Analysis of an Ordered, Comprehensive STM Mutant Library in Infectious Borrelia burgdorferi: Insights into the Genes Required for Mouse Infectivity

Abstract

The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM) analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease.

Phosphoenolpyruvate Phosphotransferase System Components Modulate Gene Transcription and Virulence of Borrelia burgdorferi

“…1). Previous studies in our laboratory generated transposon mutants with mutations in these carbohydrate transporters (26). Individual PEP-PTS and cyaB STM mutant clones were examined by PCR using primers flanking the insertion sites to confirm the transposon insertion and thus ascertain that each clone represented a pure culture.…”

“…S1 in the supplemental material); subsequently these clones were separated as pure cultures by replating. PCR analyses showed that two of the mutant clones previously determined (26) to have transposon insertions in ptsH or chbB did not have inserts in these regions (see Fig. S1 in the supplemental material); these clones were hence excluded from further experimentation.…”

“…The PEP-PTS and cyaB mutants were inactivated by transposon-mediated mutagenesis as part of an STM study in our laboratory in which 4,479 Himar1 mutants of B. burgdorferi 5A18NP1 were generated (26). All mutant clones were confirmed by PCR analysis using primers flanking the insertion site determined previously; the primers are listed in Table S1 in the supplemental material.…”

“…The plasmid content of each clone was determined as described previously (30). The parental B. burgdorferi 5A18NP1strain and all transposon mutant progeny lack lp28-4 and lp56 plasmids (26).…”

“…Recent signature-tagged mutagenesis (STM) analyses indicated that mutations in PTS carbohydrate transporter genes of B. burgdorferi exhibited a low-to no-infectivity phenotype (26). In the present study, we have analyzed in greater detail the mouse infectivity of mutants of PEP-PTS-associated carbohydrate transporters by needle and tick inoculation.…”