Polyethylene Glycol Impacts Conformation and Dynamics of Escherichia coli Prolyl-tRNA Synthetase Via Crowding and Confinement Effects.

Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have modest effects on the conformational dynamics of Ec ProRS but impact the catalytic function primarily via the excluded volume effect; they form large clusters blocking the active site pocket. In contrast, the larger molecular weight PEGs in dilute to semidilute regimes have a significant impact on the protein's conformational dynamics; they wrap on the protein surface through noncovalent interactions. Thus, lower-molecular-weight PEG molecules impact protein dynamics and function via crowding effects, whereas larger PEGs induce confinement effects. These results have implications for the development of inhibitors for protein targets in a crowded cellular environment.

Structural basis of tRNAPro acceptor stem recognition by a bacterial trans-editing domain.

High fidelity tRNA aminoacylation by aminoacyl-tRNA synthetases is essential for cell viability. ProXp-ala is a trans-editing protein that is present in all three domains of life and is responsible for hydrolyzing mischarged Ala-tRNAPro and preventing mistranslation of proline codons. Previous studies have shown that, like bacterial prolyl-tRNA synthetase, Caulobacter crescentus ProXp-ala recognizes the unique C1:G72 terminal base pair of the tRNAPro acceptor stem, helping to ensure deacylation of Ala-tRNAPro but not Ala-tRNAAla. The structural basis for C1:G72 recognition by ProXp-ala is still unknown and was investigated here. NMR spectroscopy, binding, and activity assays revealed two conserved residues, K50 and R80, that likely interact with the first base pair, stabilizing the initial protein-RNA encounter complex. Modeling studies are consistent with direct interaction between R80 and the major groove of G72. A third key contact between A76 of tRNAPro and K45 of ProXp-ala was essential for binding and accommodating the CCA-3' end in the active site. We also demonstrated the essential role that the 2'OH of A76 plays in catalysis. Eukaryotic ProXp-ala proteins recognize the same acceptor stem positions as their bacterial counterparts, albeit with different nucleotide base identities. ProXp-ala is encoded in some human pathogens; thus, these results have the potential to inform new antibiotic drug design.

Adaptive Immune Response to Vaccinia Virus LIVP Infection of BALB/c Mice and Protection against Lethal Reinfection with Cowpox Virus.

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 105 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.

Editing Domain Motions Preorganize the Synthetic Active Site of Prolyl-tRNA Synthetase.

Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate tRNAs, an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the "prokaryotic-like" ProRSs are distinguished from "eukaryotic-like" ProRSs by the presence of an editing domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal, highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic Pro-AMP formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations, as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme's catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.

Fine-scale spatial genetic structure in predominantly selfing plants with limited seed dispersal: A rule or exception?

Gene flow at a fine scale is still poorly understood despite its recognized importance for plant population demographic and genetic processes. We tested the hypothesis that intensity of gene flow will be lower and strength of spatial genetic structure (SGS) will be higher in more peripheral populations because of lower population density. The study was performed on the predominantly selfing Avena sterilis and included: (1) direct measurement of dispersal in a controlled environment; and (2) analyses of SGS in three natural populations, sampled in linear transects at fixed increasing inter-plant distances. We found that in A. sterilis major seed dispersal is by gravity in close (less than 2 m) vicinity of the mother plant, with a minor additional effect of wind. Analysis of SGS with six nuclear SSRs revealed a significant autocorrelation for the distance class of 1 m only in the most peripheral desert population, while in the two core populations with Mediterranean conditions, no genetic structure was found. Our results support the hypothesis that intensity of SGS increases from the species core to periphery as a result of decreased within-population gene flow related to low plant density. Our findings also show that predominant self-pollination and highly localized seed dispersal lead to SGS at a very fine scale, but only if plant density is not too high.

Multi-approaches analysis reveals local adaptation in the emmer wheat (Triticum dicoccoides) at macro- but not micro-geographical scale.

