The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non‐functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes ≥300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic ailment of humans that is spread through the bite of certain species of Ixodes ticks (13, 61). As a spirochete, B. burgdorferi is a member of a morphologically and phylogenetically distinct order of eubacteria (11,28,(42)(43)(44)70). Although classically defined as gram-negative organisms (28), recent phylogenetic studies based on rRNA sequences indicated that the spirochetes are as distantly related to the gramnegative Escherichia coli as they are to gram-positive bacteria (42,70).One of the most striking differences between B. burgdorferi and other bacteria is its unusual genome, which includes a small (approximately 1 Mb) linear chromosome (12,16,19,21,23,41) and numerous linear and circular plasmids, sometimes approaching 20 different plasmids in one bacterium (7,8,10,22,27,30,35,57,65,71). A curious feature of these different plasmids is that they often appear to share regions of homologous DNA (58,60,65,71,73,74). Homologs of DNA sequences mapped to circular plasmids have even been found on linear plasmids (74), although in a related bacterium, Borrelia hermsii, at least one plasmid exists in both linear and circular forms (24), blurring the distinction between these two forms of DNA. Several of the plasmids that these bacteria harbor appear to be present in all natural isolates; therefore, the term minichromosome may be a more apt description of their nature. For example, a 49-to 54-kb linear plasmid and a 26-kb circular plasmid, which carry the outer surface protein genes ospAB and ospC, respectively, are ubiquitous (7,34,37,52,54,63,71). It is not known whether members of either of these two plasmid families have similar overall gene orders in different bacterial isolates, but their nearly invariant sizes support the idea that they...

SummaryBacteria of the spirochaete genus Borrelia have linear chromosomes about 950 kbp in size. We report here that these linear chromosomes have covalently closed hairpin structures at their termini that are similar but not identical to those reported for linear plasmids carried by these organisms. Nucleotide sequence analysis of the chromosomal telomeric regions indicates that unique, apparently functional genes lie within a few hundred bp of each of the telomeres, and that there is an imperfect 26 bp inverted repeat at the two telomeres. In addition, we characterize a major chromosomal length polymorphism within the right telomeric regions of various Borrelia isolates, and show that sequences similar to those near the right telomere are often found on linear plasmids in B. burgdorferi (sensu stricto) isolates from nature. Sequences similar to a number of other regions of the chromosome, including those near the left telomere, were not found on B. burgdorferi plasmids. These observations suggest that there has been historical exchange of genetic information between the linear plasmids and the right end of the linear chromosome.

We have mapped the genes encoding the antigenic lipoproteins OspE and OspF to an approximately 18-kb circular plasmid in Borrelia burgdorferi N40. Sequencing and restriction mapping have revealed that this plasmid, cp18, is homologous to an 18-kb region of the cp32 circular plasmids found in the Lyme disease spirochetes. Our data show that cp18 may have arisen from an ancestral cp32 plasmid by deletion of a 14-kb region of DNA, indicating that a significant portion of the cp32 plasmid is not essential in cis for plasmid maintenance. These findings suggest that a relatively small recombinant plasmid capable of being stably maintained in B. burgdorferi could be constructed from a cp32 plasmid.The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle between mammals and certain Ixodes ticks (10, 40). B. burgdorferi isolates cultured from host animals generally carry large numbers of both circular and linear plasmids (4,5,11,12,15,18,21,26,27,30,32,43,47), and the functions of these plasmids and their gene products are major foci of studies of the biology of B. burgdorferi and the pathology of Lyme disease. A number of antigenic surface proteins are encoded on the extrachromosomal elements (2,6,11,12,19,21,22,26,30,43,45), and expression of at least some of these antigens appears to be specific to particular stages of the mammal-tick transmission-infection cycle (2,8,12,14,24,35,42,44,45). Long-term cultivation of B. burgdorferi in laboratory media is frequently accompanied by a loss of both extrachromosomal DNA and ability of the bacteria to infect mammals (4,34,36,48), suggesting that there may be essential virulence factors encoded by these plasmids. Plasmid loss has also been associated with altered synthesis of bacterial proteins (30). All isolates of B. burgdorferi and nearly all isolates of related borreliae have been found to contain at least one member of the cp32 circular plasmid family (11,37,43), suggesting that these plasmids may play important roles in the life cycle of B. burgdorferi.Within the first few weeks of infection, mammals produce antibodies that recognize a small number of B. burgdorferi antigens (1,13,16,42,46). Lam and colleagues (19) cloned a gene from B. burgdorferi N40 that encodes a membrane-bound lipoprotein, OspE, which is recognized by sera from infected humans and laboratory animals (19,25,42). The ospE gene is followed by the ospF gene, which encodes another lipoprotein (19), and these two genes may form an operon. The initial characterization of the ospEF locus reported it to be located on a 45-kb linear plasmid (19). B. burgdorferi B31 contains seven different loci, designated erp, that are related to the B. burgdorferi N40 ospEF locus (11,43). Each erp locus is located on a separate, related 32-kb circular plasmid, called cp32-1 through cp32-7, all of which can apparently be maintained within a single B. burgdorferi bacterium (11, 43). B. burgdorferi N40 also contains several erp-ospEF-related genes, including ...

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An association between (unculturable) gastrospirillum-like organisms (GLO) and ulcerative lesions in the pars oesophagea in stomachs of swine has been claimed. In dogs GLO detected by microscopy may represent several Helicobacter species or subspecies. Therefore we investigated which Helicobacter spp. are present in stomachs of swine and their possible association with ulcerative lesions of the pars oesophagea. The presence ofHelicobacter spp. in the antrum and pars oesophagea in 122 stomachs of slaughter swine was determined by microscopy (n = 122), by culture on selective and nonselective media (n = 112), and by a genus-specific 16S ribosomal DNA (rDNA) PCR (n = 80). GLO could not be cultured. Phylogenetic analysis of 43 16S rDNA fragments (out of 54 PCR-positive biopsy specimens) revealed the presence of Helicobacter heilmannii type 1 in 42 of them. This correlated with the presence of bacteria with GLO morphology. Helicobacter bilis 16S rDNA was amplified directly from one sample harboring bacteria with H. bilis morphology. The association betweenHelicobacter spp. and gastric lesions was investigated with a second group of 41 pigs with (n = 21 cases) or without (n = 20 controls) gastric lesions. Fifteen of the 21 cases were positive by PCR or microscopy, compared to 7 of 20 of the controls (P = 0.03). 16S rDNA sequence analysis of 7 of 14 PCR-positive cases revealed the presence of H. heilmannii type 1. Microscopy showed bacteria with GLO morphology. One sample (cases) was culture negative but PCR positive for Helicobacter pullorum-related 16S rDNA. In conclusion, our findings indicate that H. heilmannii type 1 is the predominant Helicobacter spp. in the stomachs of pigs and that its presence is associated with ulcerative lesions in the pars oesophagea.

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