Title

Transcriptional regulation of the plasmid-encoded urease genes found in members of the family Enterobacteriaceae

Date of Award

1994

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Carleen M. Collins, Committee Chair

Abstract

Urease, a nickel metalloenzyme, catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Urease production contributes to the virulence of uropathogenic bacteria by generating an alkaline microenvironment that is advantageous for growth and persistence. In Providencia stuartii, and in some Escherichia coli and Salmonella species, urease is encoded on large plasmids. Hybridization experiments showed that the urease genes on these plasmids were highly similar, thus we designated this locus the plasmid-encoded urease gene cluster. Like Proteus species, Enterobacteriaceae that carry this locus produce urease activity only in the presence of urea. Eight urease genes were shown to be required for production of a functional enzyme. Seven of these genes, located immediately adjacent to one another (ureDABCEFG), encode urease structural subunits and accessory polypeptides required for the incorporation of nickel ions. The eighth gene, ureR, is transcribed divergently from ureDABCEFC and is located 414 bp upstream of ureD. ureR codes for a 34 kDa protein with similarity to the AraC family of transcriptional activators.Complementation analyses of polar mutations in ureD and ureE suggested that three operons exist within the plasmid-encoded ureDABCEFG gene cluster: ureDABC, ureEF, and ureG. Transcriptional fusion constructs were used to identify urea-inducible promoters upstream of ureR (ureRp), ureD (ureDp), and ureG (ureGp). Transcription from these promoters, which required the ureR gene product, was increased at least 20-fold during growth with urea. A weak constitutive promoter was found upstream of ureE (ureEp). Northern blot hybridizations confirmed the presence of ureR, ureDABC, ureEF, and ureG-specific mRNA transcripts. Urea-induced transcripts that appeared to initiate at ureDp were also detected with a ureEF-specific probe, suggesting that transcriptional read through can occur from ureDp to ureF. Thus, increased expression of all eight plasmid-encoded urease genes was observed after growth in urea.Three urea-inducible promoters (ureRp, ureDp, and ureGp) were also identified in the chromosomally-encoded Proteus mirabilis urease locus. The plasmid-encoded UreR activated transcription from the P. mirabilis promoters, and the P. mirabilis UreR was able to activate the plasmid-encoded promoters, suggesting that these two urea-inducible urease loci have similar mechanisms of gene expression.

Keywords

Biology, Molecular; Biology, Microbiology

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9519727