Evan D. Brutinel

Shuttling happens: soluble flavin mediators of extracellular electron transfer in Shewanella

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.

Control of gene expression by type III secretory activity

The bacterial flagellum and the highly related injectisome (or needle complex) are among the most complicated multi-protein structures found in Gram-negative microorganisms. The assembly of both structures is dependent upon a type III secretion system. An interesting regulatory feature unique to these systems is the coordination of gene expression with type III secretory activity. This means of regulation ensures that secretion substrates are expressed only when required during the assembly process or upon completion of the fully functional structure. Prominent within the regulatory scheme are secreted proteins and type III secretion chaperones that exert effects on gene expression at the transcriptional and post-transcriptional levels. Although the major structural components of the flagellum and injectisome systems are highly conserved, recent studies reveal diversity in the mechanisms used by secretion substrates and chaperones to control gene expression.

Characterization of ExsA and of ExsA-dependent promoters required for expression of the Pseudomonas aeruginosa type III secretion system

Expression 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 DNA‐binding 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 lower‐mobility 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 ExsA‐dependent 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.

Translocation of ExsE into Chinese Hamster Ovary Cells Is Required for Transcriptional Induction of the Pseudomonas aeruginosa Type III Secretion System

ABSTRACT Transcription of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced under Ca2+-limiting growth conditions or following the contact of the bacteria with host cells. The regulatory response to low Ca2+ levels is initiated by the T3SS-mediated secretion of ExsE, a negative regulatory protein that prevents T3SS gene transcription. In the present study, we demonstrated that ExsE plays an analogous role in transcriptional induction following host cell contact. By using a flow cytometry assay, the host contact-dependent induction of T3SS gene expression was found to be dependent upon the presence of functional type III translocation machinery. Using three independent assays, we demonstrated that ExsE was translocated into Chinese hamster ovary cells in a T3SS-dependent manner. Deletion mapping experiments indicated that the amino terminus of ExsE is required both for secretion under Ca2+-limiting growth conditions and for translocation into host cells. A P. aeruginosa mutant expressing an exsE allele lacking codons 3 through 20 was deficient in ExsE secretion and translocation and showed constitutive repression of T3SS gene expression under Ca2+-limiting growth conditions. The mutant also failed to induce T3SS gene expression following host cell contact and demonstrated a significant reduction in T3SS-dependent cytotoxicity towards Chinese hamster ovary cells, indicating that the translocation of ExsE is required for the host contact-dependent induction of T3SS gene expression.

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.

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 approximately 100-amino acid carboxy-terminal domain (CTD) with two helix-turn-helix DNA binding motifs and an approximately 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 P(exsC) 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 P(exsC) 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.

ExsD Inhibits Expression of the Pseudomonas aeruginosa Type III Secretion System by Disrupting ExsA Self-Association and DNA Binding Activity

Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to damage eukaryotic host cells and evade phagocytosis. Transcription of the T3SS regulon is controlled by ExsA, a member of the AraC/XylS family of transcriptional regulators. ExsA-dependent transcription is coupled to type III secretory activity through a cascade of three interacting proteins (ExsC, ExsD, and ExsE). Genetic data suggest that ExsD functions as an antiactivator by preventing ExsA-dependent transcription, ExsC functions as an anti-antiactivator by binding to and inhibiting ExsD, and ExsE binds to and inhibits ExsC. T3SS gene expression is activated in response to low-calcium growth conditions or contact with host cells, both of which trigger secretion of ExsE. In the present study we reconstitute the T3SS regulatory cascade in vitro using purified components and find that the ExsD.ExsA complex lacks DNA binding activity. As predicted by the genetic data, ExsC addition dissociates the ExsD.ExsA complex through formation of an ExsD.ExsC complex, thereby releasing ExsA to bind T3SS promoters and activate transcription. Addition of ExsE to the purified system results in formation of the ExsE.ExsC complex and prevents ExsC from dissociating the ExsD.ExsA complex. Although purified ExsA is monomeric in solution, bacterial two-hybrid analyses demonstrate that ExsA can self-associate and that ExsD inhibits self-association of ExsA. Based on these data we propose a model in which ExsD regulates ExsA-dependent transcription by inhibiting the DNA-binding and self-association properties of ExsA.

Anomalies of the anaerobic tricarboxylic acid cycle in Shewanella oneidensis revealed by Tn‐seq

The availability of increasingly inexpensive sequencing combined with an ever‐expanding molecular biology toolbox has transported classical bacterial genetics into the 21st century. Whole genome genetic fitness analysis using transposon mutagenesis combined with next‐generation high‐throughput sequencing (Tn‐seq) promises to revolutionize systems level analysis of microbial metabolism. Tn‐seq measures the frequency of actual members of a heterogeneous mutant pool undergoing purifying selection to determine the contribution of every non‐essential gene in the genome to the fitness of an organism under a given condition. Here we use Tn‐seq to assess gene function in the Gram negative γ‐proteobacterium Shewanella oneidensis strain MR‐1. In addition to being a model environmental organism, there is considerable interest in using S. oneidensis as a platform organism for bioremediation and biotechnology, necessitating a complete understanding of the metabolic pathways that may be utilized. Our analysis reveals unique aspects of S. oneidensis metabolism overlooked by over 30 years of classical genetic and systems level analysis. We report the utilization of an alternative citrate synthase and describe a dynamic branching of the S. oneidensis anaerobic tricarboxylic acid cycle, unreported in any other organism, which may be a widespread strategy for microbes adept at dissipating reducing equivalents via anaerobic respiration.

Biochemical Characterization of a Regulatory Cascade Controlling Transcription of the Pseudomonas aeruginosa Type III Secretion System

Many Gram-negative pathogens utilize type III secretion systems (T3SS) to translocate effector proteins into eukaryotic host cells. Expression of T3SS genes is highly regulated and is often coupled to type III secretory activity. Transcription of the Pseudomonas aeruginosa T3SS genes is coupled to secretion by a cascade of interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is an activator of type III gene transcription, ExsD binds ExsA to inhibit transcription, ExsC inhibits ExsD activity, and ExsE inhibits ExsC activity. The entire process is coupled to secretion by virtue of the fact that ExsE is a secreted substrate of the T3SS. Changes in the intracellular concentration of ExsE are thought to govern formation of the ExsC-ExsE, ExsC-ExsD, and ExsD-ExsA complexes. Whereas formation of the ExsC-ExsE complex allows ExsD to bind ExsA and transcription of the T3SS is repressed, formation of the ExsC-ExsD complex sequesters ExsD from ExsA and transcription of the T3SS is induced. In this study, we characterized the self-association states of ExsC, ExsD, and ExsE and the binding interactions of ExsC with ExsE and ExsD. ExsC exists as a homodimer and binds one molecule of ExsE substrate. Dimeric ExsC also interacts directly with ExsD to form a heterotetrameric complex. The difference in binding affinities between the ExsC-ExsE (Kd 1 nm) and ExsC-ExsD (Kd 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

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 Kd of approximately 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.