Type-Specific Contributions to Chromosome Size Differences in Escherichia coli

The Escherichia coli genome varies in size from 4.5 to 5.5 Mb. It is unclear whether this variation may be distributed finely throughout the genome or is concentrated at just a few chromosomal loci or on plasmids. Further, the functional correlates of size variation in different genome copies are largely unexplored. We carried out comparative macrorestriction mapping using rare-restriction-site alleles (made with the Tn10dRCP2 family of elements, containing the NotI, BlnI, I-CeuI, and ultra-rare-cutting I-SceI sites) among the chromosomes of laboratory E. coli K-12, newborn-sepsis-associated E. coli RS218, and uropathogenic E. coli J96. These comparisons showed just a few large accessory chromosomal segments accounting for nearly all strain-to-strain size differences. Of 10 sepsis-associated and urovirulence genes, previously isolated from the two pathogens by scoring for function, all were colocalized exclusively with one or more of the accessory chromosomal segments. The accessory chromosomal segments detected in the pathogenic strains from physical, macrorestriction comparisons may be a source of new virulence genes, not yet isolated by function.     

Molecular cloning of a genetic determinant for Congo red binding ability which is essential for the virulence of Shigella flexneri.

A DNA sequence of about 1.0 kilobase (kb) derived from a 230-kb (140-megadalton) plasmid in a fully virulent Shigella flexneri 2a strain, YSH6000, was cloned into Escherichia coli K-12 by selecting for the ability to bind Congo red (Pcr+ phenotype). It was mapped and localized within the SalI restriction fragment F on the plasmid. This clone converted an S. flexneri strain which is avirulent as a result of a small deletion in the plasmid to full virulence but did not convert those without the 230-kb plasmid or with a plasmid bearing a more extensive deletion. This indicates that there are more than two virulence determinants on the plasmid. Thus, this sequence contains a genetic determinant which is essential but not sufficient for full virulence and expression of the Pcr+ phenotype in S. flexneri but is essential and sufficient for expression of the Pcr+ phenotype in E. coli K-12. We noted that there exist some other regions on the 230-kb plasmid which express the Pcr+ phenotype in E. coli K-12. Although these regions express the Pcr+ phenotype less markedly than the region cloned in the present study, they do hybridize with it.     

Identification of regions on a 230-kilobase plasmid from enteroinvasive Escherichia coli that are required for entry into HEp-2 cells.

Certain strains of Escherichia coli can cause an invasive diarrheal disease in humans which clinically resembles shigellosis. These strains share with Shigella species the ability to enter and replicate within colonic epithelial cells and the ability to bind Congo red dye in vitro when grown at 37 degrees C. Like shigellae, they contain a large plasmid essential for virulence. A 230-kilobase (kb) plasmid from enteroinvasive E. coli was genetically marked with a transposon and mobilized into an E. coli K-12 background. This plasmid conferred upon E. coli K-12 the ability to enter and multiply within cultured epithelial cells, as well as the ability to bind Congo red. Expression of these phenotypes required growth at 37 degrees C. Transposon mutagenesis was used to identify regions on the 230-kb plasmid required for virulence. All transposon insertions which resulted in loss of the ability to enter epithelial cells, as well as the ability to bind Congo red dye, were mapped to a single 25-kb BamHI fragment. Subclones from this 25-kb region were tested for the ability to complement invasion in noninvasive derivatives. A subclone containing about 8 kb of the left end of the 25-kb BamHI fragment was capable of complementing noninvasive mutants with Tn5 insertions in this region and restored to these noninvasive mutants the ability to enter epithelial cells.     

Molecular characterization of Salmonella enterica subsp. enterica serovar choleraesuis field isolates and differentiation from homologous live vaccine strains suisaloral and SC-54.

