Bacteria in the spirochete genus Borrelia
cause arthropod-borne human diseases such as Lyme disease and relapsing fever, as well as a number of diseases of veterinary importance 
. They are obligate parasites that are only found in their vertebrate or arthropod hosts and are rather difficult to study in the laboratory. Only quite recently have their biology, genetics and molecular pathogenesis begun to become accessible to experimentation 
. The determination and analysis of the first Borrelia
genome sequence, that of the Borrelia burgdorferi
type strain B31, stimulated significant progress in this arena. Its unusual genome was found to comprise a 910 Kbp linear chromosome and twenty-one (twelve linear and nine circular) plasmids that contain over 600 Kbp of DNA 
(two additional plasmids are now thought to have been lost between the isolation of strain B31 and its genome sequence determination 
). This work confirmed Barbour's 
original observations, and many other studies have shown that Borrelia
isolates universally harbor numerous linear and circular plasmids (e. g.
). The B31 chromosome carries 815 predicted genes (our re-annotation, below) that encode largely housekeeping functions. These functions include a minimal metabolic capability that cannot synthesize amino acids, nucleotides or lipids de novo
One circular plasmid, cp26, carries genes that encode several nucleotide metabolism enzymes 
, small molecule transporters 
and the enzyme that creates the unique closed hairpin telomeres present on the Borrelia
linear replicons 
. The other plasmids have very few metabolic or housekeeping genes, but do encode numerous lipoproteins, many of which have been shown to be present on the cell surface when they are expressed (e. g.
and references therein). The plasmids have a number of interesting features in addition to bearing lipoprotein genes. (i) A number of the linear plasmids have an unusually low protein coding density for prokaryotic DNA and carry numerous pseudogenes that appear to be in a state of genetic decay 
. (ii) Several of the circular plasmids in strain B31 (the cp32s) are homologous nearly throughout their lengths 
. (iii) There are unusually large numbers of paralogous genes on the plasmids. The vast majority of strain B31 plasmid genes have plasmid-borne paralogs, and in strain B31 107 of the paralogous gene families (PFams) include mostly plasmid genes. (vi) The highly paralogous nature of the plasmids, along with the apparent mutational decay of some members of PFams, suggests a history of duplicative rearrangements followed by decay of damaged and redundant genes 
. (v) Up to eleven of the B31 plasmids appear to be prophages or are prophage-related 
. (vi) Most of the plasmids, probably all but cp26, can be lost without affecting growth in culture (e. g.
). (vii) And finally, several plasmids have been shown to be required for growth in mice or in Ixodes
ticks, and/or encode proteins that interact with host components (details below). Thus, the plasmids appear to be largely involved in the interactions of Borrelia
with its hosts.
All members of the Borrelia
genus that have been analyzed carry linear chromosomes that are similar in size to the strain B31 chromosome. These chromosomes appear to be quite evolutionarily stable, since their sizes do not vary greatly and recent sequences of the chromosomes of additional Lyme agent B. burgdorferi
sensu stricto species 
and related species B. garinii
, B. afzelii
, B. “bavariensis”
, B. “finlandensis”
, B. valaisiana
, B. spielmanii
and B. bissettii
, show that they are all essentially co-linear with the chromosome of B. burgdorferi
B31, and that there are only a very small number of chromosomal gene content differences among these species (with the exception of B. burgdorferi
extreme right-end differences 
and the larger but still relatively modest differences between Lyme agent and relapsing fever Borrelia
). Directed analyses have shown that B. burgdorferi
plasmids cp26 
, lp54 
and the cp32s 
have largely conserved structures and are present in all isolates that have been studied. Other plasmids appear to have conserved structures but are only present in a subset of strains (e. g.
, B31-like cp9 
and lp38 
), while still others such as lp5, lp21, lp36, and lp56 are less frequently present and/or have highly variable sizes and presumably variable structures 
. The similar sizes of different plasmids (which are not separable in electrophoresis gels) and the highly paralogous nature of the plasmids has made unambiguous assembly and analysis of plasmid sequences complex and difficult 
. Thus, studies of bacteria in the Borrelia
genus are in an unenviable position in which determination of all the plasmids present in any new isolate requires that a complete (non-draft) genome sequence be determined.
Comparison of whole genome nucleotide sequences both within and between species is a powerful and critical part of gaining a true understanding of the organization, diversity and evolution of bacterial genomes. This strategy reveals the invariant features of the compared genomes and allows discovery of more variable sequences that (i) correlate with specific host disease features, (ii) permit tracking of sub-types within species, and (iii) give critical insight into evolutionary mechanisms. In addition, comparison of closely related genomes can often illuminate inaccuracies in the prediction of genes and other features in genomes. In this report we discuss the plasmids present in the B. burgdorferi
genomes of isolates N40, JD1 and 297 and compare their genetic contents and organizations with the previously known strain B31 genome. More global and less gene oriented comparisons of the twenty-two B. burgdorferi
sensu lato genomes that we have sequenced 
will be presented in subsequent publications.