the human respiratory pathogen Mycoplasma pneumoniae. ACS Synth Biol 2020; 9:2737–2748 [View Article] [PubMed]
     [Google Scholar]
  48. Thiaucourt F, Breard A, Lefèvre PC, Mebratu GY. Contagious caprine pleuropneumonia in Ethiopia. Vet Rec 1992; 131:585 [PubMed]
     [Google Scholar]
  49. Dupuy V, Thiaucourt F. Complete genome sequence of Mycoplasma capricolum subsp. capripneumoniae strain 9231-abomsa. Genome Announc 201401067-14 [View Article]
     [Google Scholar]
  50. Bonnefois T, Vernerey M-S, Rodrigues V, Totté P, Puech C et al. Development of fluorescence expression tools to study host-mycoplasma interactions and validation in two distant mycoplasma clades. J Biotechnol 2016; 236:35–44 [View Article] [PubMed]
     [Google Scholar]
  51. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 2013; 41:4336–4343 [View Article] [PubMed]
     [Google Scholar]
  52. Waites KB, Bade DJ, Bébéar C, Brown SD, Davidson MK. Methods for Antimicrobial Susceptibility Testing for Human Mycoplasmas; Approved Guideline. Report No. Document M43-A Clinical and Laboratory Standards Institute; 2011
     [Google Scholar]
  53. Ruiz E, Talenton V, Dubrana M-P, Guesdon G, Lluch-Senar M et al. CReasPy-Cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system. ACS Synth Biol 2019; 8:2547–2557 [View Article] [PubMed]
     [Google Scholar]
  54. Kouprina N, Larionov V. Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat Protoc 2008; 3:371–377 [View Article] [PubMed]
     [Google Scholar]
  55. Shaw D, Serrano L, Lluch-Senar M. Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments. Microbiology 2021; 167:1–11 [View Article] [PubMed]
     [Google Scholar]
  56. Gietz RD, Schiestl RH, Willems AR, Woods RA. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 1995; 11:355–360 [View Article] [PubMed]
     [Google Scholar]
  57. Lartigue C, Glass JI, Alperovich N, Pieper R, Parmar PP et al. Genome transplantation in bacteria: changing one species to another. Science 2007; 317:632–638 [View Article] [PubMed]
     [Google Scholar]
  58. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol 1983; 166:557–580 [View Article] [PubMed]
     [Google Scholar]
  59. King KW, Dybvig K. Plasmid transformation of Mycoplasma mycoides subspecies mycoides is promoted by high concentrations of polyethylene glycol. Plasmid 1991; 26:108–115 [View Article] [PubMed]
     [Google Scholar]
  60. Dybvig K, Voelker LL. Molecular biology of Mycoplasmas. Annu Rev Microbiol 1996; 50:25–57 [View Article] [PubMed]
     [Google Scholar]
  61. Ganter S, Miotello G, Manso-Silván L, Armengaud J, Tardy F et al. Proteases as secreted exoproteins in Mycoplasmas from ruminant lungs and their impact on surface-exposed proteins. Appl Environ Microbiol 2019; 85:e01439-19 [View Article] [PubMed]
     [Google Scholar]
  62. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018; 34:i884–i890 [View Article] [PubMed]
     [Google Scholar]
  63. Li H, Durbin R. Fast and accurate long-read alignment with burrows-wheeler transform. Bioinformatics 2010; 26:589–595 [View Article] [PubMed]
     [Google Scholar]
  64. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010; 20:1297–1303 [View Article] [PubMed]
     [Google Scholar]
  65. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The sequence alignment/Map format and SAMtools. Bioinformatics 2009; 25:2078–2079 [View Article] [PubMed]
     [Google Scholar]
  66. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 2012; 6:80–92 [View Article] [PubMed]
     [Google Scholar]
  67. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Cold Spring Harb Lab Press 2017; 27:722–736 [View Article] [PubMed]
     [Google Scholar]
