Exopolyphosphatase of Pseudomonas aeruginosa is essential for the production of virulence factors, and its expression is controlled by NtrC and PhoB acting at two interspaced promoters.

The exopolyphosphatase (Ppx) of Pseudomonas aeruginosa is encoded by the PA5241 gene (ppx). Ppx catalyses the hydrolysis of inorganic polyphosphates to orthophosphate (Pi). In the present work, we identified and characterized the promoter region of ppx and its regulation under environmental stress conditions. The role of Ppx in the production of several virulence factors was demonstrated through studies performed on a ppx null mutant. We found that ppx is under the control of two interspaced promoters, dually regulated by nitrogen and phosphate limitation. Under nitrogen-limiting conditions, its expression was controlled from a σ(54)-dependent promoter activated by the response regulator NtrC. However, under Pi limitation, the expression was controlled from a σ(70) promoter, activated by PhoB. Results obtained from the ppx null mutant demonstrated that Ppx is involved in the production of virulence factors associated with both acute infection (e.g. motility-promoting factors, blue/green pigment production, C6-C12 quorum-sensing homoserine lactones) and chronic infection (e.g. rhamnolipids, biofilm formation). Molecular and physiological approaches used in this study indicated that P. aeruginosa maintains consistently proper levels of Ppx regardless of environmental conditions. The precise control of ppx expression appeared to be essential for the survival of P. aeruginosa and the occurrence of either acute or chronic infection in the host.

A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.

Through the use of molecular and biochemical experiments and bioinformatic tools, this work demonstrates that the PA4921 gene of the Pseudomonas aeruginosa PAO1 genome is a gene responsible for cholinesterase (ChoE) activity. Similar to the acetylcholinesterase (AchE) of Zea mays, this ChoE belongs to the SGNH hydrolase family. In mature ChoE, i.e., without a signal peptide, (18)Ser, (78)Gly, (127)N, and (268)H are conserved aminoacyl residues. Acetylthiocholine (ATC) and propionylthiocholine (PTC) are substrates of this enzyme, but butyrylcholine is an inhibitor. The enzyme also catalyzes the hydrolysis of the artificial esters p-nitrophenyl propionate (pNPP) and p-nitrophenyl butyrate (pNPB) but with lower catalytic efficiency with respect to ATC or PTC. The second difference is that pNPP and pNPB did not produce inhibition at high substrate concentrations, as occurred with ATC and PTC. These differences plus preliminary biochemical and kinetic studies with alkylammonium compounds led us to propose that this enzyme is an acetylcholinesterase (AchE) or propionylcholinesterase. Studies performed with the purified recombinant enzyme indicated that the substrate saturation curves and the catalytic mechanism are similar to those properties described for mammalian AchEs. Therefore, the results of this work suggest that the P. aeruginosa ChoE is an AchE that may also be found in Pseudomonas fluorescens.

Choline catabolism, σ54 factor and NtrC are required for the full expression of the Pseudomonas aeruginosa phosphorylcholine phosphatase gene.

Choline favors the pathogenesis of Pseudomonas aeruginosa because hemolytic phospholipase C and phosphorylcholine phosphatase (PchP) are synthesized as a consequence of its catabolism. The experiments performed here resulted in the identification of the factors that regulate both the catabolism of choline and the gene coding for PchP. We have also identified and characterized the promoter of the pchP gene, its transcriptional organization and the factors that affect its expression. Deletion analyses reveal that the region between -188 and -68 contains all controlling elements necessary for pchP expression: a hypothetical -12/-24 promoter element, a consensus sequence for the integration host factor (-141/-133), and a palindromic sequence resembling a binding site for a potential enhancer binding protein (-190/-174). Our data also demonstrate that choline catabolism and NtrC (nitrogen regulatory protein) are necessary for the full expression of pchP and is partially dependent on σ(54) factor.