bPseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ⌬plcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The ⌬anr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence.

Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1 %) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Danr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions. INTRODUCTIONPseudomonas aeruginosa squanders few opportunities to colonize a vulnerable human host. In addition to engendering a range of nosocomial infections, including keratitis (Green et al., 2008), burn wounds (de Macedo & Santos, 2005), endocarditis (Baddour et al., 2005) and implanted medical devices (Hidron et al., 2008), it also colonizes the lungs of 80 % of persons with the heritable disease cystic fibrosis (CF) (Rajan & Saiman, 2002;Rosenfeld et al., 2003). Colonization by P. aeruginosa contributes to the decline of lung function in this patient population (Rajan & Saiman, 2002), due in part to its wide array of virulence factors that promote its growth and persistence within the host. One such virulence factor is the well-characterized exotoxin haemolytic phospholipase C (PlcH), which is secreted by P. aeruginosa when in association with eukaryotic hosts. PlcH is essential for virulence in co-culture with fungal filaments, on Arabidopsis leaves, in Galleria mellonella larvae and in mammalian hosts (Hogan & Kolter, 2002;Jander et al., 2000;Rahme et al., 2000), highlighting its importance in diverse models of infection.PlcH degrades the abundant phospholipids phosphatidylcholine (PC) and sphingomyelin, which are found in eukaryotic membranes and in lung surfactant (Berka & Vasil, 1982;Vasil, 2006). PC degradation releases both fatty acids and choline-containing c...