Functional Domains of ExsA, the Transcriptional Activator of the Pseudomonas aeruginosa Type III Secretion System ▿

The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to evade phagocytosis and damage eukaryotic cells. Transcription of the T3SS regulon is controlled by ExsA, a member of the AraC/XylS family of transcriptional regulators. These family members generally consist of an ∼100-amino acid carboxy-terminal domain (CTD) with two helix-turn-helix DNA binding motifs and an ∼200-amino acid amino-terminal domain (NTD) with known functions including oligomerization and ligand binding. In the present study, we show that the CTD of ExsA binds to ExsA-dependent promoters in vitro and activates transcription from ExsA-dependent promoters both in vitro and in vivo. Despite possessing these activities, the CTD lacks the cooperative binding properties observed for full-length ExsA at the PexsC promoter. In addition, the CTD is unaffected by the negative regulatory activity of ExsD, an inhibitor of ExsA activity. Binding studies confirm that ExsD interacts directly with the NTD of ExsA. Our data are consistent with a model in which a single ExsA molecule first binds to a high-affinity site on the PexsC promoter. Protein-protein interactions mediated by the NTD then recruit an additional ExsA molecule to a second site on the promoter to form a complex capable of stimulating wild-type levels of transcription. These findings provide important insight into the mechanisms of transcriptional activation by ExsA and inhibition of ExsA activity by ExsD

Characterization of ExsC and ExsD Self-Association and Heterocomplex Formation

Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced by calcium depletion and is positively regulated by the ExsA transcriptional activator and negatively regulated by the ExsD antiactivator. Under conditions permissive for expression of the T3SS, the negative regulatory activity of ExsD is antagonized by a direct binding interaction with ExsC. In the present study, the ExsC-ExsD binding interaction was characterized. Individually, both ExsC and ExsD form self-associated complexes, as judged by bacterial monohybrid and gel filtration experiments. A mixture of purified ExsC and ExsD readily formed a complex that elutes from gel filtration medium as a single included peak. The calculated molecular weight of the ExsC-ExsD complex is consistent with a complex containing multiple copies of ExsC and ExsD. Isothermic titration calorimetry experiments found formation of the ExsC-ExsD complex to be thermodynamically favorable, with a K(d) of ∼18 nM and a likely binding ratio of 1:1. To identify amino acid residues important for the regulatory activities of ExsC and ExsD, self-association, and complex formation, charged-cluster mutagenesis was performed. Two of the resulting ExsD charged-cluster mutants (DM2 and DM3) demonstrated a hyperrepressive phenotype for expression of the T3SS. By two-hybrid and copurification assays, the DM3 mutant was found to be impaired in its interaction with ExsC. This finding demonstrates that the binding of ExsC to ExsD is required for transcriptional induction of the T3SS under calcium-limiting growth conditions

In Vitro and In Vivo Characterization of the Pseudomonas aeruginosa Cyclic AMP (cAMP) Phosphodiesterase CpdA, Required for cAMP Homeostasis and Virulence Factor Regulation▿ †

Cyclic AMP (cAMP) is an important second messenger signaling molecule that controls a wide variety of eukaryotic and prokaryotic responses to extracellular cues. For cAMP-dependent signaling pathways to be effective, the intracellular cAMP concentration is tightly controlled at the level of synthesis and degradation. In the opportunistic human pathogen Pseudomonas aeruginosa, cAMP is a key regulator of virulence gene expression. To better understand the role of cAMP homeostasis in this organism, we identified and characterized the enzyme CpdA, a putative cAMP phosphodiesterase. We demonstrate that CpdA possesses 3′,5′-cAMP phosphodiesterase activity in vitro and that it utilizes an iron-dependent catalytic mechanism. Deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of P. aeruginosa virulence traits. Further, we demonstrate that the cAMP-dependent transcription factor Vfr directly regulates cpdA expression in response to intracellular cAMP accumulation, thus providing a feedback mechanism for controlling cAMP levels and fine-tuning virulence factor expression

NosP Signaling Modulates the NO/H-NOX-Mediated Multicomponent c-Di-GMP Network and Biofilm Formation in Shewanella oneidensis

