Relationship of the transcription factor MexT to quorum sensing and virulence in Pseudomonas aeruginosa.

IMPORTANCE: Pseudomonas aeruginosa is an opportunistic bacterial pathogen. Many of its virulence genes are regulated by quorum sensing (QS), a form of cell-to-cell communication. P. aeruginosa QS consists of three interlinked circuits, LasI-R, Rhl-R, and Pseudomonas quinolone signal (PQS). Additionally, its QS system is interconnected with other regulatory networks, which help optimize gene expression under variable conditions. The numbers of genes regulated by QS differ substantially among P. aeruginosa strains. We show that a regulatory factor MexT, which is activated in response to certain antibiotics, downregulates the RhlI-R circuit and in turn measurably lowers virulence in a nematode worm infection model. Our findings help understand how existing and future therapeutic interventions for P. aeruginosa infections may impact this bacterium's gene regulation and physiology.

Evolution of the Pseudomonas aeruginosa quorum-sensing hierarchy.

The bacterial pathogen Pseudomonas aeruginosa activates expression of many virulence genes in a cell density-dependent manner by using an intricate quorum-sensing (QS) network. QS in P. aeruginosa involves two acyl-homoserine-lactone circuits, LasI-LasR and RhlI-RhlR. LasI-LasR is required to activate many genes including those coding for RhlI-RhlR. P. aeruginosa causes chronic infections in the lungs of people with cystic fibrosis (CF). In these infections, LasR mutants are common, but rhlR-rhlI expression has escaped LasR regulation in many CF isolates. To better understand the evolutionary trajectory of P. aeruginosa QS in chronic infections, we grew LasR mutants of the well-studied P. aeruginosa strain, PAO1, in conditions that recapitulate an environment where QS signal synthesis by other bacteria might still occur. When QS is required for growth, addition of the RhlI product butyryl-homoserine lactone (C4-HSL), or bacteria that produce C4-HSL, to LasR mutants results in the rapid emergence of a population with a LasR-independent RhlI-RhlR QS system. These evolved populations exhibit subsequent growth without added C4-HSL. The variants that emerge have mutations in mexT, which codes for a transcription factor that controls expression of multiple genes. LasR-MexT mutants have a competitive advantage over both the parent LasR mutant and a LasR-MexT-RhlR mutant. Our findings suggest a plausible evolutionary trajectory for QS in P. aeruginosa CF infections where LasR mutants arise during infection, but because these mutants are surrounded by C4-HSL-producing P. aeruginosa, variants rewired to have a LasR-independent RhlIR system quickly emerge.

Modulation of Pseudomonas aeruginosa Quorum Sensing by Glutathione.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels.IMPORTANCEPseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling.

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼ 10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.

A distinct model of synergism between a processive endocellulase (TfCel9A) and an exocellulase (TfCel48A) from Thermobifida fusca.

Lignocellulosic biomass is digested in nature by the synergistic activities of enzymes with complementary properties, and understanding synergistic interactions will improve the efficiency of industrial biomass use for sustainable fuels and chemicals. Cel9A and Cel48A from a model bacterium, Thermobifida fusca (TfCel9A and TfCel48A, respectively), are two cellulases with different properties and have previously been shown to synergize well with each other. TfCel9A is a processive endocellulase with relatively high activity on crystalline cellulose. TfCel48A is a reducing end-directed exocellulase with very low activity on crystalline cellulose. Neither enzyme fits its respective role in the classical synergism model of enzymatic cellulose digestion. Using the results of time course, endpoint, and sequential addition activity assays, we propose a model of synergistic cooperation between the two cellulases. TfCel9A is most effective on fresh bacterial cellulose with a presumably uniform surface at the molecular level. Its processive activity likely erodes the surface and thus reduces its own activity. TfCel48A is able to hydrolyze the TfCel9A-modified substrate efficiently and replenish the uniform surface required by TfCel9A, creating a feedback mechanism. The model of synergistic interactions is comparable to an earlier proposed model for Trichoderma reesei Cel7A and Cel7B, but the roles of endo- and exocellulases are reversed, a finding which suggests that bacteria and fungi may have evolved different approaches to efficient biomass degradation.

