Genome-Wide Mutagenesis in Borrelia burgdorferi
Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector. STM procedure combined with Luminex Flex®Map™ technology and next-generation sequencing (e.g., Tn-seq) are the powerful high-throughput tools for the determination of Borrelia burgdorferi virulence determinants. The assessment of multiple tissue sites and two DNA resources at two different time points using Luminex Flex®Map™ technology provides a robust data set. B. burgdorferi transposon mutant screening indicates that a high proportion of genes are the novel virulence determinants that are required for mouse and tick infection. In this protocol, an effective signature-tagged Himar1-based transposon suicide vector was developed and used to generate a sequence-defined library of nearly 4800 mutants in the infectious B. burgdorferi B31 clone. In STM, signature-tagged suicide vectors are constructed by inserting unique DNA sequences (tags) into the transposable elements. The signature-tagged transposon mutants are generated when transposon suicide vectors are transformed into an infectious B. burgdorferi clone, and the transposable element is transposed into the 5′-TA-3′ sequence in the B. burgdorferi genome with the signature tag. The transposon library is created and consists of many sub-libraries, each sub-library has several hundreds of mutants with same tags. A group of mice or ticks are infected with a mixed population of mutants with different tags, after recovered from different tissues of infected mice and ticks, mutants from output pool and input pool are detected using high-throughput, semi-quantitative Luminex® FLEXMAP™ or next-generation sequencing (Tn-seq) technologies. Thus far, we have created a high-density, sequence-defined transposon library of over 6600 STM mutants for the efficient genome-wide investigation of genes and gene products required for wild-type pathogenesis, host–pathogen interactions, in vitro growth, in vivo survival, physiology, morphology, chemotaxis, motility, structure, metabolism, gene regulation, plasmid maintenance and replication, etc. The insertion sites of 4480 transposon mutants have been determined. About 800 predicted protein-encoding genes in the genome were disrupted in the STM transposon library. The infectivity and some functions of 800 mutants in 500 genes have been determined. Analysis of these transposon mutants has yielded valuable information regarding the genes and gene products important in the pathogenesis and biology of B. burgdorferi and its tick vectors.
Key wordsLyme disease Borrelia burgdorferi Signature-tagged mutagenesis (STM) Luminex® FLEXMAP™ technology Next-generation sequencing Microbial pathogenesis Host–pathogen interactions Bacterial physiology Mouse and tick infection models
We thank Drs. P.E. Stewart and P.A. Rosa (Rocky Mountain Laboratories, National Institutes of Health, Hamilton, MN) for providing the plasmid pGKT. Studies described in this article were supported by the United States National Institutes of Health, Institute of Allergy and Infectious Diseases through grants R01 AI059048 (Steven J. Norris and Tao Lin).
- 7.Lin T, Gao L, Zhang C, Odeh E, Jacobs MB, Coutte L, Chaconas G, Philipp MT, Norris SJ (2012) Analysis of an ordered, comprehensive STM mutant library in infectious Borrelia burgdorferi: insights into the genes required for mouse infectivity. PLoS One 7:e47532CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Lehoux DE, Sanschagrin F, Levesque RC (1999) Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria. BioTechniques 26(473–478):480Google Scholar
- 20.Fraser CM, Casjens S, Huang WM, Sutton GG, Clayton R, Lathigra R, White O, Ketchum KA, Dodson R, Hickey EK, Gwinn M, Dougherty B, Tomb JF, Fleischmann RD, Richardson D, Peterson J, Kerlavage AR, Quackenbush J, Salzberg S, Hanson M, van Vugt R, Palmer N, Adams MD, Gocayne J, Weidman J, Utterback T, Watthey L, McDonald L, Artiach P, Bowman C, Garland S, Fujii C, Cotton MD, Horst K, Roberts K, Hatch B, Smith HO, Venter JC (1997) Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580–586CrossRefPubMedGoogle Scholar
- 22.Escalante A, Salinas Cervantes A, Gosset G, Bolivar F (2012) Current knowledge of the Escherichia coli phosphoenolpyruvate-carbohydrate phosphotransferase system: peculiarities of regulation and impact on growth and product formation. Appl Microbiol Biotechnol 94:1483–1494CrossRefPubMedGoogle Scholar
- 32.von Lackum K, Babb K, Riley SP, Wattier RL, Bykowski T, Stevenson B (2006) Functionality of Borrelia burgdorferi LuxS: the Lyme disease spirochete produces and responds to the pheromone autoinducer-2 and lacks a complete activated-methyl cycle. Int J Med Microbiol 296(Suppl 40):92–102CrossRefGoogle Scholar