Title

Characterization of UreR interaction with the urea- and UreR-dependent promoters of the urease gene clusters

Date of Award

1998

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Carleen M. Collins, Committee Chair

Abstract

Urease, is a primary virulence factor of organisms infecting the urinary tract and gastroduodenal region. Urease catalyzes the hydrolysis of urea to release carbon dioxide and two molecules of ammonia. Within Enterobacteriaceae , genes encoding for urease can be found either on the chromosome or on large plasmids. Urease can be either expressed constitutively, induced under nitrogen starvation, or induced by urea. The plasmid-encoded gene cluster found in organisms such as Providencia stuartii and the chromosomally-encoded gene cluster of Proteus mirabilis are regulated by urea. These loci share similar genetic organization with the genes ureDABCEFG encoding for the structural subunits and accessory proteins of the enzyme. The divergently transcribed ureR encodes for a regulatory protein. Three promoters (ureRp, Dp, and Gp) are active in the presence of urea and UreR. The divergently transcribed ureRp and ureDp are situated within a 414 bp and 494 bp ureRp-Dp intergenic region of the plasmid-encoded and P. mirabilis gene clusters respectively. The minimal promoter sequence requirement for full UreR- and urea-dependent activation of ureRp and ureDp was localized to the 147 bp separating the ureR and ureD transcription start sites in the plasmid-encoded gene cluster. P. mirabilis contains a similar region of 145 bps. -10 and -35 consensus RNA polymerase binding hexamers relative to the start of ureR transcription and a -10 consensus hexamer relative to the start of ureD transcription were identified.UreR is an AraC-like transcriptional activator essential for urea-induced activation of the urease gene cluster. UreR from the plasmid-encoded and the P. mirabilis urease gene clusters share 70% amino acid identity and are functionally interchangeable. To elucidate the role of UreR in the urea-induced activation of the urease gene cluster, a purified recombinant (rUreR) protein from the plasmid-encoded gene cluster was isolated and assessed for DNA binding capability in the presence and absence of urea. Electrophoretic mobility shift assays and DNase I footprint analysis reveal that rUreR can bind specifically to the ureRp and ureDp from the plasmid-encoded and P. mirabilis urease gene clusters. In contrast, ureGp was not bound by rUreR under similar reaction conditions. Promoter interaction of rUreR is independent of urea. However, addition of urea increased the DNA interaction by 2-fold. Comparison of the DNA binding sites did not reveal a clear consensus sequence. A 13 bp sequence of TPuPyNNPyTNNATTG was identified -46 to -34 relative to the start of ureR transcription and -49 to -37 relative to the start of ureD transcription. Dissociation rate constants, which could only be determined in the presence of urea, revealed that the ureDp site has a higher affinity for rUreR. ureRp demonstrated preferential binding to RNA polymerase in vitro. In addition, specific transcription from the ureD promoter was observed only in the presence of rUreR and urea. These data demonstrate that UreR mediates transcription by direct interaction with the ureD and ureR promoters. Urea might enhance the UreR-dependent regulation of the urease gene cluster by stabilizing the UreR-DNA interaction.

Keywords

Biology, Molecular; Biology, Microbiology

Link to Full Text

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