Two-component Response Research Articles

A Strategy for Antagonizing Quorum Sensing

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ∼60 Å, twice the ∼30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multidomain proteins.

Two-component dynamic responses of vortex matter near the vortex melting line in superconductingLa1.86Sr0.14CuO4 single crystals

We report the dynamics of moving vortices inLa1.86Sr0.14CuO4 single crystals studied via a transient voltage response (TVR) near the vortex meltingtransition. In the TVR signal, we could identify two types of relaxation caused byrearrangement of the vortex configuration. Based on careful temperature- andfield-dependent measurements of these two components, we suggest that the fastcomponent results from moving of the stable ordered vortex phase, while the slow one isgenerated during the annealing process of the metastable disordered vortex phase, which isinjected through the surface. As the temperature is lowered across the melting line, themetastable disordered phase in the heterogeneous mixture gets a longer life time dueto the reduced thermal fluctuations, occupying a larger portion of the sample.Consistent with this picture, the boundary of the slow relaxation is coincident withthe irreversibility line, which is known to be a property of the surface barriers.

The Effect of Pedaling Cadence on the Kinetics of Oxygen Uptake During Severe Intensity Exercise

During exhaustive severe intensity exercise, the oxygen uptake (VO2) increases exponentially, with a time constant of ∼30 s. After ∼1 to 2 min, a slow component emerges and drives the VO2 to its maximum. Pedaling cadence contributes to the metabolic demand at a given work rate and affects several responses to cycling exercise. PURPOSE: To determine the effect of pedaling cadence on parameters of the two-component VO2 response profile during severe intensity exercise. METHODS: Eight women (mean ± SD: age 22 ± 1 yr, height 161 ± 6 cm, and weight 58.8 ± 2.3 kg) and 10 men (age 23 ± 1 yr, height 180 ± 6 cm, and weight 82.9 ± 4.4 kg) performed exhaustive constant-power cycle ergometer tests using pedaling cadences of 60 rpm, 80 rpm, and 100 rpm. RESULTS: Times to exhaustion were smaller at higher cadences (220 ± 85 < 299 ± 118 < 368 ± 168 s), whereas VO2max values were the same at all cadences (2786 ± 729 = 2768 ± 749 = 2774 ± 732 ml/min). The mean response time of the primary response was faster at higher pedaling cadences (27 ± 5 < 32 ± 5 1890 ± 493 = 1899 ± 515 ml/min); and the time delay before the slow component was smaller at higher cadences (85 ± 11 < 105 ± 17 < 118 ± 19 s). CONCLUSION: These results demonstrate that pedaling cadence affects the VO2 response profile. The higher cadences speed the primary or fundamental response and hasten the emergence of the slow component. This may have implications for the sport of cycling and should be considered when evaluating cardio-respiratory and metabolic responses to cycle ergometer exercise.

Pharmacological analysis of the spatiotemporal activity pattern in the chicken optic tectum

