RESUMO
The opportunistic pathogen Pseudomonas aeruginosa relies upon type IV pili (Tfp) for host colonization and virulence. Tfp are retractile surface appendages that promote adherence to host tissue and mediate twitching motility, a form of surface-associated translocation. Tfp are composed of a major structural pilin protein (PilA), several less abundant, fiber-associated pilin-like proteins (FimU, PilV, PilW, PilX, and PilE), and a pilus-associated tip adhesin and surface sensor (PilY1). Several proteins critical for Tfp biogenesis and surface sensing are encoded by the fimU-pilVWXY1Y2E operon. Tfp biogenesis is regulated by the global transcription factor Vfr and its allosteric effector, cyclic AMP (cAMP). Our investigation into the basis for reduced Tfp production in cAMP/vfr mutants revealed a defect in the expression of the fimU operon. We found that cAMP/Vfr activation of the fimU operon occurs via direct binding of Vfr to a specific fimU promoter sequence. We also refined the role of the AlgZ/AlgR two-component system in fimU regulation by demonstrating that phosphorylation of the response regulator AlgR is required for maximal binding to the fimU promoter region in vitro. Vfr also regulates expression of the algZR operon, revealing an indirect regulatory loop affecting fimU operon transcription. Overall, these results demonstrate that two linked but independent regulatory systems couple the expression of Tfp biogenesis and surface sensing genes and highlight the regulatory complexity governing expression of P. aeruginosa virulence factors. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections. An extensive repertoire of virulence factors aid in P. aeruginosa pathogenesis. Type IV pili (Tfp) play a critical role in host colonization and infection by promoting adherence to host tissue, facilitating twitching motility and mediating surface-associated behaviors. The fimU operon encodes several pilus-associated proteins that are essential for proper Tfp function and surface sensing. In this study, we report that linked but independent regulatory systems dictate Tfp biogenesis. We also demonstrated the importance of different phosphorylation states of the AlgZ/AlgR two-component system and its role in Tfp biogenesis. Overall, this study furthers our understanding of the complex regulatory mechanisms that govern the production of a critical and multifaceted virulence factor.
Assuntos
Proteínas de Fímbrias , Pseudomonas aeruginosa , Proteínas de Fímbrias/genética , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/genética , Fatores de Virulência/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/análogos & derivados , Óxido Nítrico/metabolismo , Shewanella/fisiologia , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Shewanella/genética , Transdução de SinaisRESUMO
UNLABELLED: Shewanella oneidensis strain MR-1 is a facultative anaerobe that thrives in redox-stratified environments due to its ability to utilize a wide array of terminal electron acceptors. Conversely, the electron donors utilized by S. oneidensis are more limited and include products of primary fermentation such as lactate, pyruvate, formate, and hydrogen. Lactate, pyruvate, and hydrogen metabolisms inS. oneidensis have been described previously, but little is known about the role of formate oxidation in the ecophysiology of these bacteria. Formate is produced by S. oneidensis through pyruvate formate lyase during anaerobic growth on carbon sources that enter metabolism at or above the level of pyruvate, and the genome contains three gene clusters predicted to encode three complete formate dehydrogenase complexes. To determine the contribution of each complex to formate metabolism, strains lacking one, two, or all three annotated formate dehydrogenase gene clusters were generated and examined for growth rates and yields on a variety of carbon sources. Here, we report that formate oxidation contributes to both the growth rate and yield of S. oneidensis through the generation of proton motive force. Exogenous formate also greatly accelerated growth on N-acetylglucosamine, a carbon source normally utilized very slowly by S. oneidensis under anaerobic conditions. Surprisingly, deletion of all three formate dehydrogenase gene clusters enabled growth of S. oneidensis using pyruvate in the absence of a terminal electron acceptor, a mode of growth never before observed in these bacteria. Our results demonstrate that formate oxidation is a fundamental strategy under anaerobic conditions for energy conservation inS. oneidensis. IMPORTANCE: Shewanella species have garnered interest in biotechnology applications for their ability to respire extracellular terminal electron acceptors, such as insoluble iron oxides and electrodes. While much effort has gone into studying the proteins for extracellular electron transport, how electrons generated through the oxidation of organic carbon sources enter this pathway remains understudied. Here, we quantify the role of formate oxidation in the anaerobic physiology of Shewanella oneidensis Formate oxidation contributes to both the growth rate and yield on a variety of carbon sources through the generation of proton motive force. Advances in our understanding of the anaerobic metabolism of S. oneidensis are important for our ability to utilize and engineer this organism for applications in bioenergy, biocatalysis, and bioremediation.
