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AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site.

ABSTRACT: Strong transcriptional activation of algD, a key event in the overproduction of alginate and establishment of mucoidy in Pseudomonas aeruginosa, depends on the functional algR gene. The predicted gene product of algR shows homologies to response regulators from bacterial signal transduction systems. The algR gene was overexpressed in Escherichia coli, its product (AlgR) was purified by utilizing its apparent affinity for heparin, and its sequence was verified by partial amino acid sequence analysis. AlgR was found to interact directly with the algD promoter. Deletion mapping analysis, in conjunction with mobility shift DNA-binding assays, indicated the presence of three regions within the algD promoter capable of specifically binding AlgR. A relatively weak interaction was observed with the algD promoter fragment containing the region immediately upstream of the algD mRNA start site (-144 to +11). However, when fragments spanning regions located very far upstream from the algD mRNA initiation site (-533 and -332) were used, strong specific binding was observed. These regions were separated by a DNA segment not binding AlgR and spanning positions -332 to -144. DNase I footprinting analysis further established the presence of discrete AlgR binding sites overlapping with FUS, the far-upstream sites required for full induction of algD transcription and its environmental modulation. There were two distinct binding sites: RB1, spanning nucleotides -479 to -457, and RB2, spanning nucleotides -400 to -380. Both of these sequences shared a highly conserved core region, ACCGTTCGTC. These results established a direct interaction of AlgR with the algD promoter and revealed an arrangement of binding sites highly unusual for response regulators of the AlgR type.

SUBMITTER: Mohr CD

PROVIDER: S-EPMC208205 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

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AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site.

Mohr C D CD Hibler N S NS Deretic V V

Journal of bacteriology 19910801 16

Strong transcriptional activation of algD, a key event in the overproduction of alginate and establishment of mucoidy in Pseudomonas aeruginosa, depends on the functional algR gene. The predicted gene product of algR shows homologies to response regulators from bacterial signal transduction systems. The algR gene was overexpressed in Escherichia coli, its product (AlgR) was purified by utilizing its apparent affinity for heparin, and its sequence was verified by partial amino acid sequence analy ...[more]

PMID: 1907266

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Project description:Activation of algD by AlgR is essential for mucoidy, a virulence factor expressed by Pseudomonas aeruginosa in cystic fibrosis. Two AlgR-binding sites, RB1 and RB2, located far upstream from the algD mRNA start site, are essential for the high-level activity of algD. However, the removal of RB1 and RB2 does not completely abolish inducibility of algD in response to environmental signals. In this work, a third binding site for AlgR, termed RB3, near the algD mRNA start site was characterized. Deletion of RB3 abrogated both the AlgR-binding ability and the residual inducibility of the algD promoter. DNase I footprinting analysis of RB3 resulted in a protection pattern spanning nucleotides -50 to -30. Eight of 10 residues encompassing a continuous region of protection within RB3 (positions -45 to -36) matched in the inverted orientation the conserved core sequence (ACCGTTCGTC) of RB1 and RB2. Quantitative binding measurements of AlgR association with RB1, RB2, and RB3 indicated that AlgR had significantly lower affinity for RB3 than for RB1 and RB2, with differences in the free energy of binding of 1.05 and 0.93 kcal/mol (4.39 and 3.89 kJ/mmol), respectively. Altering the core of RB2 to match the core of RB3 significantly reduced AlgR binding. Conversely, changing the core of RB3 to perfectly match the core of RB2 (mutant site termed RB3*) improved AlgR binding, approximating the affinity of RB2. RB3*, in the absence of the far upstream sites, showed an increase in activity, approaching the levels observed with the full-size algD promoter. Changing 4 nucleotides in two different combinations within the core of RB3 abolished the binding of AlgR to this site and resulted in a significant reduction of promoter activity in the presence of the far upstream sites. Thus, (i) the core sequence is essential for AlgR binding; (ii) the three binding sites, RB1, RB2, and RB3, are organized as an uneven palindrome with symmetrical sequences separated by 341 and 417 bp; and (iii) all three sites participate in algD activation.

Dataset Information

AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site.

Publications

AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site.

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