Cel48A from Thermobifida fusca: structure and site directed mutagenesis of key residues.

Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Cellulases from the glycoside hydrolase (GH) family 48 constitute an important component of bacterial biomass degrading systems and structures of three enzymes from this family have been previously published. We report a new crystal structure of TfCel48A, a reducing end directed exocellulase from Thermobifida fusca, which shows that this enzyme shares important structural features with the other members of the GH48 family. The active site tunnel entrance of the known GH48 exocellulases is enriched in aromatic residues, which are known to interact well with anhydroglucose units of cellulose. We carried out site-directed mutagenesis studies of these aromatic residues (Y97, F195, Y213, and W313) along with two non-aromatic residues (N212 and S311) also located around the tunnel entrance and a W315 residue inside the active site tunnel. Only the aromatic residues located around the tunnel entrance appear to be important for the ability of TfCel48A to access individual cellulose chains on bacterial cellulose (BC), a crystalline substrate. Both Trp residues were found to be important for the processivity of TfCel48A on BC and phosphoric acid swollen cellulose (PASC), but only W313 appears to play a role in the ability of the enzyme to access individual cellulose chains in BC. When acting on BC, reduced processivity was found to correlate with reduced enzyme activity. The reverse, however, is true when PASC is the substrate. Presumably, higher density of available cellulose chain ends and the amorphous nature of PASC explain the increased initial activity of mutants with lower processivity.

Two-parameter kinetic model based on a time-dependent activity coefficient accurately describes enzymatic cellulose digestion.

Lignocellulosic biomass is a potential source of renewable, low-carbon-footprint liquid fuels. Biomass recalcitrance and enzyme cost are key challenges associated with the large-scale production of cellulosic fuel. Kinetic modeling of enzymatic cellulose digestion has been complicated by the heterogeneous nature of the substrate and by the fact that a true steady state cannot be attained. We present a two-parameter kinetic model based on the Michaelis-Menten scheme ( Michaelis, L., and Menten, M. L. ( 1913 ) Biochem. Z. , 49 , 333 - 369 ) with a time-dependent activity coefficient analogous to fractal-like kinetics formulated by Kopelman ( Kopelman, R. ( 1988 ) Science 241 , 1620 - 1626 ). We provide a mathematical derivation and experimental support to show that one of the parameters is a total activity coefficient and the other is an intrinsic constant that reflects the ability of the cellulases to overcome substrate recalcitrance. The model is applicable to individual cellulases and their mixtures at low-to-medium enzyme loads. Using biomass degrading enzymes from cellulolytic bacterium Thermobifida fusca , we show that the model can be used for mechanistic studies of enzymatic cellulose digestion. We also demonstrate that it applies to the crude supernatant of the widely studied cellulolytic fungus Trichoderma reesei ; thus it can be used to compare cellulases from different organisms. The two parameters may serve a similar role to Vmax, KM, and kcat in classical kinetics. A similar approach may be applicable to other enzymes with heterogeneous substrates and where a steady state is not achievable.

Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization.

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble substrate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate. These findings are relevant for kinetic modeling and microscopy studies of modular glycoside hydrolases.

Determination of the catalytic base in family 48 glycosyl hydrolases.

The catalytic base in family 48 glycosyl hydrolases has not been previously established experimentally. Based on structural and modeling data published to date, we used site-directed mutagenesis and azide rescue activity assays to show definitively that the catalytic base in Thermobifida fusca Cel48A is aspartic acid 225. Of the tested mutants, only Cel48A with the D225E mutation retained partial activity on soluble and insoluble substrates. In azide rescue experiments, only the D225G mutation, in the smallest residue tested, showed an increase in activity with added azide.

Dynamics of cheater invasion in a cooperating population of Pseudomonas aeruginosa.