Assuntos
Proteínas de Bactérias/metabolismo , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/genética , Formiato Desidrogenases/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Óperon , Filogenia , Shewanella/genéticaRESUMO
Shewanella oneidensis strain MR-1 is a dissimilatory metal-reducing bacterium frequently found in aquatic sediments. In the absence of oxygen, S. oneidensis can respire extracellular, insoluble oxidized metals, such as iron (hydr)oxides, making it intimately involved in environmental metal and nutrient cycling. The reduction of ferric iron (Fe(3+)) results in the production of ferrous iron (Fe(2+)) ions, which remain soluble under certain conditions and are toxic to cells at higher concentrations. We have identified an inner membrane protein in S. oneidensis, encoded by the gene SO_4475 and here called FeoE, which is important for survival during anaerobic iron respiration. FeoE, a member of the cation diffusion facilitator (CDF) protein family, functions to export excess Fe(2+) from the MR-1 cytoplasm. Mutants lacking feoE exhibit an increased sensitivity to Fe(2+). The export function of FeoE is specific for Fe(2+), as an feoE mutant is equally sensitive to other metal ions known to be substrates of other CDF proteins (Cd(2+), Co(2+), Cu(2+), Mn(2+), Ni(2+), or Zn(2+)). The substrate specificity of FeoE differs from that of FieF, the Escherichia coli homolog of FeoE, which has been reported to be a Cd(2+)/Zn(2+) or Fe(2+)/Zn(2+) exporter. A complemented feoE mutant has an increased growth rate in the presence of excess Fe(2+) compared to that of the ΔfeoE mutant complemented with fieF. It is possible that FeoE has evolved to become an efficient and specific Fe(2+) exporter in response to the high levels of iron often present in the types of environmental niches in which Shewanella species can be found.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Compostos Ferrosos/metabolismo , Ferro/metabolismo , Shewanella/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Oxirredução , Shewanella/metabolismoRESUMO
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.
Assuntos
Elementos de DNA Transponíveis/genética , Genoma Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Redes e Vias Metabólicas/genética , Shewanella/genética , Shewanella/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/genética , Redes Reguladoras de Genes/genética , Genômica , Mutagênese Sítio-DirigidaRESUMO
Riboflavin (vitamin B2) is the precursor of flavin mononucleotide and flavin adenine dinucleotide, which are cofactors essential for a host of intracellular redox reactions. Microorganisms synthesize flavins de novo to fulfill nutritional requirements, but it is becoming increasingly clear that flavins play a wider role in cellular physiology than was previously appreciated. Flavins mediate diverse processes beyond the cytoplasmic membrane, including iron acquisition, extracellular respiration, and interspecies interactions. While investigating the regulation of flavin electron shuttle biosynthesis in the Gram-negative gammaproteobacterium Shewanella oneidensis, we discovered that a riboflavin biosynthetic gene (ribBA) annotated as encoding a bifunctional 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase/GTP cyclohydrolase II does not possess both functions. The novel gene, renamed ribBX here, encodes an amino-terminal DHBP synthase domain. The carboxy-terminal end of RibBX not only lacks GTP cyclohydrolase II activity but also has evolved a different function altogether in S. oneidensis, regulating the activity of the DHBP synthase domain. Phylogenetic analysis revealed that the misannotation of ribBX as ribBA is rampant throughout the phylum Proteobacteria (40% of 2,173 annotated ribBA genes) and that ribBX emerged early in the evolution of this group of microorganisms. We examined the functionality of representative ribBX genes from Beta-, Gamma-, and Epsilonproteobacteria and found that, consistent with sequence-based predictions, the encoded GTP cyclohydrolase II domains lack catalytic activity. The persistence of ribBX in the genomes of so many phylogenetically divergent bacterial species lends weight to the argument that ribBX has evolved a function which lends a selective advantage to the host.