Event Abstract Back to Event Pharmacological analysis of the spatiotemporal activity pattern in the chicken optic tectum Stefan Weigel1* and Harald Luksch1 1 Technische Universität München, Zoology, Germany The midbrain is an important processing area for sensory information in vertebrates. The optic tectum (TeO) and its mammalian counterpart, the superior colliculus, receive multimodal, topographic information and contain a sensory map which plays a role in spatial attention and in orientation movements. In avian, the TeO consists of 15 layers including separated input and output areas making it an ideal model system to study multimodal integration. Previous studies have mostly focused on characterization of particular cell types or cytochemistry. Only few studies have investigated network activation throughout the depth of the tectum. Our aim is to analyze the architecture and function of tectal local neuronal circuitry as well as the interplay between the TeO and modulatory midbrain areas e.g. the nuclei isthmi. To understand those complex interactions, we need to explore the spatiotemporal activity patterns with respect to the general cytoarchitecture of the TeO. We use an optical imaging approach with voltage sensitive dyes (RH795) to investigate population responses at a high temporal and spatial resolution in a chicken midbrain slice preparation. Thus, we can match activity patterns to the general layout of the TeO based on histological data. Based on histological data we can roughly link our findings with the general layout of the avian TeO. Here, we show inter- and intralaminar neuronal activities under different pharmacological regimes. The TeO section was activated by electrical stimulation of afferent layers, which mimicked the input from retinal ganglion cells. This evoked a two-component neuronal response consisting of a short (~10 ms) and a long-lasting component (up to several hundred ms) extending (i) in layer 5, where horizontally aligned neurons are located, and (ii) into deeper layers along radial oriented neurons. Ca2+-free solution and AMPA receptor restricted activity to the site of stimulation, indicating that most of the signal was postsynaptic and glutamatergic. Disinhibition enhanced signal amplitudes and spatial extent of the excitation. Interestingly, cholinergic transmitters were important in retention of the inhibition in the slice preparation indicating complex local networks. We will propose a simple model for the circuitry in the TeO. Keywords: avian, brain slice, midbrain, multimodal, optic tectum, visual, voltage sensitive dye imaging Conference: BC11 : Computational Neuroscience & Neurotechnology Bernstein Conference & Neurex Annual Meeting 2011, Freiburg, Germany, 4 Oct - 6 Oct, 2011. Presentation Type: Poster Topic: sensory processing (please use "sensory processing" as keyword) Citation: Weigel S and Luksch H (2011). Pharmacological analysis of the spatiotemporal activity pattern in the chicken optic tectum. Front. Comput. Neurosci. Conference Abstract: BC11 : Computational Neuroscience & Neurotechnology Bernstein Conference & Neurex Annual Meeting 2011. doi: 10.3389/conf.fncom.2011.53.00136 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Aug 2011; Published Online: 04 Oct 2011. * Correspondence: Dr. Stefan Weigel, Technische Universität München, Zoology, Freising, 85354, Germany, stefan.weigel@wzw.tum.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Stefan Weigel Harald Luksch Google Stefan Weigel Harald Luksch Google Scholar Stefan Weigel Harald Luksch PubMed Stefan Weigel Harald Luksch Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Histidine kinase two-component response regulator proteins regulate reproductive development, virulence, and stress responses of the fungal cereal pathogens Cochliobolus heterostrophus and Gibberella zeae.

Histidine kinase (HK) phosphorelay signaling is a major mechanism by which fungi sense their environment. The maize pathogen Cochliobolus heterostrophus has 21 HK genes, 4 candidate response regulator (RR) genes (SSK1, SKN7, RIM15, REC1), and 1 gene (HPT1) encoding a histidine phosphotransfer domain protein. Because most HKs are expected to signal through RRs, these were chosen for deletion. Except for pigment and slight growth alterations for rim15 mutants, no measurable altered phenotypes were detected in rim15 or rec1 mutants. Ssk1p is required for virulence and affects fertility and proper timing of sexual development of heterothallic C. heterostrophus. Pseudothecia from crosses involving ssk1 mutants ooze masses of single ascospores, and tetrads cannot be found. Wild-type pseudothecia do not ooze. Ssk1p represses asexual spore proliferation during the sexual phase, and lack of it dampens asexual spore proliferation during vegetative growth, compared to that of the wild type. ssk1 mutants are heavily pigmented. Mutants lacking Skn7p do not display any of the above phenotypes; however, both ssk1 and skn7 mutants are hypersensitive to oxidative and osmotic stresses and ssk1 skn7 mutants are more exaggerated in their spore-type balance phenotype and more sensitive to stress than single mutants. ssk1 mutant phenotypes largely overlap hog1 mutant phenotypes, and in both types of mutant, the Hog1 target gene, MST1, is not induced. ssk1 and hog1 mutants were examined in the homothallic cereal pathogen Gibberella zeae, and pathogenic and reproductive phases of development regulated by Ssk1 and Hog1 were found to mirror, but also vary from, those of C. heterostrophus.

