Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.
Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids.
Gherardini
F C
FC
Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599-7290.
Hobbs
M M
MM
Stamm
L V
LV
Bassford
P J
PJ
D015139:Blotting, Southern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D011392:Proline / Q000096:biosynthesis* / Q000235:genetics; D011762:Pyrroline Carboxylate Reductases / Q000032:analysis / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis; D011995:Recombination, Genetic; D014210:Treponema pallidum / Q000235:genetics*
D011994:Recombinant Proteins; D011392:Proline; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011762:Pyrroline Carboxylate Reductases