Transcriptional regulation of Proteus mirabilis urease by UreR

Abstract

Proteus mirabilis is the most frequent cause of infection-induced bladder and kidney stones. P. mirabilis urease catalyzes the hydrolysis of urea thereby raising the pH of urine and initiating formation of urinary stones. The enzyme is critical for kidney colonization and the development of acute pyelonephritis. Urease is induced by urea and is not controlled by the nitrogen regulatory system (ntr) or catabolite repression. Genes necessary for urease activity, ureD,A,B,C,E,F, have previously been cloned and sequenced (Jones, B. D. and H. L. T. Mobley. 1989. J. Bacteriol. 171:6414-6422). Purified whole cell RNA from induced and uninduced cultures of P. mirabilis and Escherichia coli harboring cloned urease sequences probed with a 4.2 kb Bg/I fragment from within the urease operon revealed a 4.2-fold and 5.8-fold increase in urease-specific mRNA upon induction with urea. Northern blot analysis of whole-cell RNA from wild-type P. mirabilis HI4320, probed with the same 4.2 kb DNA segment revealed significantly higher amounts of mRNA transcripts of 2.8 and 3.6 kb when induced with urea which correspond to the predicted lengths of mRNA encoding transcripts of ureABCE and ureDABCE, respectively. However, the presence of putative promoters preceding both ureD and ureA makes this interpretation difficult. A 1.2 kb EcoRV-BamHI restriction fragment upstream of the urease gene sequences confers inducibility upon the operon in trans. Nucleotide sequencing of this fragment revealed a single open reading frame 400 bp upstream of ureD consisting of 882 nucleotides, designated ureR, which is transcribed in the opposite direction of the urease structural and accessory genes, and encodes a 293 amino acid polypeptide predicted to be 33,415 Da. Autoradiographs of SDS-polyacrylamide gels of ({dollar}\sp{lcub}35{rcub}{dollar}S) -methionine-labeled polypeptides obtained by in vitro transcription-translation of the PCR fragments carrying only ureR yielded a single band of 32 kDa apparent molecular size; fragments carrying an in-frame deletion within ureR synthesized a truncated product. The predicted UreR amino acid sequence shares amino acid similarity to a number of DNA binding proteins including E. coli regulatory proteins for acid phosphatase synthesis (AppY), porin synthesis (EnvY), and rhamnose utilization (RhaR). Subsequent analysis of this sequence revealed a potential helix-turn-helix motif and an AraC family signature (M. D. Island and H. L. T. Mobley, personal communication). These data suggest that UreR governs inducibility of P. mirabilis urease. To study the specificity of induction of urease, the ability of urea analogs and urease inhibitors to induce the enzyme was studied by constructing a fusion of ureA (urease subunit gene) and lacZ ({dollar}\beta{dollar}-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 with anti-{dollar}\beta{dollar}-galactosidase antiserum. Induction was specific for urea as no structural analog of urea induced fusion protein activity. These data suggest that induction by UreR, the protein that governs regulation of the urease operon, is exquisitely specific for urea and does not respond to closely related structural analogs.