Detecting local adaptation and its spatial scale is one of the most important questions of evolutionary biology. However, recognition of the effect of local selection can be challenging when there is considerable environmental variation across the distance at the whole species range. We analyzed patterns of local adaptation in emmer wheat, Triticum dicoccoides, at two spatial scales, small (inter-population distance less than one km) and large (inter-population distance more than 50 km) using several approaches. Plants originating from four distinct habitats at two geographic scales (cold edge, arid edge and two topographically dissimilar core locations) were reciprocally transplanted and their success over time was measured as 1) lifetime fitness in a year of planting, and 2) population growth four years after planting. In addition, we analyzed molecular (SSR) and quantitative trait variation and calculated the QST/FST ratio. No home advantage was detected at the small spatial scale. At the large spatial scale, home advantage was detected for the core population and the cold edge population in the year of introduction via measuring life-time plant performance. However, superior performance of the arid edge population in its own environment was evident only after several generations via measuring experimental population growth rate through genotyping with SSRs allowing counting the number of plants and seeds per introduced genotype per site. These results highlight the importance of multi-generation surveys of population growth rate in local adaptation testing. Despite predominant self-fertilization of T. dicoccoides and the associated high degree of structuring of genetic variation, the results of the QST - FST comparison were in general agreement with the pattern of local adaptation at the two spatial scales detected by reciprocal transplanting.

Feedback effects of host-derived adenosine on enteropathogenic Escherichia coli.

Enteropathogenic E. coli (EPEC) is a common cause of diarrhea in children in developing countries. After adhering to intestinal cells, EPEC secretes effector proteins into host cells, causing cell damage and eventually death. We previously showed that EPEC infection triggers the release of ATP from host cells and that ATP is broken down to ADP, AMP, and adenosine. Adenosine produced from the breakdown of extracellular ATP triggers fluid secretion in intestinal monolayers and may be an important mediator of EPEC-induced diarrhea. Here we examined whether adenosine has any effects on EPEC bacteria. Adenosine stimulated EPEC growth in several types of media in vitro. Adenosine also altered the pattern of EPEC adherence to cultured cells from a localized adherence pattern to a more diffuse pattern. Adenosine changed the expression of virulence factors in EPEC, inhibiting the expression of the bundle-forming pilus (BFP) and enhancing expression of the EPEC secreted proteins (Esps). In vivo, experimental manipulations of adenosine levels had strong effects on the outcome of EPEC infection in rabbit intestinal loops. In addition to its previously reported effects on host tissues, adenosine has strong effects on EPEC bacteria, stimulating EPEC growth, altering its adherence pattern, and changing the expression of several important virulence genes. Adenosine, like noradrenaline, is a small, host-derived molecule that is utilized as a signal by EPEC.

Effect of zinc in enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5'-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5'-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5'-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5'-nucleotidase in vitro than the nucleotide analog alpha,beta-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than alpha,beta-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5'-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host.

Ecto-5'-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5'-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5'-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5'-AMP. EPEC infection triggered a release of ecto-5'-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5'-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5'-nucleotidase was susceptible to inhibition by zinc acetate and by alpha,beta-methylene-adenosine diphosphate (alpha,beta-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5'-AMP. In addition, alpha,beta-methylene-ADP and zinc blocked the ability of 5'-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5'-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5'-nucleotidase, such as alpha,beta-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.

Conversion of active promoter-RNA polymerase complexes into inactive promoter bound complexes in E. coli by the transcription effector, ppGpp.

Guanosine tetraphosphate (ppGpp) is a signal of nutritional stress that regulates transcription. An RNA polymerase rudder mutant rpoC (Delta 312-315) is found to suppress ppGpp deficiency phenotypes by restoring both negative and positive activities of promoter fusions in vivo, as if ppGpp were present. Measurements of defects in transcription of the PargT tRNA promoter with mutant RNA polymerase reveal that the mutant enzyme quantitatively mimics the presence of added ppGpp. DNaseI footprints and mobility shifts under RNA polymerization conditions reveal that the promoter-specific transcription defect of the mutant enzyme can be ascribed to the presence of inactive dead-end promoter complexes with features similar to those of a stable closed complex. We propose that formation of such inactive complexes represents an alternative explanation of "stringent RNA polymerase" mutant behavior to those currently published, and it represents a newly discovered mode of action of ppGpp.