Genome-Wide Mutagenesis in Borrelia burgdorferi.
Genome-Wide Mutagenesis in Borrelia burgdorferi.
复制标题
伯氏疏螺旋体的全基因组诱变。
DOI：
10.1007/978-1-4939-7383-5_16
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Gao,Lihui
中科院分区：
文献类型：
--
作者：
Lin,Tao;Gao,Lihui
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 ofBorrelia 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 ofBorrelia burgdorferivirulence 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. burgdorferitransposon 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-taggedHimar1-based transposon suicide vector was developed and used to generate a sequence-defined library of nearly 4800 mutants in the infectiousB. burgdorferiB31 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 infectiousB. burgdorfericlone, and the transposable element is transposed into the 5′-TA-3′ sequence in theB. burgdorferigenome 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 ofB. burgdorferi and its tick vectors.
