The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression

Westfall, Landon W.; Luna, A.M.; Francisco, Michael San; Diggle, Stephen P.; Worrall, Kathryn E.; Williams, Paul; Cámara, Miguel; Hamood, Abdul N.

doi:10.1099/mic.0.27270-0

Cited by 26 publications

(23 citation statements)

References 53 publications

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“…MvaT and MvaU are global transcriptional regulators belonging to the H-NS family. Involvement of these two homologous proteins in several aspects of bacterial physiology in P. aeruginosa has been reported previously (3,5,27,28,33,34). However, most of the previous studies involved genetic analysis of mutants devoid of either mvaT or mvaU but not genetic analysis of mvaT mvaU double mutants.…”

Section: Discussionmentioning

confidence: 86%

The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in Pseudomonas aeruginosa PAO1

Wally

Miller

et al. 2009

J Bacteriol

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The MvaT and MvaU proteins belonging to the H-NS family were identified as DNA-binding proteins that interact with the regulatory region of the aotJQMOP-argR operon for arginine uptake and regulation. Recombinant MvaT and MvaU proteins were purified, and binding of these purified proteins to the aotJ regulatory region was demonstrated using electromobility shift assays. Polyclonal antibodies against purified MvaT and MvaU were prepared and employed in supershift assays to support these observations. Knockout mutations resulting in a single lesion in mvaT or mvaU, as well as knockout mutations resulting in double lesions, were constructed using biparental conjugation, and the absence of MvaT and MvaU in the resulting mutants was confirmed by immunoblot analysis. Using measurements of the ␤-galactosidase activities from aotJ::lacZ fusions in the mutants and the parental strain, it was found that MvaT and MvaU serve as repressors in control of aotJ expression. The effects of MvaT and MvaU on pyocyanin synthesis and CupA fimbrial expression in these mutants were also analyzed. Pyocyanin synthesis was induced in the single mutants but was completely abolished in the double mutant, suggesting that there is a complicated regulatory scheme in which MvaT and MvaU are essential elements. In comparison, MvaT had a more profound role than MvaU as a repressor of cupA expression; however, a combination of MvaT depletion and MvaU depletion had a strong synergistic effect on cupA. Moreover, prophage Pf4 integrated into the chromosome of Pseudomonas aeruginosa PAO1 was activated in an mvaT mvaU double mutant but not in a single mutant. These results were supported by purification and nucleotide sequencing of replicative-form DNA and by the release of phage particles in plaque assays. In summary, the mvaT mvaU double mutant was viable, and depletion of MvaT and MvaU had serious effects on a variety of physiological functions in P. aeruginosa.The MvaT and MvaU proteins of Pseudomonas aeruginosa belong to the H-NS family of small DNA-binding proteins. MvaT was initially identified in P. mevalonii as a positive regulator for mevalonate catabolism (25). Subsequently, MvaT homologues have been identified in other pseudomonads based on structural and functional similarities; five homologues have been identified in P. putida, three homologues have been identified in P. fluorescens, four homologues have been identified in P. syringae, and two homologues have been identified in P. aeruginosa. In P. putida, the TurA protein represses the Pu promoter of the TOL plasmid in a temperature-dependent manner (24). In P. fluorescens, the MvaT and MvaV proteins regulate the expression of two biocontrol exoproducts, 2,4-diacetyl phloroglucinol and pyoluteorin (1). In P. aeruginosa, MvaT is involved in quorum-sensing responses and biofilm formation. Inactivation of mvaT resulted in increased production of PA-IL lectin and the toxic exoproduct pyocyanin, reduced biofilm formation, increased drug resistance, and reduced swarming motility (5, 33). In ad...

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Section: Discussionmentioning

confidence: 86%

The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in Pseudomonas aeruginosa PAO1

Wally

Miller

et al. 2009

J Bacteriol

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“…The upstream and downstream fragments of pqsR were amplified by PCR from PAO1 chromosomal DNA using the primers pairs FW pqsR Up-RV pqsR Up and FW pqsR Down-RV pqsR Down, respectively ( Table S2 ), introduced into pDM4 [49] and the resulting Δ pqsR mutants obtained by allelic exchange [50]. The pqsA promoter fused to the luxCDABE reporter operon was introduced into both the Δ pqsR and Δ pqsA Δ pqsH Δ pqsR mutants using the miniCTX pqsA - lux plasmid as described previously [23].…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for Native Agonist and Synthetic Inhibitor Recognition by the Pseudomonas aeruginosa Quorum Sensing Regulator PqsR (MvfR)

et al. 2013

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Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

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“…In P. aeruginosa, ptxS has been identified as involved in the control of the toxA gene and of its own VOL. 190, 2008 CONTROL OF PERIPHERAL GLUCOSE PATHWAYS IN P. PUTIDA 2337 synthesis (35,40). In P. aeruginosa, the target of PtxS is a 14-bp dyad sequence whose disruption in front of this gene resulted in its overexpression.…”

Section: Discussionmentioning

confidence: 99%

A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate

2008

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Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism.

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The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression

Cited by 26 publications

References 53 publications

The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in Pseudomonas aeruginosa PAO1

The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in Pseudomonas aeruginosa PAO1

Structural Basis for Native Agonist and Synthetic Inhibitor Recognition by the Pseudomonas aeruginosa Quorum Sensing Regulator PqsR (MvfR)

A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate

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