Phenotypic characterization and genetic mapping of a new gene required for male and female gametophyte development in rice

Gametophyte development is an important process in flowering plants. Seeds are the primary organs for harvesting, and therefore spikelet fertility is especially vital for rice yield. Here, we report the characterization and genetic mapping of a novel rice male and female sterile mutant, mfs1, which was generated from a transgenic line of japonica rice variety Zhonghua 9. Anther transverse sections indicate that the microspores in these plants were abnormally degenerated at the meiotic leptotene stage, while the tapetum layer was not degenerated and remained present; therefore, no functional pollen grains formed in the mutant anther locules. In addition, the majority of mutant embryo sacs did not undergo differentiation or the differentiation was incomplete, resulting in the absence of functional embryo sac formation. Genetic analysis of three F2 populations indicated that the mutant phenotype was controlled by a single recessive gene. The mutant phenotype was confirmed by linkage analysis to be not due to a foreign T-DNA insertion. A map-based cloning strategy was employed to map the mutant gene, and, finally, the mfs1 locus was mapped to a 99-kb physical interval between simple sequence repeat markers RM12585 and D051 on rice chromosome 2. Sequence analysis indicates that 11 rice candidate genes are located in this region, including three encoded putative proteins and eight encoded known proteins, such as transcription factors having WRKY and zinc finger domains, a ubiquitin fusion degradation protein, chaperone protein clpB 1, a two-component response regulator, the ZOS2-04-C2H2 zinc finger protein, a peptidase of the T1 family and a protein kinase.

Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens

BackgroundOsmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions.ResultsA C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain.ConclusionsThis work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

Acetosyringone induces the accumulation of a set of RNAs in the Arbuscular Mycorrhiza fungus Glomus intraradices

Plant root exudates contain a range of low molecular weight metabolites, which are believed to trigger the structural and physiological changes associated with the progression and establishment of the mycorrhizal symbiosis. Glomus intraradices spores were incubated with acetosyringone (AS), and an overall increase in the hyphal respiration was observed, indicating a physiological response triggered by this plant regulator. A G. intraradices cDNA library was then screened with a total cDNA probe obtained from the AS-treated spores and mycelium. The cDNAs from different functional categories were identified as induced in G. intraradices when exposed to AS, such as protein synthesis, membrane transport, signal transduction, general metabolism, without assigned function, and no identity. Interestingly, a cDNA coding for a fragment of a histidine kinase was also induced by AS, suggesting a two-component mediated response. We also demonstrated the differential accumulation of a cruciform DNA-binding protein mRNA, termed as GiBP1. Time-course experiments demonstrated its rapid accumulation within 2h of induction with AS. These results indicate the presence of a set of fungal genes that are induced by the presence of the inducer. These findings are discussed in terms of the possible molecular events that follow the exchange of signals between the mycorrhizal symbionts.

Convergence of Ser/Thr and Two-component Signaling to Coordinate Expression of the Dormancy Regulon in Mycobacterium tuberculosis*[S

Signal transduction in Mycobacterium tuberculosis is mediated primarily by the Ser/Thr protein kinases and the two-component systems. The Ser/Thr kinase PknH has been shown to regulate growth of M. tuberculosis in a mouse model and in response to NO stress in vitro. Comparison of a pknH deletion mutant (ΔpknH) with its parental M. tuberculosis H37Rv strain using iTRAQ enabled us to quantify >700 mycobacterial proteins. Among these, members of the hypoxia- and NO-inducible dormancy (DosR) regulon were disregulated in the ΔpknH mutant. Using kinase assays, protein-protein interactions, and mass spectrometry analysis, we demonstrated that the two-component response regulator DosR is a substrate of PknH. PknH phosphorylation of DosR mapped to Thr(198) and Thr(205) on the key regulatory helix α10 involved in activation and dimerization of DosR. PknH Thr phosphorylation and DosS Asp phosphorylation of DosR cooperatively enhanced DosR binding to cognate DNA sequences. Transcriptional analysis comparing ΔpknH and parental M. tuberculosis revealed that induction of the DosR regulon was subdued in the ΔpknH mutant in response to NO. Together, these results indicate that PknH phosphorylation of DosR is required for full induction of the DosR regulon and demonstrate convergence of the two major signal transduction systems for the first time in M. tuberculosis.

