Abstract
Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology. This approach is particularly useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. In this review, progress to date in the application of transposon mutagenesis to the study of Borrelia burgdorferi is reported. An effective Himar1-based transposon vector has been developed and used to acquire a sequence-defined library of nearly 4500 mutants in the infectious, moderately transformable B. burgdorferi B31 derivative 5A18NP1. Analysis of these transposon mutants using signature-tagged mutagenesis (STM) and Tn-seq approaches has begun to yield valuable information regarding the genes important in the pathogenesis and biology of this organism.
Highlights
Edited by: Catherine Ayn Brissette, University of North Dakota School of Medicine and Health Sciences, USA
Since its initial discovery and culture in 1981, B. burgdorferi has been the subject of intensive study in an attempt to better understand the biology of this organism and thereby identify properties useful in the diagnosis, treatment, or prevention of Lyme disease
Fewer than 100 of the 1739 open reading frames (ORFs) in infectious B. burgdorferi have been subjected to site-directed mutagenesis despite intensive efforts by several laboratories
Summary
Edited by: Catherine Ayn Brissette, University of North Dakota School of Medicine and Health Sciences, USA. Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology This approach is useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. A modified version of pMarGent called pGKT (Figure 1) was later developed to include a second selectable marker (KanR) in the non-transposed “backbone” in addition to the gentamycin resistance gene present in the transposable element (Stewart and Rosa, 2008) This modification greatly increases the stability of the vector in E. coli and facilitates additional alterations (such as the addition of signature tags). In both pMarGent and pGKT, the transposable element consists of the B. burgdorferi constitutive promoter flgBP coupled with the gentamicin resistance cassette aacC1 and the ColE1 origin of replication flanked by two Himar inverted
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