New antibiotic resistance cassettes suitable for genetic studies in Borrelia burgdorferi.
In this report we describe two distinct approaches to develop new antibiotic resistance cassettes that allow for efficient selection of Borrelia burgdorferi transformants. The first approach utilizes fusions of borrelial flagellar promoters to antibiotic resistance markers from other bacteria. The AACC1 gene, which encodes a gentamicin acetyltransferase, conferred a high level of gentamicin resistance in B. Burfdorferi when expressed from these promoters. No cross-resistance occurred between this cassette and the kanamycin resistance cassette, which was previously developed in an analogous fashion. A second and different approach was taken to develop an efficient selectable marker that confers resistance to the antibiotic coumermycin A1. A synthetic gene was designed from the GYRB301 allele of the coumermycin-resistant B. Burgdorferi strain B31-NGR by altering the coding sequence at the wobble position. The resulting gene, GYRB(SYN), encodes a protein identical to the product of GYRB301, but the genes share only 66% nucleotide identity. The nucleotide sequence of GYRB(SYN)is sufficiently divergent from the endogenous B. Burgdorferi GYRB gene to prevent recombination between them. The cassettes described in this paper improve our repertoire of genetic tools in B. Burgdorferi. These studies also provide insight into parameters governing recombination and gene expression in B. Burgdorferi.
Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Mont., USA.
Abdallah F
AF
Elias
James L
JL
Bono
John J
JJ
Kupko
Philip E
PE
Stewart
Jonathan G
JG
Krum
Patricia A
PA
Rosa
D000595:Amino Acid Sequence; D001483:Base Sequence; D025065:Borrelia burgdorferi / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D005819:Genetic Markers