Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum.
We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.
Isaacs
R D
RD
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
Hanke
J H
JH
Guzman-Verduzco
L M
LM
Newport
G
G
Agabian
N
N
Norgard
M V
MV
Lukehart
S A
SA
Radolf
J D
JD
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D005407:Flagella / Q000276:immunology; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012995:Solubility; D014210:Treponema pallidum / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C061551:endoflagellar sheath protein, Treponema pallidum