Cloning, sequencing, and expression of two class B endoflagellar genes of Treponema pallidum subsp. pallidum encoding the 34.5- and 31.0-kilodalton proteins.
Two structural endoflagellar genes of Treponema pallidum that encode the 34.5- and 31.0-kilodalton (kDa) polypeptides as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were cloned, sequenced, and expressed. We designated these genes flaB1 and flaB3. A DNA sequence analysis of flaB1 and flaB3 showed that each gene possesses a single open reading frame that encodes a polypeptide; these polypeptides have molecular masses of 31.1 and 31.0 kDa, respectively. Shine-Dalgarno ribosome-binding sequences were identified upstream from the initiation codons of each gene. In addition, a single consensus promoter sequence was identified 121 base pairs upstream from the initiation codon of flaB1, suggesting polycistronic transcription of flaB1 and flaB3. Computer-induced alignment showed that the FlaB1 amino acid sequence was identical at 206 positions (72%) to the FlaB3 sequence. Both genes were subcloned into pATH vectors and were expressed under the control of the trpE promoter. The expression products of flaB1 and flaB3 revealed fusion proteins having molecular masses of 61.0 and 59.0 kDa, respectively, which were identified on immunoblots by using specific anti-T. pallidum endoflagellar serum.
Champion
C I
CI
Department of Microbiology and Immunology, University of California, Los Angeles 90024.
Miller
J N
JN
Lovett
M A
MA
Blanco
D R
DR
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D005407:Flagella; D005408:Flagellin; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D015183:Restriction Mapping; D014210:Treponema pallidum / Q000235:genetics* / Q000648:ultrastructure
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C064192:flaB1 protein, bacteria; C064193:flaB3 protein, Treponema pallidum; D005408:Flagellin