Assuntos
Vias Biossintéticas/genética , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Proteobactérias/enzimologia , Proteobactérias/genética , Riboflavina/biossíntese , Evolução Molecular , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Shewanella oneidensis couples oxidation of lactate to respiration of many substrates. Here we report that llpR (L-lactate-positive regulator, SO_3460) encodes a positive regulator of L-lactate utilization distinct from previously studied regulators. We also demonstrate D-lactate inhibition of L-lactate utilization in S. oneidensis, resulting in preferential utilization of the D isomer.
Assuntos
Ácido Láctico/química , Ácido Láctico/metabolismo , Shewanella/metabolismo , Fatores de Transcrição/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Oxirredução , EstereoisomerismoRESUMO
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.
Assuntos
Ciclo do Ácido Cítrico , Regulação Bacteriana da Expressão Gênica , Shewanella/genética , Shewanella/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , MutaçãoRESUMO
Transcription of the Pseudomonas aeruginosa type III secretion system is controlled by ExsA, a member of the AraC/XylS family of regulators. Each ExsA-dependent promoter contains two adjacent binding sites for monomeric ExsA. The promoter-proximal site (binding site 1) consists of highly conserved GnC and TGnnA sequences that are individually recognized by the two helix-turn-helix (HTH) DNA-binding motifs of an ExsA monomer. While the GnC and TGnnA sequences are important for binding to site 1, the promoter-distal binding sites (site 2) lack obvious similarity among themselves or with binding site 1. In the present study, we demonstrate that site 2 in the P(exsC) promoter region contains a GnC sequence that is functionally equivalent to the GnC in site 1 and recognized by the first HTH motif of an ExsA monomer. Likewise, the second HTH interacts with an adenine residue in binding site 2. Although several candidate GnC sequences are also present in site 2 of the P(exsD), P(exoT), and P(pcrG) promoters, the GnC sequences were not required for ExsA-dependent transcription or ExsA binding. A comparison of hybrid promoters composed of binding site 2 from one promoter fused to binding site 1 derived from another promoter indicates that ExsA-binding affinity, promoter strength, and the degree of promoter bending are properties that are largely determined by binding site 2. Based on these data, we propose that the manner in which ExsA interacts with binding site 2 at the P(exsC) promoter is distinct from the interactions occurring at other promoters.
Assuntos
Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas/genética , Pseudomonas aeruginosa/metabolismo , Transativadores/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutagênese , Mutação , Ligação Proteica , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genéticaRESUMO
ExsA is a transcriptional activator of the Pseudomonas aeruginosa type III secretion system (T3SS) and a member of the AraC/XylS protein family. Each of the 10 ExsA-dependent promoter regions that define the T3SS regulon has two adjacent binding sites for monomeric ExsA. Whereas the promoter-proximal sites (binding site 1) contain highly conserved GnC and TGnnA sequences that are separated by â¼10 bp, the promoter-distal sites (binding site 2) share no obvious sequence similarity to each other or to the binding site 1 consensus. In the present study, we used footprinting with Fe-BABE (a protein-labeling reagent that can be conjugated to cysteine residues) to demonstrate that the two ExsA monomers bind to the P(exsC), P(exsD), P(exoT), and P(pcrG) promoters in a head-to-tail orientation. The footprinting data further indicate that the conserved GnC and TGnnA sequences constitute binding site 1. When bound to site 1, the first helix-turn-helix (HTH) motif of ExsA interacts with the conserved GnC sequence, and the second HTH interacts at or near the TGnnA sequences. Genetic data using the P(exoT) promoter indicate that residues L198 and T199 in the first HTH motif of ExsA contact the guanine in the GnC sequence and that residue K202, also in the first HTH motif, contacts the cytosine. Likewise, evidence is presented that residues Q248, Y250, T252, and R257 located in the second HTH motif contribute to the recognition of the TGnnA sequence. These combined data define interactions of ExsA with site 1 on the P(exoT) promoter and provide insight into the nature of the interactions involved in recognition of binding site 2.