  68. Marçais G, Delcher AL, Phillippy AM, Coston R, Salzberg SL et al. MUMmer4: A fast and versatile genome alignment system. PLoS Comput Biol 2018; 14:e1005944 [View Article] [PubMed]
     [Google Scholar]
  69. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang R-Y et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 2010; 329:52–56 [View Article] [PubMed]
     [Google Scholar]
  70. Lartigue C, Blanchard A, Renaudin J, Thiaucourt F, Sirand-Pugnet P. Host specificity of mollicutes oriC plasmids: functional analysis of replication origin. Nucleic Acids Res 2003; 31:6610–6618 [View Article] [PubMed]
     [Google Scholar]
  71. Sharma S, Citti C, Sagné E, Marenda MS, Markham PF et al. Development and host compatibility of plasmids for two important ruminant pathogens, Mycoplasma bovis and Mycoplasma agalactiae. PLoS One 2015; 10:e0119000 [View Article] [PubMed]
     [Google Scholar]
  72. Algire MA, Lartigue C, Thomas DW, Assad-Garcia N, Glass JI et al. New selectable marker for manipulating the simple genomes of Mycoplasma species. Antimicrob Agents Chemother 2009; 53:4429–4432 [View Article] [PubMed]
     [Google Scholar]
  73. Pettersson B, Leitner T, Ronaghi M, Bölske G, Uhlen M et al. Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons. J Bacteriol 1996; 178:4131–4142 [View Article] [PubMed]
     [Google Scholar]
  74. Pettersson B, Bölske G, Thiaucourt F, Uhlén M, Johansson KE. Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes. J Bacteriol 1998; 180:2350–2358 [View Article] [PubMed]
     [Google Scholar]
  75. Dupuy V, Verdier A, Thiaucourt F, Manso-Silván L. A large-scale genomic approach affords unprecedented resolution for the molecular epidemiology and evolutionary history of contagious caprine pleuropneumonia. Vet Res 2015; 46:74 [View Article] [PubMed]
     [Google Scholar]
  76. Loire E, Ibrahim AI, Manso-Silván L, Lignereux L, Thiaucourt F. A whole-genome worldwide molecular epidemiology approach for contagious caprine pleuropneumonia. Heliyon 2020; 6:e05146 [View Article] [PubMed]
     [Google Scholar]
  77. Guesdon G, Gourgues G, Rideau F, Ipoutcha T, Manso-Silván L et al. Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast. bioRxiv 2023; 12:3252–3266 [View Article] [PubMed]
     [Google Scholar]
  78. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 2016; 34:184–191 [View Article] [PubMed]
     [Google Scholar]
  79. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013; 31:827–832 [View Article] [PubMed]
     [Google Scholar]
  80. Hutchison CA, Chuang R-Y, Noskov VN, Assad-Garcia N, Deerinck TJ et al. Design and synthesis of a minimal bacterial genome. Science 2016; 351:6253 [View Article] [PubMed]
     [Google Scholar]
  81. Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination. I. recombination between loxP sites. J Mol Biol 1981; 150:467–486 [View Article] [PubMed]
     [Google Scholar]
  82. Cordova CMM, Lartigue C, Sirand-Pugnet P, Renaudin J, Cunha RAF et al. Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids. J Bacteriol 2002; 184:5426–5435 [View Article] [PubMed]
     [Google Scholar]
  83. Bertin C, Pau-Roblot C, Courtois J, Manso-Silván L, Tardy F et al. Highly dynamic genomic loci drive the synthesis of two types of capsular or secreted polysaccharides within the Mycoplasma mycoides cluster. Appl Environ Microbiol 2015; 81:676–687 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001423
Loading
A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae
Microbiology 170, 001423 (2024); https://doi.org/10.1099/mic.0.001423
/content/journal/micro/10.1099/mic.0.001423
/content/journal/micro/10.1099/mic.0.001423
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL

Supplementary material 3

EXCEL

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error