Structure of the Borrelia Bacteriophage φBB1 Procapsid.

Bacteriophages of Borrelia burgdorferi are a biologically important but under-investigated feature of the Lyme disease-causing spirochete. No virulent borrelial viruses have been identified, but all B. burgdorferi isolates carry a prophage φBB1 as resident circular plasmids. Like its host, the φBB1 phage is quite distinctive and shares little sequence similarity with other known bacteriophages. We expressed φBB1 head morphogenesis proteins in Escherichia coli which resulted in assembly of homogeneous prolate procapsid structures and used cryo-electron microscopy to determine the three-dimensional structure of these particles. The φBB1 procapsids consist of 415 copies of the major capsid protein and an equal combined number of three homologous capsid decoration proteins that form trimeric knobs on the outside of the particle. One of the end vertices of the particle is occupied by a portal assembled from twelve copies of the portal protein. The φBB1 scaffolding protein is entirely α-helical and has an elongated shape with a small globular domain in the middle. Within the tubular section of the procapsid, the internal scaffold is built of stacked rings, each composed of 32 scaffolding protein molecules, which run in opposite directions from both caps with a heterogeneous part in the middle. Inside the portal-containing cap, the scaffold is organized asymmetrically with ten scaffolding protein molecules bound to the portal. The φBB1 procapsid structure provides better insight into the vast structural diversity of bacteriophages and presents clues of how elongated bacteriophage particles might be assembled.

Multipartite Genome of Lyme Disease Borrelia: Structure, Variation and Prophages.

All members of the Borrelia genus that have been examined harbour a linear chromosome that is about 900 kbp in length, as well as a plethora of both linear and circular plasmids in the 5-220 kbp size range. Genome sequences for 27 Lyme disease Borrelia isolates have been determined since the elucidation of the B. burgdorferi B31 genome sequence in 1997. The chromosomes, which carry the vast majority of the housekeeping genes, appear to be very constant in gene content and organization across all Lyme disease Borrelia species. The content of the plasmids, which carry most of the genes that encode the differentially expressed surface proteins that interact with the spirochete's arthropod and vertebrate hosts, is much more variable. Lyme disease Borrelia isolates carry between 7-21 different plasmids, ranging in size from 5-84 kbp. All strains analyzed to date harbor three plasmids, cp26, lp54 and lp17. The plasmids are unusual, as compared to most bacterial plasmids, in that they contain many paralogous sequences, a large number of pseudogenes, and, in some cases, essential genes. In addition, a number of the plasmids have features indicating that they are prophages. Numerous methods have been developed for Lyme disease Borrelia strain typing. These have proven valuable for clinical and epidemiological studies, as well as phylogenomic and population genetic analyses. Increasingly, these approaches have been displaced by whole genome sequencing techniques. Some correlations between genome content and pathogenicity have been deduced, and comparative whole genome analyses promise future progress in this arena.

Transgenic expression of RecA of the spirochetes Borrelia burgdorferi and Borrelia hermsii in Escherichia coli revealed differences in DNA repair and recombination phenotypes.

After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B. burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli. As a control, E. coli RecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage lambda. Overall, we found that transgenic cells with recA genes of B. burgdorferi, B. hermsii, and L. biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B. burgdorferi recA and, most notably, B. hermsii recA were significantly less capable than cells with L. biflexa recA or E. coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E. coli.