Bacterial Cyclic Diguanylate Signaling Networks Sense Temperature

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria

Regulation of biofilm formation in Pseudomonasand Burkholderias pecies

In the present review, we describe and compare the molecular mechanisms that are involved in the regulation of biofilm formation by Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Burkholderia cenocepacia. Our current knowledge suggests that biofilm formation is regulated by cyclic diguanosine-5′-monophosphate (c-di-GMP), small RNAs (sRNA) and quorum sensing (QS) in all these bacterial species. The systems that employ c-di-GMP as a second messenger regulate the production of exopolysaccharides and surface proteins which function as extracellular matrix components in the biofilms formed by the bacteria. The systems that make use of sRNAs appear to regulate the production of exopolysaccharide biofilm matrix material in all these species. In the pseudomonads, QS regulates the production of extracellular DNA, lectins and biosurfactants which all play a role in biofilm formation. In B. cenocepacia QS regulates the expression of a large surface protein, lectins and extracellular DNA that all function as biofilm matrix components. Although the three regulatory systems all regulate the production of factors used for biofilm formation, the molecular mechanisms involved in transducing the signals into expression of the biofilm matrix components differ between the species. Under the conditions tested, exopolysaccharides appears to be the most important biofilm matrix components for P. aeruginosa, whereas large surface proteins appear to be the most important biofilm matrix components for P. putida, P. fluorescens, and B. cenocepacia

The Cyclic AMP-Vfr Signaling Pathway in <i>Pseudomonas aeruginosa</i> Is Inhibited by Cyclic Di-GMP

The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels.Published versio

Strains and plasmids used in this study.

<p>Tc^r, tetracycline; Cb^r, carbenicillin.</p><p>Strains and plasmids used in this study.</p

Bacterial aggregation in mouse pneumonia requires the T3SS translocon but does not require T3SS effectors.

<p>BALB/c mice were injected intranasally with PAK, PAKΔ<i>STY</i>, or PAKΔ<i>popB</i> and lungs were isolated, sectioned, and stained at 3 hours post-infection. (A) Widefield epifluorescent imaging showed PAK (red) bound to lung epithelium (green). Representative image from 67 images is shown. Scale bar, 100 µm. (B) The amount of aggregation by PAK and T3SS mutants was quantified and plotted against bacterial density (n≥60 images for each strain). Linear regression lines were applied to each bacterial strain. (C) Comparisons of linear regression lines showed no differences in slope but significant differences in the intercept of PAKΔ<i>popB</i> compared to PAK or PAKΔ<i>STY</i>. Statistics in Supplemental Statistical Analysis (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004479#ppat.1004479.s010" target="_blank">Text S1</a>).</p

Cell-associated aggregation requires the T3SS translocon but does not require T3SS effectors.

<p>MDCK cells stably transfected with PH-Akt-GFP (green) were infected with mCherry-expressing (A) PAK, (B) PAKΔ<i>STY</i> (lacks the known T3SS effectors), or (C) PAKΔ<i>popB</i> (has the functional needle apparatus but lacks the translocon) for 60 minutes, fixed, and stained for actin (blue). Cell-associated aggregates (red) were visible with PAK (A) and PAKΔ<i>STY</i> (B) and were accompanied by formation of membranous protrusions (white arrows) containing PH-Akt-GFP. PAK<i>ΔpopB</i> (C) bound individually or as groups of 2 to 3 bacteria. Representative confocal images from three independent experiments are shown. Scale bars, 10 µm. (D) Cell-associated aggregation by PAK and T3SS mutants was quantified using spinning disk confocal microscopy. Shown is the number of aggregates (≥10 bacteria) normalized to PAK (n≥3 independent experiments). Data are mean ± SEM. ***p<0.001 compared to PAK. Statistics in Supplemental Statistical Analysis (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004479#ppat.1004479.s010" target="_blank">Text S1</a>).</p