Characterization of the CpxRA Regulon in Haemophilus ducreyi

The Haemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in Escherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.

Intralaminar and interlaminar activity within the rodent superior colliculus visualized with voltage imaging.

The superior colliculus (SC) is a midbrain structure that plays a role in converting sensation into action. Most SC research focuses on either in vivo extracellular recordings from behaving monkeys or patch-clamp recordings from smaller mammals in vitro. However, the activity of neuronal circuits is necessary to generate behavior, and neither of these approaches measures the simultaneous activity of large populations of neurons that make up circuits. Here, we describe experiments in which we measured changes in membrane potential across the SC map using voltage imaging of the rat SC in vitro. Our results provide the first high temporal and spatial resolution images of activity within the SC. Electrical stimulation of the SC evoked a characteristic two-component optical response containing a short latency initial-spike and a longer latency after-depolarization. Single-pulse stimulation in the superficial SC evoked a pattern of intralaminar and interlaminar spread that was distinct from the spread evoked by the same stimulus applied to the intermediate SC. Intermediate layer stimulation produced a more extensive and more ventrally located activation of the superficial layers than did stimulation in the superficial SC. Together, these results indicate the recruitment of dissimilar subpopulations of circuitry depending on the layer stimulated. Field potential recordings, pharmacological manipulations, and timing analyses indicate that the patterns of activity were physiologically relevant and largely synaptically driven. Therefore, voltage imaging is a powerful technique for the study of spatiotemporal dynamics of electrical signaling across neuronal populations, providing insight into neural circuits that underlie behavior.

Redox sensor SsrB Cys 203 enhances Salmonella fitness against nitric oxide generated in the host immune response to oral infection

We show herein that the Salmonella pathogenicity island 2 (SPI2) response regulator SsrB undergoes S-nitrosylation upon exposure of Salmonella to acidified nitrite, a signal encountered by this enteropathogen in phagosomes of macrophages. Mutational analysis has identified Cys(203) in the C-terminal dimerization domain of SsrB as the redox-active residue responding to nitric oxide (NO) congeners generated in the acidification of nitrite. Peroxynitrite and products of the autooxidation of NO in the presence of oxygen, but not hydrogen peroxide, inhibit the DNA-binding capacity of SsrB, demonstrating the selectivity of the reaction of Cys(203) with reactive nitrogen species (RNS). These findings identify the two-component response regulator SsrB Cys(203) as a thiol-based redox sensor. A C203S substitution protects SsrB against the attack of RNS while preserving its DNA-binding capacity. When exposed to SPI2-inducing conditions, Salmonella expressing the wild-type ssrB allele or the ssrB C203S variant sustain transcription of the sifA, sspH2, and srfJ effector genes. Nonetheless, compared with the strain expressing a redox-resistant SsrB C203S variant, wild-type Salmonella bearing the NO-responsive allele exhibit increased fitness when exposed to RNS in an NRAMP(R), C3H/HeN murine model of acute oral infection. Given the widespread occurrence of the wild-type allele in Salmonella enterica, these findings indicate that SsrB Cys(203) increases Salmonella virulence by serving as a redox sensor of NO resulting from the host immune response to oral infection.

Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43

BackgroundThe cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria.MethodsIn this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen Klebsiella pneumoniae were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of pmr genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and in vitro phosphor-transfer assay.ResultsDeletion of the rcsB, which encoded an activator for the production of capsular polysaccharide, had a minor effect on K. pneumoniae resistance to polymyxin B. On the other hand, deletion of ugd or pmrF gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in Salmonella, expression of pmrD in K. pneumoniae was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of pmr for the resistance to polymyxin B.ConclusionsThe study reports a role of the capsular polysaccharide level and the pmr genes for K. pneumoniae resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of pmr expression was demonstrated. In comparison to the pmr regulation in Salmonella, PhoP in K. pneumoniae plays a major regulatory role in polymyxin B resistance.

