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Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi.

ABSTRACT: Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A1-resistant transformants in which gyrB had interrupted the targeted site on the 26-kb plasmid via homologous recombination with the flanking sequences. Antibiotic resistance conferred by the mutated gyrB gene on the plasmid is dominant, and transformed spirochetes carrying this plasmid do not contain any unaltered copies of the plasmid. Coumermycin A1 resistance can be transferred to naive B. burgdorferi by transformation with borrelial plasmid DNA from the initial transformants. This work represents the first example of a directed mutation in B. burgdorferi whereby a large segment of heterologous DNA (gyrB) has been inserted via homologous recombination with flanking sequences, thus demonstrating the feasibility of specific gene inactivation by allelic exchange.

SUBMITTER: Rosa P

PROVIDER: S-EPMC178451 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi.

Rosa P P Samuels D S DS Hogan D D Stevenson B B Casjens S S Tilly K K

Journal of bacteriology 19961001 20

Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which thi ...[more]

PMID: 8830691

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Project description:Plasmid cp8.3 of Borrelia afzelii IP21 carries several open reading frames (ORFs) and a 184-bp inverted repeat (IR) element. It has been speculated that this plasmid may encode factors involved in virulence or infectivity. In this report, we have characterized the distribution, molecular variability, and organization of ORFs 1, 2, and 4 and the IR elements among isolates of the Borrelia burgdorferi sensu lato complex. ORFs 1 and 2 are contained within a segment of cp8.3 that is bordered by the IR elements, while ORF 4 resides just outside of the IR-bordered region. By PCR, ORF 4 was amplified from most isolates while ORFs 1 and 2 were amplified from only some B. afzelii isolates. However, Southern hybridization analyses with ORF 1, 2, and 4 probes detected related sequences even in some isolates that were PCR negative. The ORF restriction fragment length polymorphism patterns varied widely even among isolates of the same species. Two-dimensional contour-clamped homogeneous electric field-pulsed-field gel electrophoresis and Southern hybridization detected ORF 1-, 2-, and 4-related sequences on linear and circular plasmids. In addition, an ORF 4-related sequence was detected on a previously uncharacterized, circular plasmid that is greater than 70 kb in size. The IR elements originally identified on plasmid cp8.3 of B. afzelii IP21 were also analyzed by Southern hybridization. Related sequences were detected in some but not all B. burgdorferi sensu lato isolates. These sequences are carried on plasmids in addition to cp8.3 in some isolates. Single-primer PCR analyses demonstrated that in some isolates these sequences exist with IR orientation. The data presented here demonstrate that the IR elements and the ORF 1-, 2-, and 4-related sequences are multicopy and are variable in organization and in genomic location among isolates of the B. burgdorferi sensu lato complex. These analyses provide additional evidence for the highly variable organization of the plasmid component of the B. burgdorferi sensu lato genome.

Project description:Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing, and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multicolor imaging in B. burgdorferi We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Last, we describe a set of promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete.IMPORTANCE Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium's transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

Project description:Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi.We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes).Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.

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Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi.

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Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi.

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