Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli.
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 from at least two different treponemal promoters. 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. coli, we concluded that a similar proteolytic processing takes place in both E. coli and T. pallidum. Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent than tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front of the structural genes. The nucleotide sequence of the T. pallidum tmpA gene was established. This is the first T. pallidum gene sequenced. Codon usage and the nature of transcriptional and translational signals are discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a signal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having beta-galactosidase enzyme activity as well as TmpA epitopes. The enzyme-linked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage lambda were used. In this case the protein produced was a sandwich protein consisting of 21 amino acids of the lambda cro protein, 204 amino acids of the T. pallidum TmpA protein, and 1,020 amino acids of the E. coli lambda-galactosidase. The potential use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed.
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000032:analysis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D009876:Operon; D013588:Syphilis Serodiagnosis; D014158:Transcription, Genetic; D014210:Treponema pallidum / Q000235:genetics*; D001616:beta-Galactosidase / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D003062:Codon; D008565:Membrane Proteins; D001616:beta-Galactosidase