Proteome of the phytopathogen Xanthomonas citri subsp. citri: a global expression profile

BackgroundCitrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS).ResultsIn order to gain insight into the metabolism of Xac, cells were grown on two different media (NB - Nutrient Broth and TSE - Tryptone Sucrose broth enriched with glutamic acid), and proteins were proteolyzed with trypsin and examined by 2D LC-MS/MS. Approximately 39% of all predicted proteins by annotation of Xac were identified with their component peptides unambiguously assigned to tandem mass spectra. The proteins, about 1,100, were distributed in all annotated functional categories.ConclusionsThis is the first proteomic reference map for the most aggressive strain of Xanthomonas pathogen of all orange varieties. The compilation of metabolic pathways involved with bacterial growth showed that Xac expresses a complete central and intermediary metabolism, replication, transcription and translation machineries and regulation factors, distinct membrane transporters (ABC, MFS and pumps) and receptors (MCP, TonB dependent and metabolites acquisition), two-component systems (sensor and regulatory components) and response regulators. These data corroborate the growth curve in vitro and are the first reports indicating that many of these genome annotated genes are translated into operative in Xac. This proteomic analysis also provided information regarding the influence of culture medium on growth and protein expression of Xac.

The two-component sensor response regulator RoxS/RoxR plays a role in Pseudomonas aeruginosa interactions with airway epithelial cells

Pseudomonas aeruginosa is an opportunistic pathogen that infects the lungs of patients with cystic fibrosis causing aberrant and destructive neutrophil (PMN)-dominated inflammation of airways. Interaction of P. aeruginosa with the lung epithelial barrier resulting in trans-epithelial PMN migration likely represents a key event during PMN recruitment. To investigate bacterial factors involved in interactions with lung epithelial cells, a mutant library of two-component system response regulators was evaluated to identify mutants exhibiting defects in the ability to induce PMN trans-epithelial migration. Of forty-eight mutants, five reproducibly demonstrated a reduced PMN trans-epithelial migration response. All five mutants also exhibited a decreased ability to interact with lung epithelial cells. One mutant identified lacks the response regulator gene roxR, which has not previously been reported to be involved regulating factors that facilitate interactions with lung epithelial cells. This finding suggests that RoxR likely regulates genes with relevance to P. aeruginosa mediated lung disease.

Investigation of the dependence of the coherent dynamics of a bismuth lattice on the crystal excitation level

Fully symmetric coherent phonons in bismuth have been investigated in a wide range of pumppulse energies by the pump-probe method using femtosecond laser pulses. It has been shown that in the linear regime, which is implemented only for low pump intensities, the coherent amplitude is proportional to the pump intensity, whereas the relaxation rate of the coherent state and its frequency remain unchanged. In the nonlinear regime, which can be divided into superlinear and sublinear regions, the relaxation rate of a photoinduced lattice state can be approximated by a two-component response, where only one component depends strongly on the pump intensity. The nature of the coherence of the phonon subsystem, which is created by an ultrashort laser pulse, is discussed on the basis of the analysis of the data.