Four independent molecular methods were used to characterize the Salmonella enterica subsp. enterica serovar choleraesuis live vaccine strains SC-54 and Suisaloral and to differentiate them from S. choleraesuis field isolates. Plasmid analysis revealed the presence of seven plasmid profiles. A virulence plasmid of 52-kbp was identified by hybridization with an spvB-spvC gene probe in each of the S. choleraesuis field isolates and in the Suisaloral vaccine strain, but not in the SC-54 vaccine strain. Ribotyping, performed with a gene probe that recognized 23S, 16S, and 5S rRNA genes, resulted in three closely related hybridization patterns. IS200 elements were not detected in the field isolates or in the two S. choleraesuis live vaccine strains. Macrorestriction analysis with the enzymes XbaI, SpeI, NotI, and SfiI differentiated the 29 S. choleraesuis strains included in this study into 10, 13, 8, and 13 different fragment patterns, respectively. While the Suisaloral vaccine strain showed a unique XbaI macrorestriction pattern, the fragment patterns of the SC-54 strain obtained with the different enzymes were shared by 2 to 18 S. choleraesuis field strains. A combination of plasmid analysis and macrorestriction analysis proved to be most suitable for the molecular typing of S. choleraesuis and the differentiation of both live vaccine strains from field isolates of this serovar.     

Complete genome sequence of the marine fish pathogen Vibrio anguillarum harboring the pJM1 virulence plasmid and genomic comparison with other virulent strains of V. anguillarum and V. ordalii

We dissected the complete genome sequence of the O1 serotype strain Vibrio anguillarum 775(pJM1) and determined the draft genomic sequences of plasmidless strains of serotype O1 (strain 96F) and O2β (strain RV22) and V. ordalii. All strains harbor two chromosomes, but 775 also harbors the virulence plasmid pJM1, which carries the anguibactin-producing and cognate transport genes, one of the main virulence factors of V. anguillarum. Genomic analysis identified eight genomic islands in chromosome 1 of V. anguillarum 775(pJM1) and two in chromosome 2. Some of them carried potential virulence genes for the biosynthesis of O antigens, hemolysins, and exonucleases as well as others for sugar transport and metabolism. The majority of genes for essential cell functions and pathogenicity are located on chromosome 1. In contrast, chromosome 2 contains a larger fraction (59%) of hypothetical genes than does chromosome 1 (42%). Chromosome 2 also harbors a superintegron, as well as host "addiction" genes that are typically found on plasmids. Unique distinctive properties include homologues of type III secretion system genes in 96F, homologues of V. cholerae zot and ace toxin genes in RV22, and the biofilm formation syp genes in V. ordalii. Mobile genetic elements, some of them possibly originated in the pJM1 plasmid, were very abundant in 775, resulting in the silencing of specific genes, with only few insertions in the 96F and RV22 chromosomes.     

Acquisition of avian pathogenic Escherichia coli plasmids by a commensal E. coli isolate enhances its abilities to kill chicken embryos, grow in human urine, and colonize the murine kidney

We have found an avian pathogenic Escherichia coli (APEC) plasmid, pAPEC-O2-ColV, which contains many of the genes associated with APEC virulence and also shows similarity in content to a plasmid and pathogenicity island of human uropathogenic E. coli (UPEC). To test the possible role of this plasmid in virulence, it was transferred by conjugation along with a large R plasmid, pAPEC-O2-R, into a commensal avian E. coli strain. The transconjugant was compared to recipient strain NC, UPEC strain HE300, and donor strain APEC O2 using various assays, including lethality for chicken embryos, growth in human urine, and ability to cause urinary tract infection in mice. The transconjugant killed significantly more chicken embryos than did the recipient. In human urine, APEC O2 grew at a rate equivalent to that of UPEC strain HE300, and the transconjugant showed significantly increased growth compared to the recipient. The transconjugant also significantly outcompeted the recipient in colonization of the murine kidney. These findings suggest that APEC plasmids, such as pAPEC-O2-ColV, contribute to the pathogenesis of avian colibacillosis. Moreover, since avian E. coli and their plasmids may be transmitted to humans, evaluation of APEC plasmids as possible reservoirs of urovirulence genes for human UPEC may be warranted.     