Biofilms form when bacteria aggregate in a self-secreted exopolysaccharide matrix; they are resistant to antibiotics and implicated in disease. Nitric oxide (NO) is known to mediate biofilm formation in many bacteria via ligation to H-NOX (heme-NO/oxygen binding) domains. Most NO-responsive bacteria, however, lack H-NOX domain-containing proteins. We have identified another NO-sensing protein (NosP), which is predicted to be involved in two-component signaling and biofilm regulation in many species. Here, we demonstrate that NosP participates in the previously described H-NOX/NO-responsive multicomponent c-di-GMP signaling network in; Shewanella oneidensis; . Strains lacking either; nosP; or its co-cistronic kinase; nahK; (previously; hnoS; ) produce immature biofilms, while; hnoX; and; hnoK; (kinase responsive to NO/H-NOX) mutants result in wild-type biofilm architecture. We demonstrate that NosP regulates the autophosphorylation activity of NahK as well as HnoK. HnoK and NahK have been shown to regulate three response regulators (HnoB, HnoC, and HnoD) that together comprise a NO-responsive multicomponent c-di-GMP signaling network. Here, we propose that NosP/NahK adds regulation on top of H-NOX/HnoK to modulate this c-di-GMP signaling network, and ultimately biofilm formation, by governing the flux of phosphate through both HnoK and NahK. In addition, it appears that NosP and H-NOX act to counter each other in a push-pull mechanism; NosP/NahK promotes biofilm formation through inhibition of H-NOX/HnoK signaling, which itself reduces the extent of biofilm formation. Addition of NO results in a reduction of c-di-GMP and biofilm formation, primarily through disinhibition of HnoK activity

The Pseudomonas aeruginosa Vfr Regulator Controls Global Virulence Factor Expression through Cyclic AMP-Dependent and -Independent Mechanisms▿ †

Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments

Genome-scale metabolic network validation of Shewanella oneidensis using transposon insertion frequency analysis.

Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA). TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1) previous genome-wide direct gene-essentiality assignments; and, 2) flux balance analysis (FBA) predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions

(A) 50 out of 273 essential genes were associated with a fitness value.

<p>The circumference of the circles represents the logarithmic read counts of each insertion, and the color of the circles represents the gene location of the insertion: first 10% (magenta), last 10% (cyan), and middle 80% (black). 22 essential genes contained ≥2 insertions in the middle. The spread in fitness and read counts was very large, suggesting different causes for the existence of insertions in essential genes. (B) Transposon insertion locations, fitnesses, and projected ribosome binding site (RBS) strengths associated with intra-gene start codons. Black dots show all TA locations, and top row diamonds show the observed insertions with color coded fitness. The bottom diamonds represent alternative start codons in each gene. The color scales logarithmically with the associated RBS strengths. Each couple of rows represents the RBS strengths of all intra-gene start codons for both possible insertion orientations for each mutant, with the first row representing no insertion.</p

Three-way comparison between FBA predictions and TIFA and DEC essentiality calls.

<p>(A) Comparison between FBA and TIFA. (B) Comparison between FBA and direct gene essentiality calls (DEC). The sectors of the comparison matrix show the intersections of FBA essential genes (E), FBA nonessential genes (N) and genes not in the MR-1 model (NA) with TIFA and DEC calls for essential genes (E), nonessential genes (N) and uncalled genes (U). The TIFA essential gene calls that could be alternatively explained by polar operon effects are shown in brackets.</p

Comparison of gene essentiality calls between TIFA and DEC.

<p>The six essentiality classes (columns) that were previously defined <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003848#pcbi.1003848-Deutschbauer1" target="_blank">[18]</a> for the direct essentiality calls (DECs), were cross compared to the three TIFA essentiality classes (rows). Columns (1,2) represent DEC essential gene calls; columns (3,4,5) DEC nonessential gene calls. The excluded TIFA essential gene calls that could be explained alternatively by operon polar effects are listed between brackets.</p>1<p>Essential orthologs;</p>2<p>essential gene calls with conflicting or absent corroborating evidence from orthologs,</p>3<p>nonessential orthologs;</p>4<p>nonessential gene calls with conflicting evidence from orthologs.</p>5<p>nonessential gene calls without corroborating evidence from <i>E. coli</i> orthologs.</p><p>A detailed description of the six DEC categories is given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003848#pcbi.1003848.s003" target="_blank">Text S1</a> of reference <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003848#pcbi.1003848-Deutschbauer1" target="_blank">[18]</a>.</p><p>Comparison of gene essentiality calls between TIFA and DEC.</p