Pseudomonas aeruginosa quorum sensing (QS) regulates expression of dozens of genes in a cell density-dependent manner. Many QS-regulated genes code for production of extracellular factors, "public goods" that can benefit the entire population. This cooperation encourages individuals to cheat by using but not producing public goods. QS also controls expression of a limited number of genes encoding "private" cellular enzymes like Nuh, an enzyme involved in adenosine catabolism. Growth of P. aeruginosa on casein requires QS-regulated production of an extracellular protease and is an example of cooperative behavior. When P. aeruginosa is transferred daily on casein, QS mutants emerge. These cheaters have mutations in lasR, which encodes the primary QS transcription factor. When growth is on casein and adenosine, cheater emergence is constrained. Here, we report the dynamics of LasR mutant invasion during growth on casein or casein plus adenosine. We show that LasR mutants have the greatest advantage during early to mid-logarithmic growth on casein. Addition of adenosine to casein medium constrains cheaters throughout growth. Our data support the view that co-regulation of the public protease and the private nucleosidase by QS stabilizes cooperation, and the data are not consistent with other proposed alternate hypotheses.

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor.

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies.

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.

Experimental and modeling studies of an unusual water-filled pore structure with possible mechanistic implications in family 48 cellulases.

Molecular dynamics simulations were used to study the possible catalytic role of an unusual conserved water-filled pore structure in the family 48 cellulase enzyme Cel48A from Thermobifida fusca. It was hypothesized that this pore serves as the pathway for the water molecules consumed in the hydrolysis catalyzed by the enzyme to reach the active site in a continuous stream to participate in the processive reactions. Theoretical mutants of this enzyme were created in which all of the residues lining the pore were made hydrophobic, which had the effect in molecular dynamics simulations of emptying the pore of water molecules and preventing any from passing through the pore on the simulation time scale. Mutants with smaller numbers of substitutions of this nature, which could be created experimentally by site-directed mutagenesis, were also identified from simulations, and these proteins were subsequently produced in Escherichia coli, expressed and purified, but were found to not fold in a manner similar to the wild type protein, preventing the determination of the importance of the water pore for activity. It is possible that the presence of a small vacuum in the pore was responsible for the instability of the mutants. In addition, alternate pathways were observed in the simulations that would allow water molecules to reach the active site of the enzyme, suggesting that the hypothesis that the pore has functional significance might be incorrect.

Cellulase processivity.

There are two types of processive cellulases, exocellulases and processive endoglucanases. There are also two classes of exocellulases, ones that attack the reducing ends of cellulose chains and ones that attack the nonreducing ends. There are a number of ways of assaying processivity but none of them are ideal. It appears that exocellulases, all of which have their active sites in a tunnel, couple movement along a cellulose chain with cleavage of cellobiose from the end of the cellulose molecule. There are two sets of structures that suggest how an exocellulase might move along a cellulose chain. For family 48 exocellulases there are two different ways that a chain can be bound in the active site while for family 6 exocellulases there are several different ligand-bound structures. Site-directed mutagenesis of Thermobifida fusca exocellulases Cel48A and Cel6B and the processive endoglucanase Cel9A have identified some mutations that increase processivity and some that decrease processivity. In addition a mutation in Cel6B was identified that appears to allow the mutant enzyme to move along a cellulose chain in the absence of cleavage.

Mixed Co/Fe oxide nanoparticles in block copolymer micelles.