Tn1725 transposon mutagenesis of 9-18 delta 7, an EcoRI deletion derivative of Salmonella dublin lane plasmid pSDL2.

A 37.5-kb derivative of the Salmonella dublin virulence plasmid pSDL2 was subjected to mutagenesis with the transposon Tn1725. Fifty-two insertions were mapped, and the mutants were tested for their ability to restore virulence to a plasmid-free strain of S. dublin. Twenty-nine of these inserts could not restore full virulence and thus define nine regions of the plasmid essential for virulence. Deletion of a 4.5-kb region by Bal31 nuclease resulted in a 33-kb derivative that maintained full virulence.     

Two pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning and sample sequencing.

Many of the virulence genes of pathogenic strains of Escherichia coli are carried in large multigene chromosomal segments called pathogenicity islands (PAIs) that are absent from normal fecal and laboratory K-12 strains of this bacterium. We are studying PAIs in order to better understand factors that govern virulence and to assess how such DNA segments are gained or lost during evolution. The isolation and sample sequencing of a set of 11 cosmid clones that cover all of one and much of a second large PAI in the uropathogenic E. coli J96 are described. These PAIs were mapped to the 64- and 94-min regions of the E. coli K-12 chromosome, which differ from the locations of three PAIs identified in other pathogenic E. coli strains. Analysis of the junction sequences with E. coli K-12-like DNAs showed that the insert at 94 min is within the 3' end of a phenylalanine tRNA gene, pheR, and is flanked by a 135-bp imperfect direct repeat. Analysis of the one junction recovered from the insert at 64 min indicated that it lies near another tRNA gene, pheV. To identify possible genes unique to these PAIs, 100 independent subclones of the cosmids were made by PstI digestion and ligation into a pBS+ plasmid and used in one-pass sample DNA sequencing from primer binding sites at the cloning site in the vector DNA. Database searches of the J96 PAI-specific sequences identified numerous instances in which the cloned DNAs shared significant sequence similarities to adhesins, toxins, and other virulence determinants of diverse pathogens. Several likely insertion sequence elements (IS100, IS630, and IS911) and conjugative R1 plasmid and P4 phage genes were also found. We propose that such mobile genetic elements may have facilitated the spread of virulence determinants within PAIs among bacteria.     

Simultaneous analysis of multiple enzymes increases accuracy of pulsed-field gel electrophoresis in assigning genetic relationships among homogeneous Salmonella strains

Due to a highly homogeneous genetic composition, the subtyping of Salmonella enterica serovar Enteritidis strains to an epidemiologically relevant level remains intangible for pulsed-field gel electrophoresis (PFGE). We reported previously on a highly discriminatory PFGE-based subtyping scheme for S. enterica serovar Enteritidis that relies on a single combined cluster analysis of multiple restriction enzymes. However, the ability of a subtyping method to correctly infer genetic relatedness among outbreak strains is also essential for effective molecular epidemiological traceback. In this study, genetic and phylogenetic analyses were performed to assess whether concatenated enzyme methods can cluster closely related salmonellae into epidemiologically relevant hierarchies. PFGE profiles were generated by use of six restriction enzymes (XbaI, BlnI, SpeI, SfiI, PacI, and NotI) for 74 strains each of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium. Correlation analysis of Dice similarity coefficients for all pairwise strain comparisons underscored the importance of combining multiple enzymes for the accurate assignment of genetic relatedness among Salmonella strains. The mean correlation increased from 81% and 41% for single-enzyme PFGE up to 99% and 96% for five-enzyme combined PFGE for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains, respectively. Data regressions approached 100% correlation among Dice similarities for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains when a minimum of six enzymes were concatenated. Phylogenetic congruence measures singled out XbaI, BlnI, SfiI, and PacI as most concordant for S. enterica serovar Enteritidis, while XbaI, BlnI, and SpeI were most concordant among S. enterica serovar Typhimurium strains. Together, these data indicate that PFGE coupled with sufficient enzyme numbers and combinations is capable of discerning accurate genetic relationships among Salmonella serovars comprising highly homogeneous strain complexes.     

