Penicillin-binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum.
Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically radiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125I-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 microM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.
Radolf
J D
JD
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
Moomaw
C
C
Slaughter
C A
CA
Norgard
M V
MV
D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006602:Hexosyltransferases; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000302:isolation & purification* / Q000378:metabolism; D017830:Octoxynol; D010400:Penicillin G / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan / Q000302:isolation & purification* / Q000648:ultrastructure; D010458:Peptidyl Transferases; D011092:Polyethylene Glycols; D011817:Rabbits; D014210:Treponema pallidum / Q000032:analysis* / Q000648:ultrastructure
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G