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

Abstract

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...

Basis for the Essentiality of H-NS Family Members in Pseudomonas aeruginosa

“…A previous report demonstrated that cells of P. aeruginosa strain PAO1 that contain deletions of both the mvaT and mvaU genes are viable (31). This finding appears to contradict our findings that MvaT and MvaU are essential in the absence of the partner regulator (9) (Fig.…”

“…These findings resolve the apparent discrepancy between our earlier findings obtained through protein depletion, that the loss of both MvaT and MvaU cannot be tolerated (9), and the previous demonstration that cells containing deletions of both mvaT and mvaU are viable (31). We note that, consistent with the findings reported here, activation of Pf4 prophage was observed previously in cells containing deletions of both mvaT and mvaU (31).…”

The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa

“…An mvaT mutant exhibits more production of PA-IL lectin and pyocyanin, reduced biofilm formation and swarming motility, and increased drug resistance (53,204). The observation that mvaT and mvaU single mutants increase pyocyanin synthesis while an mvaT mvaU double mutant abolishes pyocyanin and PQS production suggests that these regulators work in different ways to control pyocyanin production, with one involving PQS production and the other directly controlling pyocyanin synthesis (117). In addition to the four genes involved in PQS biosynthesis, the pqsABCDE operon contains a fifth, pqsE (PA1000), encoding a protein with a metallo-␤-lactamase fold (62) that is not required for PQS synthesis (71).…”

The Accessory Genome of Pseudomonas aeruginosa

“…Two H-NS family proteins that have been studied in P. aeruginosa are MvaT and MvaU, which act coordinately and preferentially bind regions of the P. aeruginosa chromosome corresponding to accessory genetic elements (37). Not surprisingly, then, double mutation of mvaT and mvaU results in activation of chromosomally integrated filamentous Pf4 prophage (126). The extent of H-NS-mediated repression of horizontally transferred genes has yet to be determined.…”