Swarming ofPseudomonas aeruginosaIs Controlled by a Broad Spectrum of Transcriptional Regulators, Including MetR

Pseudomonas aeruginosa exhibits swarming motility on semisolid surfaces (0.5 to 0.7% agar). Swarming is a more than just a form of locomotion and represents a complex adaptation resulting in changes in virulence gene expression and antibiotic resistance. In this study, we used a comprehensive P. aeruginosa PA14 transposon mutant library to investigate how the complex swarming adaptation process is regulated. A total of 233 P. aeruginosa PA14 transposon mutants were verified to have alterations in swarming motility. The swarming-associated genes functioned not only in flagellar or type IV pilus biosynthesis but also in processes as diverse as transport, secretion, and metabolism. Thirty-three swarming-deficient and two hyperswarming mutants had transposon insertions in transcriptional regulator genes, including genes encoding two-component sensors and response regulators; 27 of these insertions were newly identified. Of the 25 regulatory mutants whose swarming motility was highly impaired (79 to 97%), only 1 (a PA1458 mutant) had a major defect in swimming, suggesting that this regulator might influence flagellar synthesis or function. Twitching motility, which requires type IV pili, was strongly affected in only two regulatory mutants (pilH and PA2571 mutants) and was moderately affected in three other mutants (algR, ntrB, and nosR mutants). Microarray analyses were performed to compare the gene expression profile of a swarming-deficient PA3587 mutant to that of the wild-type PA14 strain under swarming conditions. PA3587 showed 63% homology to metR, which encodes a regulator of methionine biosynthesis in Escherichia coli. The observed dysregulation in the metR mutant of nine different genes required for swarming motility provided a possible explanation for the swarming-deficient phenotype of this mutant.

Molecular analysis of the copper-responsive CopRSCD of a pathogenic Pseudomonas fluorescens strain

CopRS/CopABCD is one of the known systems that control copper homeostasis in bacteria. Although CopRS/CopABCD homologues are found to exist in Pseudomonas fluorescens, the potential role of this system in P. fluorescens has not been investigated. In this study a genetic cluster, consisting of copR, S, C, and D but lacking copAB, was identified in a pathogenic P. fluorescens strain (TSS) isolated from diseased fish. The copRSCD cluster was demonstrated to be required for full copper resistance and regulated at the transcription level by Cu. Expression of copCD is regulated directly by the two-component response regulator CopR, which also regulates its own expression. Interruption of the regulated expression of copR affected bacterial growth, biofilm formation, and tissue dissemination and survival. A mutant CopR, which lacks the N-terminal signal receiver domain and is constitutively active, was found to have an attenuating effect on bacterial virulence when expressed in TSS. To our knowledge, this is the first report that suggests a link between CopR and bacterial pathogenicity in P. fluorescens.

MzrA: a novel modulator of the EnvZ/OmpR two‐component regulon

Analysis of suppressors that alleviate the acute envelope stress phenotype of a ΔbamBΔdegP strain of Escherichia coli identified a novel protein MzrA and pleiotropic envZ mutations. Genetic evidence shows that overexpression of MzrA – formerly known as YqjB and EcfM – modulates the activity of EnvZ/OmpR similarly to pleiotropic EnvZ mutants and alter porin expression. However, porin expression in strains devoid of MzrA or overexpressing it is still sensitive to medium osmolarity, pH and procaine, all of which modulate EnvZ/OmpR activities. Thus, MzrA appears to alter the output of the EnvZ/OmpR system but not its ability to receive and respond to various environmental signals. Localization and topology experiments indicate that MzrA is a type II membrane protein, with its N-terminus exposed in the cytoplasm and C-terminus in the periplasm. Bacterial two-hybrid experiments determined that MzrA specifically interacts with EnvZ but not with OmpR or the related membrane sensor kinase, CpxA. This and additional genetic and biochemical evidence suggest that the interaction of MzrA with EnvZ would either enhance EnvZ's kinase activity or reduce its phosphatase activity, thus elevating the steady state levels of OmpR∼P. Furthermore, our data show that MzrA links the two-component envelope stress response regulators, CpxA/CpxR and EnvZ/OmpR.

σSControls Multiple Pathways Associated with Intracellular Multiplication ofLegionella pneumophila

Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes sigma(S), does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that sigma(S) affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that sigma(S) affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between sigma(S) and the posttranscriptional regulator CsrA. We analyzed the network of sigma(S)-controlled genes by mutational analysis of transcriptional regulators affected by sigma(S). One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that sigma(S) is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.