GbdR Regulates Pseudomonas aeruginosa plcH and pchP Transcription in Response to Choline Catabolites

Pseudomonas aeruginosa hemolytic phospholipase C, PlcH, can degrade phosphatidylcholine (PC) and sphingomyelin in eukaryotic cell membranes and extracellular PC in lung surfactant. Numerous studies implicate PlcH in P. aeruginosa virulence. The phosphorylcholine released by PlcH activity on phospholipids is hydrolyzed by a periplasmic phosphorylcholine phosphatase, PchP. Both plcH gene expression and PchP enzyme activity are positively regulated by phosphorylcholine degradation products, including glycine betaine. Here we report that the induction of plcH and pchP transcription by glycine betaine is mediated by GbdR, an AraC family transcription factor. Mutants that lack gbdR are unable to induce plcH and pchP in media containing glycine betaine or choline and in phosphatidylcholine-rich environments, such as lung surfactant or mouse lung lavage fluid. In T broth containing choline, the gbdR mutant exhibited a 95% reduction in PlcH activity. In electrophoretic mobility shift assays, a GbdR-maltose binding protein fusion bound specifically to both the plcH and pchP promoters. Promoter mapping, alignment of GbdR-regulated promoter sequences, and analysis of targeted promoter mutants that lack GbdR-dependent induction of transcription were used to identify a region necessary for GbdR-dependent transcriptional activation. GbdR also plays a significant role in plcH and pchP regulation within the mouse lung. Our studies suggest that GbdR is the primary regulator of plcH and pchP expression in PC-rich environments, such as the lung, and that pchP and other genes involved in phosphorylcholine catabolism are necessary to stimulate the GbdR-mediated positive feedback induction of plcH

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

Pseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ΔplcHR 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 Δanr 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

Junctional conductance (<i>G</i>_<i>j</i>) of <i>C. elegans</i> body-wall muscle was significantly decreased in mutants of six innexins.

<p><b>A</b>. Diagram showing ventral body-wall muscle cells. Ventral muscles include the left and right quadrants with each quadrant consisting of two rows of muscle cells in a monolayer. The highlighted cell pairs (L1/L2 and R1/R2) represent those used for electrophysiological analyses. <b>B</b>. <i>G</i>_<i>j</i> was indistinguishable between wild type (WT) and mutants of 16 innexins. <b>C</b>. Mutants of 6 innexins showed significantly lower <i>G</i>_<i>j</i> when compared with WT, and the coupling defect was rescued completely by expressing a wild-type innexin in each corresponding mutant. The asterisk (*) indicates a statistically significant difference compared with WT. The number on each bar represents the number (<i>N</i>) of cell pairs analyzed.</p

Expression patterns of innexins functioning in <i>C. elegans</i> body-wall muscle.

<p>Expression patterns were assessed by analyzing GFP signal in live worms expressing innexin promoter and GFP transcriptional fusions. Body-wall muscle expression (indicated by arrows) was observed for four of the six innexins important to muscle electrical coupling, including <i>unc-9, inx-10, inx-11</i> and <i>inx-18</i>. The <i>unc-9</i> expression data were shown previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076877#B33" target="_blank">33</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076877#B34" target="_blank">34</a>].</p

Speculative models of muscle gap junctions.

<p>These highly speculative models are used to illustrate three possible stoichiometries out of the myriad possibilities. See the text for reasons for speculating these models.</p