SummaryExpression of the Pseudomonas aeruginosa type III secretion system (T3SS) is activated by ExsA, a member of the AraC/XylS family of transcriptional regulators. In the present study we examine the DNAbinding properties of ExsA. ExsA was purified as a histidine-tagged fusion protein (ExsAHis) and found to be monomeric in solution. ExsAHis specifically bound T3SS promoters with high affinity as determined by electrophoretic mobility shift assays (EMSA). For each promoter tested two distinct ExsA-DNA complexes were detected. Biochemical analyses indicate that the higher-mobility complex consists of a single ExsAHis molecule bound to DNA while the lowermobility complex results from the binding of two ExsAHis molecules. DNase I protection assays demonstrate that the ExsAHis binding site overlaps the -35 RNA polymerase binding site and extends upstream an additional~34 bp. An alignment of all 10 ExsAdependent promoters revealed a number of highly conserved nucleotides within the footprinted region. We find that most of the highly conserved nucleotides are required for transcription in vivo; EMSA-binding assays confirm that several of these nucleotides are essential determinants of ExsAHis binding. The combined data support a model in which two ExsAHis molecules bind adjacent sites on the promoter to activate T3SS gene transcription.

The genus Shewanella contains Gram negative γ-proteobacteria capable of reducing a wide range of substrates, including insoluble metals and carbon electrodes. The utilization of insoluble respiratory substrates by bacteria requires a strategy that is quite different from a traditional respiratory strategy because the cell cannot take up the substrate. Electrons generated by cellular metabolism instead must be transported outside the cell, and perhaps beyond, in order to reduce an insoluble substrate. The primary focus of research in model organisms such as Shewanella has been the mechanisms underlying respiration of insoluble substrates. Electrons travel from the menaquinone pool in the cytoplasmic membrane to the surface of the bacterial cell through a series of proteins collectively described as the Mtr pathway. This review will focus on respiratory electron transfer from the surface of the bacterial cell to extracellular substrates. Shewanella sp. secrete redox-active flavin compounds able to transfer electrons between the cell surface and substrate in a cyclic fashion-a process termed electron shuttling. The production and secretion of flavins as well as the mechanisms of cell-mediated reduction will be discussed with emphasis on the experimental evidence for a shuttle-based mechanism. The ability to reduce extracellular substrates has sparked interest in using Shewanella sp. for applications in bioremediation, bioenergy, and synthetic biology.

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.

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.