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Infect Immun, March 1998, p. 1149-1158, Vol. 66, No. 3
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Analysis of the Organization of Multicopy Linear- and Circular-Plasmid-Carried Open Reading Frames in Borrelia burgdorferi Sensu Lato Isolates

Jason A. Carlyon, Crystal Lavoie, Shian-Ying Sung, and Richard T. Marconi*

Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0678

Received 17 April 1997/Returned for modification 2 June 1997/Accepted 31 December 1997

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Box 980678, 1101 East Marshall St., Richmond, VA 23298-0678. Phone: (804) 828-3779. Fax: (804) 828-9946. E-mail: RMARCONI{at}HSC.VCU.EDU.




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