Regulation of Gene Expression and Cellular Localization of Cloned Klebsiella aerogenes (K. pneumoniae) Urease

Mulrooney, Scott B.; Pankratz, H. Stuart; Hausinger, Robert P.

doi:10.1099/00221287-135-6-1769

Cited by 51 publications

(54 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The relative activities of cultures on medium supplemented with L-arginine, NH4Cl, L-glutamine, urea, and L-glutamate were 100, 46, 36, 20, and 2.7%, respectively. The finding of optimal activity for cultures grown on L-arginine as the source of nitrogen appears to be consistent with urease expression in Kiebsiella aerogenes (9,28). Urease activity was not detected in cultures grown on nitrogen-rich medium.…”

Section: Materuils and Methodssupporting

confidence: 62%

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions

1992

View full text Add to dashboard Cite

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previousl shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 + 0.39 ,umol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E.coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreHl), and 19,819 (Urel). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coil, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the KiebsieUla aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes),. whereas this homology was restricted to a

show abstract

Section: Materuils and Methodssupporting

confidence: 62%

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions

1992

View full text Add to dashboard Cite

show abstract

“…These cultures were grown at 37°C in TB medium containing 100 g⅐ml Ϫ1 ampicillin (Amersham Pharmacia Biotech) to A 600 ϳ1, induced with 0.5 mM IPTG (Amersham Pharmacia Biotech), and harvested after 4 -6 h. H144*UreE and H144*UreE variant proteins were purified according to previously published procedures (23). E. coli DH5␣ cells containing pKAU17 (26) were used for production of urease apoprotein. These cultures were grown at 37°C overnight in LB medium supplemented with 100 g⅐ml Ϫ1 ampicillin, and the desired protein was purified by previously published procedures (27).…”

Section: Methodsmentioning

confidence: 99%

In Vivo and in Vitro Kinetics of Metal Transfer by the Klebsiella aerogenes Urease Nickel Metallochaperone, UreE

Colpas

Hausinger

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The genes for K. aerogenes urease were recently cloned and overexpressed such that urease accounted for over 10% of the cellular protein (20). Although the enzyme requires Ni for activity, no Ni-dependent regulation of gene expression was observed.…”

mentioning

confidence: 99%

Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme

1990

View full text Add to dashboard Cite

Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it possessed the same heteropolymeric structure as native enzyme, demonstrating that Ni is not required for intersubunit association. Ni did, however, substantially increase the stability of the intact metalloprotein (Tm = 79°C) compared with apoenzyme (Tm = 62°C), as revealed by differential scanning calorimetric analysis. An increased number of histidine residues were accessible to diethyl pyrocarbonate in apourease compared with holoenzyme, consistent with possible Ni ligation by histidinyl residues. Addition of Ni to purified apourease did not yield active enzyme; however, urease apoenzyme was very slowly activated in vivo by addition of Ni ions to Ni-free ceil cultures, even after treatment of the cells with spectinomycin to inhibit protein synthesis. In contrast, sonicated cells and cells treated with dinitrophenol or dicyclohexylcarbodiimide were incapable of activating apourease. These results indicate that apourease activation is an energy-dependent process that is destroyed by cell disruption.Urease, a nickel-containing enzyme found in many plants and microorganisms, hydrolyzes urea to yield carbonic acid and ammonia (2,18). Whereas the role of urease in plants is poorly understood (28), the microbial enzyme plays important roles in human and animal pathogenic states, in ruminant metabolism, and in environmental transformations of certain nitrogenous compounds (18). The best-characterized plant urease is that isolated from jack bean; it was the first enzyme ever crystallized (23) and the first enzyme shown to possess nickel (6). This hexameric protein contains two Ni ions per subunit (Mr = 90,770), the sequence of which was recently reported (24). The best-characterized bacterial urease is that from Klebsiella aerogenes (currently K. pneumoniae), which possesses three subunits [Mrs = 72,000 [a], 11,000 [1], and 9,000 [ry]) in an a2f34Y4 stoichiometry (25).The native enzyme contains two catalytic sites, each of which is associated with two Ni ions (26). The genes for K. aerogenes urease were recently cloned and overexpressed such that urease accounted for over 10% of the cellular protein (20). Although the enzyme requires Ni for activity, no Ni-dependent regulation of gene expression was observed.This report describes the isolation, characterization, and in vivo reconstitution of apourease from K. aerogenes. Experiments were designed to ascertain (i) whether Ni is required for intersubunit association, (ii) whether metal ions other than Ni are incorporated into urease during growth in Ni-free medium, (iii) how the presence of Ni affects the enzyme thermal stability and histidine reactivity, and (iv) the requirements for Ni to be incorporated into preformed apoenzyme. MATERIALS AND METHODSBacterial strains and growth conditions. K. aerogenes CG253 was transformed with plasmid pKAU19 (20), which possesses the urease genes from this microorgani...

show abstract

Regulation of Gene Expression and Cellular Localization of Cloned Klebsiella aerogenes (K. pneumoniae) Urease

Cited by 51 publications

References 31 publications

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions

In Vivo and in Vitro Kinetics of Metal Transfer by the Klebsiella aerogenes Urease Nickel Metallochaperone, UreE

Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme

Contact Info

Product

Resources

About