DNA fingerprinting of Mycobacterium tuberculosis has been shown to be a powerful epidemiologic tool. We propose a standardized technique which exploits variability in both the number and genomic position of IS6110 to generate strain-specific patterns. General use of this technique will permit comparison of results between different laboratories. Such comparisons will facilitate investigations into the international transmission of tuberculosis and may identify specific strains with unique properties such as high infectivity, virulence, or drug resistance.

Five different genetic elements have been found to be associated with genetic rearrangements in Mycobacterium tuberculosis complex strains. Of these elements, the insertion sequence IS6110 is presently the most frequently used genetic marker for strain differentiation ofM. tuberculosis. In the present study we compared five genetic elements for their potentials to differentiate a given cluster ofM. tuberculosis strains. Because of the presence of only a single copy of IS6110 or two IS6110 copies at the same chromosomal locus, a large number of strains could not be differentiated by IS6110 fingerprinting. Most strains, including the low-copy-number IS6110 strains, could be differentiated by fingerprinting with the 36-bp direct repeat or the polymorphic GC-rich repetitive DNA element. Less discriminative power was obtained with the major polymorphic tandem repeat and the insertion element IS1081. One strain which did not contain IS6110 DNA was encountered. Until now, this element has invariantly been found to be present in all M. tuberculosis complex strains. On the basis of classical taxonomic criteria and sequencing of the 16S rRNA gene, this strain was shown to be a genuline M. tuberculosis strain. Therefore, the use of this element as a target for polymerase chain reaction-facilitated detection of M. tuberculosis should be reconsidered.

We report the DNA sequence of a previously cloned Mycobacterium bovis BCG gene encoding an immunogenic 64-kilodalton protein. This protein, MbaA, was purified from overproducing Escherichia coli K-12 cells, and the presence of antibodies to MbaA in human sera was determined by an enzyme-linked immunosorbent assay. In about 80% of serum samples from tuberculosis patients and in about 60% of samples from BCG-vaccinated individuals, significant levels of anti-MbaA antibodies were found. Surprisingly, in about 30% of the control serum samples obtained from children, anti-MbaA antibodies were also observed. Guinea pigs sensitized with M. bovis BCG or MbaA showed a delayed-type hypersensitivity reaction after challenge with purified MbaA, supporting the previously observed strong reactivity of human T-cell clones with this, for mycobacteria, common antigen.

A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start ftom at least two different treponemal promnoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coil, we concluded that a similar proteolytic processing takes place in both E. coli and T.paflidum6 Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent thlah tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front bf the structural genes. The nucleotide sequence of the T. pallidum tmpA geite was established. This is the first T. paUidim gene sequenced. Codon usage and the nature of transcriptional and translational signals ate discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a s;gnal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having I-galactosidase enzyme activity as well as TmpA epitopes. The enzymelinked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage A were used. In this case the protein produced was a sandwich protein consisting of 21 aminio acids of the A cro protein, 204 amnino acids of the T. pallidam TmpA protein, and 1,020 amino lcids of the E. coli X-galictosidase. The potedtial use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed. Treponema pallidum is the causative agent of syphilis. Because the microorganism cannot be grown in vitro, little is known about the factors that contribute to its virulence and the immunologic response duting a syphilitic infection. For this reason various groups have cloned genes of T. pallidum that encode for immunogenic proteins (17, 24, 27, 30). These recent studies have shown that T. pallidum DNA can be expressed well in Escherichia coli, and treponemal transcriptional and translational signals appear to be recognized by the E. coli machinery for protein synthesis. Recently, we screened a cosmid library with T. pallidum DNA inserts for the production of T. pallidum antigens (27). Among the 800 clones scteened, one particular phenotype was found most frequently. Of 16 antigen-producing clones,

A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning Saul-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones. These antigens reacted also with anti-T. pallidum rabbit serum. No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum. The antigens produced by the E. coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T. pallidum bacteria, suggesting that the treponemal DNA is well expressed in E. coli K-12. Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T. pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen. Monoclonal antibodies were raised against T. pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T. pallidum but also with the antigen produced by one of the E. coli clones.

Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfatepolyacrylamide gel electrophoresis results from different laboratoriesAs the first step in a cooperative effort to standardize the identification of the polypeptides of Treponerna pallidum subsp. pallidurn, the sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 ofthe major polypeptide bands, the cross-identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two-dimensional electrophoresis provided an improved means of separating and characterizing T.pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two-dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two-dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T.pal1idum proteins. Abbreviations: CIE, crossed immunoelectrophoresis; 2DE, two-dimensional gel electrophoresis; IRS, infected rabbit serum; 2-ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; RIP, radioimmunoprecipitation: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; T, Tween 20 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0173-0835/87/0202-0077 %02.50/0 S. J. Norris et al. Electrophoresis 1987,8, 77-92 Figure 16. Reactivity of rabbit antiserum directed against cloned antigenTmpC (1301, seeFig. 6)with the 2DEpatternof T.paNidum.Theprocedure wasas described in Fig. 15. (A) Reaction with the TmpC-specific antiserum. (B) Antigenic profile following IRS counterstaining. The antiserum reacted specifically with a single band on the SDS-PAGE pattern (far left) and a single spot at the acid end of the 2DE pattern.Figure 17. Localization of cloned antigen TpD (1301, see Fig. 6) in the 2DE profile of T.pallidum, utilizing a specific rabbit antiserum prepared by affinity chromatography. (A) Reactivity of TpD-specific antiserum. Due to prolonged incubation with the color reagent, background reactivities of the antiserum to TmpC and the major 61 polypeptide were also detectable in this case. (B) Antigenic profile following IRS counterstaining. The positions of TpD and TmpC in the 2D and single dimension SDS-PAGE profiles are indicated by the arrows.

The transferability of plasmid pRI405 between various streptococci of groups A, B, and D, Streptococcus pneumoniae, and Staphylococcus aureus is described. pRI405 originated from Streptococcus faecalis and encodes for resistance to macrolides, lincomycin, and streptogramin B (MLS resistance). The host range of the well-documented streptococcal plasmid pAM/i1 was found to be similar to that of pRI405. Cleavage with restriction enzymes suggests that pRI405 belongs to a related family of MLS resistance plasmids.It is now generally accepted that resistance to various antimicrobial drugs in streptococci is mediated by plasmids. Many of these plasmids have been shown to be self-transferable (8,12,13,16,20,23,32,34) by a process that needs cellto-cell contact between donor and recipient and which resembles in several aspects conjugation (16). Non-self-transferable plasmids can be mobilized by self-transferable ones. The first reports on conjugal transfer of streptococcal plasmids involved intraspecific matings between isolates of group D streptococci (8,16,25). Later, interspecific plasmid transfer between the serogroups B and D was also reported (11, 13, 34). The plasmids used in these studies originated from group B and group D streptococci, and they encoded for resistance to macrolide antibiotics, lincomycin, and streptogramins of group B, the so-called MLS resistance phenotype (37). The host range of one particular plasmid, pAM/i1 (originating from Streptococcus faecalis), has been the subject of several studies. Le Blanc et al. (20) introduced this plasmid into a group F streptococcus by transformation and then retransferred it by mating to the oral streptococci, S. mutans, S. sanguis, and S. salivarius. Furthermore, pAM,B1 could also be transferred to members of the family of Lactobacillaceae (10). Recently, Malke (23) reported the conjugal transferability of MLS resistance plasmids originating from group A and B streptococci between streptococcal strains of groups A, D, and H. Transformation of plasmids has been demonstrated for group H and group F streptococci (11,18,19), and transduction has been reported for group A, C, and G streptococci (15,22, 29).In a preliminary study we reported intergroup transfer of an MLS resistance plasmid between streptococci of groups A, B, and D (35). This plasmid, pRI405, originated from S. faecalis. In this study we extend the earlier observations on the host range of this plasmid and show that conjugal transfer is possible also to strains of Streptococcus pneumoniae and Staphylococcus aureus.MATERIALS AND METHODS Strains. All bacterial strains used in this study are listed in Table 1. Staphylococcal phage 80 (NCTC 9788) was used in transduction experiments.Media. Streptococci, Staphylococcus aureus, and Bacillus subtilis were grown in nutrient broth or on nutrient agar as previously described (34). The media for growth of S. pneumoniae were supplemented with inactivated horse serum to a final concentration of 5%. Haemophilus influenzae was grown in nutrient broth and on nutrie...

With the purpose of determining whether the risk of infection with a particular clone of Mycobacterium tuberculosis is influenced by the human immunodeficiency virus (HIV) status of the host, we analyzed and compared 68 mycobacterial isolates obtained from HIV-seropositive patients with tuberculosis (TB) in Dar esSalaam, Tanzania, with 66 mycobacterial isolates obtained from HIV-seronegative patients with TB in the same geographical region by using both DNA fingerprinting and classical phenotyping methods. One hundred one different IS6110 fingerprinting patterns were observed in the 134 isolates. The level of diversity of the DNA fingerprints observed in the HIV-seropositive group was comparable to the level of the diversity observed in the HIV-seronegative group. Resistance to a single anti-TB drug was found in 8.8% of the tested isolates, and 3.2% of the isolates were resistant to more than one anti-TB drug. The drug susceptibility profiles were not significantly difference between the two groups of isolates compared in the present study. Phenotypic characteristics which classify M. tuberculosis strains as belonging to the Asian subgroup correlated with a low IS6110 copy number per isolate. However, the occurrence of Asian subgroup strains was not associated with the HIV status of the patients. The results of the study suggested an equal risk of infection with a defined M. tuberculosis clone for HIV-seropositive and HIV-seronegative individuals.