Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi - PubMed (original) (raw)

Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi

M Sartakova et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Molecular genetic analysis of Borrelia burgdorferi, the cause of Lyme disease, has been hampered by the absence of any means of efficient generation, identification, and complementation of chromosomal and plasmid null gene mutants. The similarity of borrelial G + C content to that of Gram-positive organisms suggested that a wide-host-range plasmid active in Gram-positive bacteria might also be recognized by borrelial DNA replication machinery. One such plasmid, pGK12, is able to propagate in both Gram-positive and Gram-negative bacteria and carries erythromycin and chloramphenicol resistance markers. pGK12 propagated extrachromosomally in B. burgdorferi B31 after electroporation but conferred only erythromycin resistance. pGK12 was used to express enhanced green fluorescent protein in B31 under the control of the flaB promoter. Escherichia coli transformed with pGK12 DNA extracted from B31 expressing only erythromycin resistance developed both erythromycin and chloramphenicol resistance, and plasmid DNA isolated from these transformed E. coli had a restriction pattern similar to the original pGK12. Our data indicate that the replicons of pGK12 can provide the basis to continue developing efficient genetic systems for B. burgdorferi together with the erythromycin resistance and reporter egfp genes.

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Figures

Figure 1

Figure 1

Agarose gel electrophoresis and Southern hybridization of plasmid DNA isolated from B. burgdorferi B31 erythromycin-resistant clones electroporated with plasmid pGK12. (a) Agarose gel electrophoresis. (b) Southern blot of agarose gel in a. Lane 1, pGK12 DNA purified from E. coli MM294; Lane 2, Plasmid DNA from wild-type B. burgdorferi B31; Lanes 3–5, Plasmid DNA of three different B. burgdorferi erythromycin-resistant electroporants containing pGK12.

Figure 2

Figure 2

Construction of plasmid pMS1 containing the egfp gene. (a) Plasmid pGK12. (b) Ligated PCR amplification products containing the flagellin B promoter-P_fla_ and egfp gene. (c) Recombinant plasmid pMS1.

Figure 3

Figure 3

Micrographs of B. burgdorferi B31 containing plasmid pMS1. (a) Phase contrast microphotograph of B. burgdorferi B31 with pMS1; (b–f) Fluorescent microphotographs of B. burgdorferi with pMS1. (g) Phase contrast microphotograph of wild-type B. burgdorferi B31; (h) Fluorescent microphotograph of wild-type B. burgdorferi B31.

Figure 4

Figure 4

Detection of EGFP and egfp mRNA in B. burgdorferi B31 containing plasmid pMS1. (a) Immunodetection of EGFP. Lane 1, protein lysate of E. coli containing pMS1. Lane 2, protein lysate of wild-type B. burgdorferi B31. Lanes 3 and 4, B. burgdorferi isolates containing pMS1. (b) RT-PCR of egfp RNA. Lane 1, molecular weight standards. Lane 2, amplification of RNA from B. burgdorferi containing pMS1 without reverse transcriptase. Lane 3, amplification of RNA from B. burgdorferi containing pMS1 with reverse transcriptase. Lanes 4 and 5, the same as 2 and 3, but with cultures of B. burgdorferi shaken in the presence of oxygen. Lane 6, RT-PCR performed without RNA.

Figure 5

Figure 5

Competitive PCR of egfp and flaB DNAs in B. burgdorferi containing pMS1. All reactions except negative controls contained 0.5 ng of total DNA from B. burgdorferi cells with pMS1. In flaB (lanes 1–7) and egfp (lanes 8–14), the competitive amplifications were performed with primers specific to the flaB and egfp genes, respectively. The PCR reactions contained 1 pg of correspondent competitor (lanes 1 and 8), 300 fg (lanes 2 and 9), 100 fg (lanes 3 and 10), 33 fg (lanes 4 and 11), 10 fg (lanes 5 and 12), and 3.3 fg (lanes 6 and 13). Lanes 7 and 14 correspond to reactions carried out without DNA.

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