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.

AlgR-binding sites within the algD promoter make up a set of inverted repeats separated by a large intervening segment of DNA

“…Protein overexpression was performed in E. coli BL21(DE3) harboring pLysS (36). The plasmid constructs used for the overexpression and purification of AlgR have been described previously (29). DNA manipulations were done according to published procedures (1).…”

“…The recent purification of AlgR identified in this laboratory, by deletion and functional analyses, as critical for the high-level transcription of algD (30). Centered around the middle of both RB1 and RB2 is a perfectly conserved 10-bp sequence (5'ACCGTTCGTC3') termed the core region (29). The core sequences of RB1 and RB2 form a pair of direct repeats with a head-to-tail separation of 66 nucleotides (29,30).…”

“…Centered around the middle of both RB1 and RB2 is a perfectly conserved 10-bp sequence (5'ACCGTTCGTC3') termed the core region (29). The core sequences of RB1 and RB2 form a pair of direct repeats with a head-to-tail separation of 66 nucleotides (29,30).…”

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

“…Because AlgR is part of a two-component system that is important for both acute and chronic infections (19,32), it was necessary to address the mechanism for AlgR activation of rsmA in order to create a framework to understand how AlgR regulation of rsmA might impact virulence gene expression. AlgR binds a consensus sequence CCGTTCGTC (21,48,49), and phosphorylation is thought to enable AlgR to bind potential binding sites that deviate from this consensus, such as the sites found in the fimU promoter (32,47). However, the rsmA promoter deviates from the AlgR-binding consensus, and AlgR phosphorylation was not required for rsmA expression.…”

“…In the case of acute infections, AlgR activates the fimU operon, enabling the production of T4P (19,21,47). In chronic infections, AlgR, AlgU, and other transcriptional regulators activate the production of alginate (22,(48)(49)(50). These and other virulence factors in P. aeruginosa are often considered mutually exclusive, depending on whether there is an acute or chronic infection (51)(52)(53).…”