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Borrelia burgdorferi is the causative agent of Lyme borreliosis, a spirochetal illness with a variety of acute clinical manifestations that may lead to debilitating neurological and arthritic complications. Diagnosis is difficult because symptoms mimic a variety of unrelated clinical conditions, spirochetes cannot always be isolated from infected patients, and current serological tests are frequently inconclusive because of the presence of cross-reacting non-B. burgdorferi antibodies. To identify antigens specific to B. burgdorferi that could be used in the serodiagnosis of Lyme borreliosis, we screened a Borrelia DNA expression library in Escherichia coli for antigens reactive with human Lyme borreliosis sera. One clone carried a 6.3-kilobase EcoRI chromosomal fragment (pSPR33), which encoded two species-specific antigens with molecular masses of 28 (P28) and 39 (P39) kilodaltons (kDa). These two antigens were immunologically distinct from OspA, OspB, and the 41-kDa flagellin. Ninety-four serum specimens from patients having Lyme borreliosis were tested for reactivity with P39. All of 33 the serum specimens with immunofluorescence assay titers of. 1:256, 13 of 17 serum specimens with titers of 1:128, and 14 of 44 serum specimens with titers of '1:64 reacted with P39. Notably, many sera reactive to P39 did not appear to react with the 41-kDa flagellin. Therefore, antibody to P39 could be mistaken for antibody to the 41-kDa flagellin in tests of human sera by Western blot (immunoblot). Twenty-five control serum specimens, which included sera from syphilitic, relapsing fever, and amyotrophic lateral sclerosis patients as well as from 10 normal individuals, did not react to P39. Our data suggest that P39 may be a useful antigen for the serological confirmation of Lyme borreliosis. Lyme borreliosis in humans is a multisystemic disorder caused by infection with the tick-borne spirochete, Borrelia burgdorferi (11, 25, 40). Since the first epidemiological investigations of this disease in south-central Connecticut (40-42), cases of Lyme borreliosis in humans have now been reported in 43 states of the United States (12), five provinces of Canada (13), numerous countries throughout Europe and Asia (1, 18, 32), and possible restricted foci in Australia (43) and Africa (20, 39). Between 1982 and 1988, reports of 13,825 cases of Lyme borreliosis were received by the Centers for Disease Control, Atlanta, Ga., from all 50 states of the United States (12), making this disease the most prevalent arthropod-borne infection in the country. With the dramatic increase in awareness, prevalence, and geographical distribution of Lyme borreliosis, a tremendous new demand has been placed on clinical laboratories to serologically confirm cases (36; L. A. Magnarelli, Editorial, J. Am. Med. Assoc. 262:3464-3465, 1989) or to rule out this disease in differential diagnoses. However, many potential problems exist with the currently available serological tests for Lyme borreliosis, which may result in either falsepositive or false-negative resul...

A partial nucleotide sequence that included 1,693 base pairs of the M12 (emml2) gene of group A streptococci (strain CS24) and adjacent upstream DNA was determined. Type 12 M protein-specific mRNA of strain CS24 is transcribed from two promoters (P, and P3) (35) it has been shown that the switch from high to low levels of M-protein expression occurs at high frequency and is reversible, which are characteristics of phase variation described for other bacterial species (7,26). In addition to phase variation of M-protein expression (35), streptococci also exhibit both antigenic variation (more than 70 strains with antigenically distinct M proteins have been described) and size variation of this protein (11). The mechanisms that control phase and antigenic variation and the relationship between them is not understood. Although the nature of M protein as an antiphagocytic molecule has been demonstrated, a role for the M-state in the survival of the organism has not been identified. Phase variation has been postulated to be a virulence factor that controls the expression of the type 1 pilus of Escherichia coli (6, 7) and the pilus of Neisseria gonorrhoeae (26 report results of a study of the role of upstream neighboring sequences in the regulation of expression of the emmi2 gene of strain CS24 in E. coli, describe the physical relationship between the deletion in strain CS64 and the 5' end of the emml2 gene, and study the transcriptional activity of the emml2 gene by Northern and primer extension analyses. We found that transcription of the emml2 gene in strain CS64 is diminished by more than 100-fold and that the deletion is more than 400 bases upstream from the transcription start sites of the emml2 gene. MATERIALS AND METHODSBacterial strains, plasmids, bacteriophage, and media. M type 12 group A streptococcal strain CS24 and variant CS64 have been described previously (3,39). Liquid cultures of group A streptococci were grown in Todd-Hewitt broth supplemented with 1% Neopeptone (Difco Laboratories, Detroit, Mich.) for 15 to 18 h at 37°C. E. coli JM83, JM103, and JM110, carrying constructs of plasmids pUC19 and pUC18, were propagated in L broth or on L agar containing ampicillin (30 ,ug/ml) and 5-bromo-4-chloro-3-indolyl-P-Dgalactoside (0.03%) at 37°C. All plasmids used and constructed in this study are listed in Fig. 1

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