Kit Tilly

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non‐infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.

Lipoprotein succession in Borrelia burgdorferi: similar but distinct roles for OspC and VlsE at different stages of mammalian infection.

Borrelia burgdorferi alternates between ticks and mammals, requiring variable gene expression and protein production to adapt to these diverse niches. These adaptations include shifting among the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. We hypothesize that these proteins carry out a basic but essential function, and that OspC and VlsE fulfil this requirement during early and persistent stages of mammalian infection respectively. Previous work by other investigators suggested that several B. burgdorferi lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In this study, we assessed whether vlsE and ospA could restore infectivity to an ospC mutant, and found that neither gene product effectively compensated for the absence of OspC during early infection. In contrast, we determined that OspC production was required by B. burgdorferi throughout SCID mouse infection if the vlsE gene were absent. Together, these results indicate that OspC can substitute for VlsE when antigenic variation is unnecessary, but that these two abundant lipoproteins are optimized for their related but specific roles during early and persistent mammalian infection by B. burgdorferi.

Cloning and expression of the Borrelia burgdorferi Ion gene

Abstract The ATP-dependent protease Lon (La) of Escherichia coli degrades abnormal proteins and is involved in the regulation of capsular polysaccharide synthesis. In addition, mutations in the E. coli Ion gene suppress temperature-sensitive mutations in other genes. The Ion gene of Borrelia burgdorferi, encoding a homolog of the Lon protease, has been cloned and sequenced. The gene encodes a protein of 806 amino acids. The deduced amino acid sequence of the B. burgdorferi Lon protease shares substantial sequence identity with those of other known Lon proteases. The transcription start point of the B. burgdorferi Ion gene was identified by primer extension analysis and the potential promoter did not show similarities to the consensus heat-shock promoter in E. coli. The 5′-end of the B. burgdorferi lon gene appears to suppress the temperature-sensitive phenotype of an E. coli lpxA mutant.

7 Genetic Methods in Borrelia and Other Spirochaetes

Publisher Summary Spirochaetes are a phylogenetically ancient bacterial group and one of the few for which classification by morphology is consistent with molecular typing based upon rRNA sequence comparisons. Although a unique spiral shape and periplasmic flagella for motility are universal among the spirochaetes, they are a diverse group of bacteria with regard to many other characteristics. Several spirochaetes represent important human and animal pathogens, with histories of disease that extend for centuries. None of the spirochaetes has been the subject of extensive genetic analysis and there is a corresponding paucity in current genetic tools and methods with which to manipulate them. All other buffers used for analytical purposes such as gel electrophoresis are prepared with deionized water. Instead of the water drawn from a Millipore system, double-distilled water can be used as well. Media for cultivating and storing bacteria generally include liquid media, solid media, storage media, and supplements. Solid media are mostly liquid media solidified by the addition of agar.

Functional Equivalence of OspA and OspB, but Not OspC, in Tick Colonization by Borrelia burgdorferi

ABSTRACT Borrelia burgdorferi, a Lyme disease agent, makes different major outer surface lipoproteins at different stages of its mouse–tick infectious cycle. Outer surface protein A (OspA) coats the spirochetes from the time they enter ticks until they are transmitted to a mammal. OspA is required for normal tick colonization and has been shown to bind a tick midgut protein, indicating that OspA may serve as a tick midgut adhesin. Tick colonization by spirochetes lacking OspA is increased when the infecting blood meal is derived from mice that do not produce antibody, indicating that OspA may protect the spirochetes from host antibody, which will not recognize tick-specific proteins such as OspA. To further study the importance of OspA during tick colonization, we constructed a form of B. burgdorferi in which the ospA open reading frame, on lp54, was replaced with the ospC gene or the ospB gene, encoding a mammal-specific or tick-specific lipoprotein, respectively. These fusions yielded a strain that produces OspC within a tick (from the fusion gene) and during early mammalian infection (from the normal ospC locus) and a strain that produces OspB in place of OspA within ticks. Here we show that the related, tick-specific protein OspB can fully substitute for OspA, whereas the unrelated, mammal-specific protein OspC cannot. These data were derived from three different methods of infecting ticks, and they confirm and extend previous studies indicating that OspA both protects spirochetes within ticks from mammalian antibody and serves an additional role during tick colonization.