Proteus mirabilis urease: use of a ureA-lacZ fusion demonstrates that induction is highly specific for urea.
Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated urinary tract infection, is the most frequent cause of infection-induced bladder and kidney stones. Urease-catalyzed urea hydrolysis initiates stone formation in urine and can be inhibited by acetohydroxamic acid and other structural analogs of urea. Since P. mirabilis urease is inducible with urea, there has been some concern that urease inhibitors actually induce urease during an active infection, thus compounding the problem of elevated enzyme activity. Quantitating induction by compounds that simultaneously inhibit urease activity has been difficult. Therefore, to study these problems, we constructed a fusion of ureA (a urease subunit gene) and lacZ (the beta-galactosidase gene) within plasmid pMID1010, which encodes an inducible urease of P. mirabilis expressed in E. coli JM103 (Lac-). The fusion protein, predicted to be 117 kDa, was induced by urea and detected on Western blots (immunoblots) with anti-beta-galactosidase antiserum. Peak beta-galactosidase activity of 9.9 mumol of ONPG (o-nitrophenyl-beta-D-galactopyranoside) hydrolyzed per min per mg of protein, quantitated spectrophotometrically, was induced at 200 mM urea. The uninduced rate was 0.2 mumol of ONPG hydrolyzed per min per mg of protein. Induction was specific for urea, as no structural analog of urea (including acetohydroxamic acid, hydroxyurea, thiourea, hippuric acid, flurofamide, or hydroxylamine) induced fusion protein activity. These data suggest that induction by inactivation of UreR, the urease repressor protein that governs regulation of the urease operon, is specific for urea and does not respond to closely related structural analogs.
Nicholson
E B
EB
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.
Concaugh
E A
EA
Mobley
H L
HL
D003001:Cloning, Molecular; D004790:Enzyme Induction / Q000187:drug effects; D007763:Lac Operon; D011513:Proteus mirabilis / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013379:Substrate Specificity; D014508:Urea / Q000494:pharmacology*; D014510:Urease / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D014508:Urea; D014510:Urease