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Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae.

ABSTRACT: Klebsiella pneumoniae can use urea as the sole source of nitrogen, thanks to a urease encoded by the ureDABCEFG operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen. Much of this nitrogen regulation of urease formation depended on the nitrogen regulatory system acting through the nitrogen assimilation control protein, NAC. In a strain deleted for the nac gene, nitrogen limitation resulted in only a 7-fold increase in the specific activity of urease, in contrast to the 70-fold increase seen in that of the wild type. The ure operon was transcribed from two promoters. The proximal promoter (P1) had an absolute requirement for NAC; little or no transcription was seen in the absence of NAC. The distal promoter (P2) was independent of NAC, but its activity increased about threefold when the growth rate of the cells was limited by the nitrogen source. Transcriptional regulation of P1 and P2 accounted for most of the changes in urease activity seen under various nitrogen conditions. However, when transcription of ureDABCEFG was less than 20% of its maximum, the amount of active urease formed per transcript of ure decreased almost linearly with decreasing transcription. This may reflect a defect in the assembly of active urease and accounted for as much as a threefold activity difference under the conditions tested here. Thus, the ure operon was transcribed from a NAC-independent promoter (P2) and the most strongly NAC-dependent promoter known (P1). Most of the regulation of urease formation was transcriptional, but when ure transcription was low, assembly of active urease also was defective.

SUBMITTER: Liu Q

PROVIDER: S-EPMC2168754 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae.

Liu Qiong Q Bender Robert A RA

Journal of bacteriology 20070824 21

Klebsiella pneumoniae can use urea as the sole source of nitrogen, thanks to a urease encoded by the ureDABCEFG operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen. Much of this nitrogen regulation of urease formation depended on the nitrogen regulatory system ...[more]

PMID: 17720785

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Project description:Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreB?1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreB?1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.

Project description:BACKGROUND:Klebsiella pneumoniae (KP) is an opportunistic pathogen that mainly causes respiratory and urinary tract infections. The frequent occurrence of simultaneously virulent and multiple drug-resistant isolates led WHO to include this species in the list of top priorities for research and development of therapeutic alternatives. The comprehensive knowledge of the molecular mechanisms underlying KP virulence may lead to the proposal of more efficient and specific drugs. One of its virulence factors is the Type VI Secretion System (T6SS), which contributes to bacterial competition, cell invasion and in vivo colonisation. Despite the few studies showing the involvement of T6SS in KP pathogenesis, little is known concerning the regulation of its expression. The understanding of regulatory mechanisms may give more clues about the function of the system and the possibilities of future interference in this process. This work aimed to standardise the annotation of T6SS genes in KP strains and identify mechanisms of their transcriptional regulation through computational predictions. RESULTS:We analyzed the genomes of Kp52.145, HS11286 and NTUH-K2044 strains to perform a broad prediction and re-annotation of T6SS genes through similarity searches, comparative and linear discriminant analysis. 38 genes were found in Kp52.145, while 29 in HS11286 and 30 in NTUH-K2044. Genes coding for iron uptake systems are encoded in adjacencies of T6SS, suggesting that KP T6SS might also play a role in ion import. Some of the T6SS genes are comprised in syntenic regions. 17 sigma 70-dependent promoter regions were identified in Kp52.145, 12 in HS11286 and 12 in NTUH-K2044. Using VirtualFootprint algorithm, binding sites for 13 transcriptional regulators were found in Kp52.145 and 9 in HS11286 and 17 in NTUH-K2044. Six of them are common to the 3 strains: OxyR, H-NS, RcsAB, GcvA, Fis, and OmpR. CONCLUSIONS:The data presented herein are derived from computational analysis. Although future experimental studies are required to confirm those predictions, they suggest that KP T6SS might be regulated in response to environmental signals that are indeed sensed by the bacteria inside the human host: temperature (H-NS), nutrition-limitation (GcvA and Fis), oxidative stress (OxyR) and osmolarity (RscAB and OmpR).

Dataset Information

Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae.

Publications

Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae.

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