Small iron oxide and Co-doped iron oxide nanoparticles (NPs) were synthesized in a commercial amphiphilic block copolymer, poly(ethylene oxide)-b-poly(methacrylic acid) (PEO 68-b-PMAA8), in aqueous solutions. The structure and composition of the micelles containing guest molecules (metal salts) or NPs (metal oxides) were studied using transmission electron microscopy, dynamic light scattering, X-ray photoelectron spectroscopy, and X-ray powder diffraction. The enlarged micelle cores after incorporation of metal salts are believed to be formed by both PMAA blocks containing metal species and penetrating PEO chains. The nanoparticle size distributions in PEO 68-b-PMAA8 were determined using small-angle X-ray scattering (SAXS) in bulk. Two independent methods for SAXS data interpretation for comprehensive analysis of volume distributions of metal oxide NPs showed presence of both small particles and larger entities containing metal species which are ascribed to organization of block copolymer micelles in bulk. The magnetometry measurements revealed that the NPs are superparamagnetic and their characteristics depend on the method of the NP synthesis. The important advantage of the PEO 68-b-PMAA8 stabilized magnetic nanoparticles described in this paper is their remarkable solubility and stability in water and buffers.

Transformations of poly(methoxy hexa(ethylene glycol) methacrylate)-b-(2-(diethylamino)ethyl methacrylate) block copolymer micelles upon metalation.

Micelle transformations upon metalation (i.e., incorporation of metal compounds and metal nanoparticle formation) in poly(methoxy hexa(ethylene glycol) methacrylate)-block-poly((2-(diethylamino)ethyl methacrylate)), PHEGMA-b-PDEAEMA, solutions have been studied using transmission electron microscopy (TEM) and photon correlation spectroscopy (PCS). Three different methods for the formation of metalated micelles are compared: (A) dissolution of the block copolymers in pure water followed by incorporation of platinic acid (H(2)PtCl(6).6H(2)O), (B) micellization in acidic molecular solutions of block copolymers induced by interaction of the protonated amino groups with the PtCl(6)(2)(-) ions, and (C) incorporation of metal species in pH-induced micelles. The latter method leads to well-defined metalated micelles of 22-25 nm diameter containing nanoparticles with diameters of 1.3-1.5 nm. No nanoparticle aggregation is observed. Good agreement is obtained for the sizes of the platinic acid-containing micelles assessed by TEM and PCS.

Core-shell nanostructures from single poly(N-vinylcaprolactam) macromolecules: stabilization and visualization.

Formation of core-shell poly(N-vinylcaprolactam) (PVCL) single-molecule nanostructures due to interaction of PVCL with metal ions was studied using transmission electron microscopy, 13C NMR, and light scattering. This study demonstrates that addition of CoCl2 to PVCL in its globular conformation yields unimolecular core-shell polymer particles with the core decorated with Co(II) ions. The crucial condition for formation of well-defined unimolecular nanostructures is the presence of stable globular aggregates in aqueous solution. Moreover, the metal ions should have a sufficiently high coordination number (higher than 2) to provide a cross-linking and stabilization of the core.

Micellization in pH-sensitive amphiphilic block copolymers in aqueous media and the formation of metal nanoparticles.

Dynamic light scattering, potentiometric titration, transmission electron microscopy and atomic force microscopy have been used to investigate the micellar behaviour and metal-nanoparticle formation in poly(ethylene oxide)-block-poly(2-vinylpyridine), PEO-b-P2VP, poly(hexa(ethylene glycol) methacrylate)-block-poly(2-(diethylamino)ethyl methacrylate), PHEGMA-b-PDEAEMA, and PEO-b-PDEAEMA amphiphilic diblock copolymers in water. The hydrophobic block of these copolymers (P2VP or PDEAEMA) is pH-sensitive: at low pH it can be protonated and becomes partially or completely hydrophilic leading to molecular solubility whereas at higher pH micelles are formed. These micelles consist of a P2VP or PDEAEMA core and a PEO or PHEGMA corona, respectively, where the core forming amine units can incorporate metal compounds due to coordination. The metal compounds (e.g., H2PtCl6, K2PtCl6) can either be introduced in a micellar solution, where they are incorporated within the micelle core via coordination with functional groups, or can be added to a unimer solution at low pH, where they lead to a metal-induced micellization. In these micellar nanoreactors, metal nanoparticles nucleate and grow upon reduction with sizes in the range of a few nanometers as observed by TEM. The effect of the metal incorporation method on the characteristics of the micelles and of the synthesized nanoparticles is investigated.