Genetic stability of Borrelia burgdorferi recovered from chronically infected immunocompetent mice.

Persistent infection with Borrelia burgdorferi in the presence of a vigorous host immune response has been demonstrated in humans and in animal models of Lyme disease. Long-term persistence of B. burgdorferi was documented recently in our studies of BALB/c and C3H mice infected with cloned and uncloned strains of B. burgdorferi. From mice inoculated with the cloned strain, 11 isolates were recovered from the skin, bladder, and blood after 1 year of infection. Analysis of the genes encoding the major outer surface proteins (OspA and OspB) by restriction digestion and DNA sequencing showed no evidence of point mutations or other small genetic alterations after 1 year. Genomic macrorestriction analysis of whole B. burgdorferi showed no loss or gross alterations of the plasmids encoding OspA, OspB, or OspC. However, in two isolates, loss of a 38-kb plasmid encoding outer surface protein D was noted. Our studies suggest that loss or alteration of the genes encoding OspA and OspB is not a common occurrence during persistent spirochetal infection and that other possible mechanisms, including invasion of immunologically privileged sites, should be actively explored.     

Macrorestriction analysis of clinical and environmental isolates of Sporothrix schenckii

Sporothrix schenckii causes sporotrichosis, a disease that most commonly presents as a subacute or chronic skin infection. An unusually high incidence of clinical cases of sporotrichosis occurred in the southwest of Western Australia over the last 5 years. Anecdotal accounts from patients implicated contact with hay prior to infection. Isolates of S. schenckii from hay and clinical cases were investigated by traditional phenotypic methods and pulsed-field gel electrophoresis (PFGE). The phenotypic evaluation separated S. schenckii from Ophiostoma spp. A DNA macrorestriction method using SfiI and NotI macrorestriction digestion by PFGE was developed to investigate the epidemiological connections. BioNumerics software was used to analyze the results. DNA macrorestriction digestion patterns for the recent Western Australian clinical isolates and four hay isolates were indistinguishable. Eastern state clinical isolates, national Quality Assurance Program isolates, and other environmental isolates gave different macrorestriction patterns. Clinical isolates from the southwest of Western Australia collected in the 1980s and 1990s were also characterized using PFGE. The patterns generated were indistinguishable from those of the recent clinical isolates. PFGE showed that the dominant strain of S. schenckii causing sporotrichosis in Western Australia is present in hay, has caused sporotrichosis for at least 15 years, and is a different strain from the strains found in other parts of Australia.     

Characterization of Salmonella enterica serovar typhimurium DT104 isolated from Denmark and comparison with isolates from Europe and the United States

