Dataset Information

A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene.

ABSTRACT: The structural gene for arylsulfatase (atsA) of Klebsiella aerogenes was cloned into a pKI212 vector in Escherichia coli. Deletion analysis showed that the atsA gene with the promoter region was located within a 3.2-kilobase cloned segment. In E. coli cells which carried the plasmid, the synthesis of arylsulfatase was repressed by various sources of sulfur; the repression was relieved, in each case, by tyramine. Transfer of the plasmid into atsA or constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsA but not of atsR. The nucleotide sequence of the 3.2-kilobase fragment was determined. Two open reading frames, the atsA gene and an unknown gene (atsB), were found. These are located between a potential promoter and a transcriptional terminator sequence. Deletion analysis suggests that atsB is a potential positive factor for the regulation of arylsulfatase. Analysis of the amino acid sequences of the first 13 amino acids from the N terminus of the purified secreted arysulfatase agrees with that of the nucleotide sequence of atsA. The leader peptide extends over 20 amino acids and has the characteristics of a signal sequence. Primer extension mapping of transcripts generated in vivo suggests that the synthesis of mRNA starts at a site 31 or 32 bases upstream from the ATG initiation codon of the atsB gene. By Northern (RNA) blot analysis of the transcripts induced by tyramine, we found a 2.7-kilobase transcript which is identical in size to the total sequence of the atsB and atsA genes. Thus, the ats operon is composed of two cistrons and is regulated by sulfur and tyramine.

SUBMITTER: Murooka Y

PROVIDER: S-EPMC208713 | biostudies-other | 1990 Apr

REPOSITORIES: biostudies-other

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A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene.

Murooka Y Y Ishibashi K K Yasumoto M M Sasaki M M Sugino H H Azakami H H Yamashita M M

Journal of bacteriology 19900401 4

The structural gene for arylsulfatase (atsA) of Klebsiella aerogenes was cloned into a pKI212 vector in Escherichia coli. Deletion analysis showed that the atsA gene with the promoter region was located within a 3.2-kilobase cloned segment. In E. coli cells which carried the plasmid, the synthesis of arylsulfatase was repressed by various sources of sulfur; the repression was relieved, in each case, by tyramine. Transfer of the plasmid into atsA or constitutive atsR mutant strains of K. aerogene ...[more]

PMID: 2180918

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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.

Dataset Information

A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene.

Publications

A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene.

Similar Datasets

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