Cloning of urease gene sequences from Providencia stuartii

Abstract

Providencia stuartii was the most prevalent isolate recovered from urine specimens taken weekly over a 1-year period from 51 nursing home patients with urinary catheters in place. Thirty percent of the isolates were urease positive. Urease, which is implicated in renal stone formation, was shown to be transmissible on an 82-kilobase conjugative plasmid in one isolate. Plasmid DNA isolated from this strain was digested with EcoRI, ligated into the EcoRI site of pBR322, and used to transform Escherichia coli HB101. Ampicillin-resistant clones were replica plated onto urea segregation agar, and a urease-positive clone, designated pMID101, was isolated. Recombinant and native urease from cell lysates had identical electrophoretic mobilities on nondenaturing polyacrylamide urease activity gels. The native enzyme was induced fourfold when cells were grown in the presence of 0.1% urea and had a K(m) of 9.4 mM and a V(max) of 3.2 μmol of NH3 per min per mg of protein. Its molecular weight was estimated to be 375,000 ± 35,000 by Sephacryl S-300 chromatography. The enzyme was cytoplasmic in P. stuartii, was inhibited in vitro by hydroxyurea, acetohydroxamic acid, and EDTA, and appears to have a complex subunit structure and a unique molecular size within genera of the Proteeae tribe.