Treponema pallidum 3-phosphoglycerate mutase is a heat-labile enzyme that may limit the maximum growth temperature for the spirochete.
In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn(2+) while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25 degrees C, retained only 50% activity after incubation for 20 min at 34 degrees C or 10 min at 37 degrees C, and was completely inactive after 10 min at 42 degrees C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42 degrees C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.
Benoit
S
S
Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA.
Posey
J E
JE
Chenoweth
M R
MR
Gherardini
F C
FC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000201:enzymology; D009876:Operon; D010736:Phosphoglycerate Mutase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013696:Temperature; D014210:Treponema pallidum / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D003470:Culture Media; D011994:Recombinant Proteins; D008345:Manganese; D000255:Adenosine Triphosphate; D010736:Phosphoglycerate Mutase; D005947:Glucose