Assuntos
ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Pseudomonas aeruginosa/metabolismo , Transativadores/metabolismo , ADP Ribose Transferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Proteínas Ativadoras de GTPase/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Regiões Promotoras Genéticas , Ligação Proteica , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genéticaRESUMO
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.
Assuntos
Flavinas/metabolismo , Shewanella/metabolismo , Elétrons , Oxirredução , Vitamina K 2/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Proteína Receptora de AMP Cíclico/genética , DNA Bacteriano , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Pseudomonas aeruginosa/genética , Fatores de Virulência/genéticaRESUMO
ExsA is a member of the AraC family of transcriptional activators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). ExsA-dependent promoters consist of two binding sites for monomeric ExsA located approximately 50 bp upstream of the transcription start sites. Binding to both sites is required for recruitment of sigma(70)-RNA polymerase (RNAP) to the promoter. ExsA-dependent promoters also contain putative -35 hexamers that closely match the sigma(70) consensus but are atypically spaced 21 or 22 bp from the -10 hexamer. Because several nucleotides located within the putative -35 region are required for ExsA binding, it is unclear whether the putative -35 region makes an additional contribution to transcription initiation. In the present study we demonstrate that the putative -35 hexamer is dispensable for ExsA-independent transcription from the P(exsC) promoter and that deletion of sigma(70) region 4.2, which contacts the -35 hexamer, has no effect on ExsA-independent transcription from P(exsC). Region 4.2 of sigma(70), however, is required for ExsA-dependent activation of the P(exsC) and P(exsD) promoters. Genetic data suggest that ExsA directly contacts region 4.2 of sigma(70), and several amino acids were found to contribute to the interaction. In vitro transcription assays demonstrate that an extended -10 element located in the P(exsC) promoter is important for overall promoter activity. Our collective data suggest a model in which ExsA compensates for the lack of a -35 hexamer by interacting with region 4.2 of sigma(70) to recruit RNAP to the promoter.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/metabolismo , Fator sigma/metabolismo , Transativadores/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano , RNA Polimerases Dirigidas por DNA/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator sigma/genética , Transativadores/genética , Transcrição GênicaRESUMO
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.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/genética , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/genética , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Western Blotting , Cromatografia em Gel , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Exotoxinas/genética , Exotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/metabolismo , Dados de Sequência Molecular , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/genética , Homologia de Sequência de Aminoácidos , Fatores de Virulência/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Proteínas de Bactérias/genética , DNA Bacteriano , Mutação , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Transativadores/genética , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transativadores/química , Transativadores/metabolismo , Ativação Transcricional , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Pseudomonas aeruginosa/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genéticaRESUMO
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 (ExsA(His)) and found to be monomeric in solution. ExsA(His) 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 ExsA(His) molecule bound to DNA while the lower-mobility complex results from the binding of two ExsA(His) molecules. DNase I protection assays demonstrate that the ExsA(His) binding site overlaps the -35 RNA polymerase binding site and extends upstream an additional approximately 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 ExsA(His) binding. The combined data support a model in which two ExsA(His) molecules bind adjacent sites on the promoter to activate T3SS gene transcription.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Pegada de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Dados de Sequência Molecular , Fases de Leitura Aberta , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transativadores/química , Transativadores/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/metabolismo , Proteínas de Bactérias/genética , Flagelos/metabolismo , Bactérias Gram-Negativas/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMO
Transcription of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced under Ca(2+)-limiting growth conditions or following the contact of the bacteria with host cells. The regulatory response to low Ca(2+) 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 Ca(2+)-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 Ca(2+)-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.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Proteínas de Bactérias/fisiologia , Células CHO , Sinalização do Cálcio/genética , Cricetinae , Cricetulus , Regulação para Baixo/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Transporte Proteico/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/fisiologia , Transativadores/genética , Transativadores/metabolismoRESUMO
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 (K(d) 1 nm) and ExsC-ExsD (K(d) 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.