A total of 136 isolates of Salmonella enterica serovar Typhimurium DT104 from Denmark (n = 93), Germany (n = 10), Italy (n = 4), Spain (n = 5), and the United Kingdom (n = 9) were characterized by antimicrobial resistance analysis, plasmid profiling, pulsed-field gel electrophoresis (PFGE) with the restriction enzymes XbaI and BlnI, and analysis for the presence of integrons and antibiotic resistance genes. The isolates from Denmark were from nine pig herds, while the isolates from other countries were both of animal and of human origin. All but 10 isolates were resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfonamides, and tetracycline. Five isolates from the United Kingdom and Spain were sensitive to all antibiotics examined, whereas four isolates from the United Kingdom and the United States were also resistant to one or more of the antibiotics, namely, gentamicin, neomycin, and trimethoprim. All but two strains had the same PFGE profiles when the XbaI restriction enzyme was used, while seven different profiles were observed when the BlnI restriction enzyme was used. Different dominating BlnI types were observed among European isolates compared with the types observed among those from the United States. All the isolates harbored common 95-kb plasmids either alone or in combination with smaller plasmids, and a total of 11 different plasmid profiles were observed. Furthermore, all but one of the multidrug-resistant isolates contained two integrons, ant (3")-Ia and pse-1. Sensitive isolates contained no integrons, and isolates that were resistant to spectinomycin, streptomycin, and sulfonamides had only one integron containing ant (3")-Ia. When restriction enzyme BlnI was used, the 14 isolates from one of the nine herds in Denmark showed unique profiles, whereas isolates from the remaining herds were homogeneous. Among isolates from seven of nine herds, the same plasmid profile (95 kb) was observed, but isolates from two herds had different profiles. Thus, either PFGE (with BlnI) or plasmid profiling could distinguish isolates from three of nine pig herds in Denmark. The epidemiological markers (antimicrobial susceptibility testing, plasmid profiling, and PFGE) applied demonstrated high in vivo stability in the Danish herds. This may indicate that some different strains of multidrug-resistant S. enterica serovar Typhimurium DT104 have been introduced into Danish food animal herds. The presence of isolates from six different countries with similar profiles by PFGE with XbaI and highly homogeneous profiles by PFGE with BlnI indicate that multidrug-resistant S. enterica serovar Typhimurium DT104 has probably been spread clonally in these countries. However, some minor variation could be observed by using plasmid profiling and profiling by PFGE with BlnI. Thus, a more sensitive technique for subtyping of strains of DT104 and a broader investigation may help in elucidating the epidemiological spread of DT104 in different parts of the world.     

Genetic diversity of canine gastric helicobacters, Helicobacter bizzozeronii and H. salomonis studied by pulsed-field gel electrophoresis.

Genetic diversity of Helicobacter bizzozeronii and H. salomonis, two recently identified canine gastric Helicobacter spp., was studied by pulsed-field gel electrophoresis (PFGE). All 15 Finnish H. bizzozeronii strains collected between 1991 and 1996 from pet dogs produced different PFGE patterns with all restriction endonucleases studied (AscI, ApaI, SpeI, NotI and PacI) suggesting significant genetic diversity. The five independent H. salomonis strains produced four different patterns with these enzymes; two strains showed identical patterns with all the enzymes. Three separate isolates from one dog had identical patterns, suggesting long-lasting infection with the same strain. H. salomonis strains had several small fragments common for all strains, suggesting relatedness. The PFGE method was shown to be useful for epidemiological studies of canine gastric helicobacter infection. Hybridisation of the DNA digests with digoxigenin-labelled ureB or 16S rRNA gene probes generated by PCR indicated conservation in the localisation of these genes in the H. salomonis genome, because the probes hybridised with similar size fragments of different strains. In contrast, the probes hybridised with different size fragments of H. bizzozeronii strains. Comparison of Southern blots of PFGE patterns digested with SpeI, ApaI and AscI indicated that each species has two 16S rRNA genes and one urease gene. Genome sizes of 11 H. bizzozeronii strains estimated from SpeI and NotI patterns were c. 1.6-1.9 Mb and those of five H. salomonis strains estimated from NotI and PacI patterns were c. 1.7-1.8 Mb.     

Evaluation and comparison of molecular techniques for epidemiological typing of Salmonella enterica subsp. enterica serovar dublin.

A total of 28 unrelated isolates of the Salmonella enterica subsp. enterica serovar dublin (S. dublin) collected during a 6-year period, as well as four samples of the S. dublin live vaccine strain Bovisaloral and its prototype strain S. dublin 442/039, were investigated by different molecular typing methods for the following reasons: (i) to find the most discriminatory method for the epidemiological typing of isolates belonging to this Salmonella serovar and (ii) to evaluate these methods for their capacity to discriminate among the live vaccine strain Bovisaloral, its prototype strain S. dublin 442/039, and field isolates of the serovar dublin. Five different plasmid profiles were observed; a virulence plasmid of 76 kbp as identified by hybridization with an spvB-spvC gene probe was present in all isolates. The detection of 16S rRNA genes and that of IS200 elements proved to be unsuitable for the epidemiological typing of S. dublin; only one hybridization pattern could be observed with each of these methods. The results obtained from macrorestriction analysis strongly depended on the choice of restriction enzyme. While the enzyme NotI yielded the lowest discriminatory index among all enzymes tested, it was the only enzyme that allowed discrimination between the Bovisaloral vaccine strain and its prototype strain. In contrast to the enzymes XbaI and SpeI, which only differentiated among the S. dublin field isolates, XhoI as well as AvrII also produced restriction fragment patterns of the Bovisaloral strain and of its prototype strain that were not shared by any of the S. dublin field isolates. Macrorestriction analysis proved to be the most discriminatory method not only for the epidemiological typing of S. dublin field isolates but also for the identification of the S. dublin live vaccine strain Bovisaloral.     

Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine.

Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.     

V(D)J recombinase mediated inter-chromosomal HPRT alterations at cryptic recombination signal sequences in peripheral human T cells

The V(D)J recombinase enzyme complex is responsible for the development of a diverse immune system by catalyzing intra-molecular rearrangements of immunoglobulin (Ig) and T cell receptor (TCR) genes at specific recombination signal sequences (RSSs). This enzyme complex has also been implicated in mediating pathologic and non-pathologic intra- and inter-molecular genomic rearrangements at cryptic (Psi) RSSs outside the immune system loci in lymphoid cells. We describe here two V(D)J recombinase mediated genomic rearrangements resulting in alterations at the HPRT locus in human T-cells. These are inter-chromosomal insertions in which DNA fragments are inserted at breakpoints generated by V(D)J recombinase cleavage at Psi RSS sites in the HPRT locus at Xq26. In the first, a TCR signal ended segment from chromosome 14q11 is inserted at a Psi RSS in intron 1 of the HPRT locus. In the second, a DNA fragment from 9q22 is integrated between the coding ends generated by a V(D)J recombinase mediated HPRT deletion. Identification of these in vivo V(D)J mediated inter-chromosomal insertions at Psi RSSs in the HPRT gene supports the accumulating evidence that V(D)J recombinase can mediate mutagenic rearrangements in humans with potential pathologic consequences.     

Molecular and evolutionary characterization of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297

In this study, we characterized seven members of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297. Complete sequence analysis of a 21-kb plasmid (cp18-2) confirmed that the strain 297 plasmids are similar in overall content and organization to their B31 counterparts. Of the 31 open reading frames (ORFs) in cp18-2, only three showed sequence relatedness to proteins with known functions, and only one, a ParA/SopA ortholog, was related to nonborrelial polypeptides. Besides the lipoproteins, none of the ORFs appeared likely to encode a surface-exposed protein. Comparison with the B31 genomic sequence indicated that paralogs for most of the ORFs in cp18-2 can be identified on other genetic elements. cp18-2 was found to lack a 9- to 10-kb fragment present in the 32-kb homologs which, by extrapolation from the B31 cp32 sequences, contains at least 15 genes presumed to be unnecessary for plasmid maintenance. Sequence analysis of the lipoprotein-encoding variable loci provided evidence that recombinatorial processes within these regions may result in the acquisition of exogenous DNA. Pairwise analysis with random shuffling revealed that the multiple lipoproteins (Mlp; formerly designated 2.9 LPs) fall into two distinct homology groups which appear to have arisen by gene fusion events similar to those recently proposed to have generated the three OspE, OspF, and Elp lipoprotein families (D. R. Akins, M. J. Caimano, X. Yang, F. Cerna, M. V. Norgard, and J. D. Radolf, Infect. Immun. 67:1526-1532, 1999). Comparative analysis of the variable regions also indicated that recombination within the loci of each plasmid may occur independently. Last, comparison of variable loci revealed that the cp32/18 plasmid complements of the B31 and 297 isolates differ substantially, indicating that the two strains have been subject to divergent adaptive pressures. In addition to providing evidence for two different types of recombinatorial events involving cp32/18 plasmids, these findings underscore the need for genetic analysis of diverse borrelial isolates in order to elucidate the Lyme disease spirochete's complex parasitic strategies.