Studies of the Protein Interaction Network Required for Enterobactin Biosynthesis in Escherichia coli

Abstract Studies of the Protein Interaction Network Required for Enterobactin Biosynthesis in Escherichia coli Sofia Khalil, Ph.D. Concordia University, 2010 Siderophores are small-molecule iron chelators that many bacteria synthesize and secrete in order to survive in iron-depleted environments. In Escherichia coli, biosynthesis of the siderophore molecule enterobactin requires the activities of six enzymes, EntA-EntF. These enzymes function sequentially to produce enterobactin molecule in the cytoplasm. The enterobactin biosynthesis pathway is divided into two modules. The first module involves the conversion of chorismate to 2,3-dihydroxybenzoic acid (2,3-DHB), and requires the activities of EntC, EntB (N-terminal domain) and EntA. The second module involves non-ribosomal peptide synthesis (NRPS) such that three molecules of 2,3-DHB are condensed with three molecules of L-serine to form the siderophore. The NRPS module requires the activities of EntE, EntB (C-terminal domain), EntD and EntF. The overall goal of my research project is characterization of the enterobactin biosynthetic enzyme EntE. EntE catalyzes the activation of 2,3-DHB via adenylation producing DHB-AMP. I am interested in addressing the following questions: (i) How does EntE bind its 2,3-DHB substrate? (ii) How does it interact with its upstream and downstream partner proteins: EntA, which produces 2,3-DHB, and EntB, which uses the EntE product (DHB-AMP) as a substrate, respectively? My thesis is divided into three research-related chapters: The first research chapter focused on the interaction of EntE with its substrate, 2,3-DHB, as well as the characterization of EntE-EntB interaction in the presence and absence of 2,3-DHB. A significant change in EntB conformation was observed upon the interaction with EntE when in the presence of 2,3-DHB. In the pull-down assay, EntB as bait protein pulled down more EntE in the presence of exogenous 2,3-DHB. We conclude from this chapter that the ligand-loaded state of the protein was necessary for efficient protein-protein interaction. The second research chapter involves the characterization of a novel interaction between EntE and its upstream partner protein EntA. A significant increase in EntE activity was observed upon adding EntA. Furthermore, EntA reduces the FRET signal of EntE-bound 2,3-DHB in a saturable manner with increasing EntA concentrations. Using this fluorescence binding assay at 20 °C revealed a positive cooperativity in EntA-EntE interaction with Hill coefficient greater than one. The AUC experiments showed that EntA conformation is highly dependent on its concentration. In conclusion, the results of this chapter suggest that EntA-EntE interaction likely induces remodeling of EntE active site, resulting in the observed increase in EntE catalysis. In the third research chapter, the EntA-EntE interaction interface was characterized using phage display. Based on the interaction interface predicted by phage display data, EntA variants containing single-site and double-site mutations were created (Q64A, A68Q, and Q64A/A68Q). Our in vitro biophysical techniques and growth phenotype experiments revealed that EntA (Q64A) and EntA (Q64A/A68Q) mutations have a more pronounced effect than EntA (A68Q) mutation on disruption of the EntA-EntE interaction interface

Ligand-Induced Conformational Rearrangements Promote Interaction between the Escherichia coli Enterobactin Biosynthetic Proteins EntE and EntB

Siderophores are small-molecule iron chelators that many bacteria synthesize and secrete in order to survive in iron-depleted environments. Biosynthesis of enterobactin, the Escherichia coli catecholate siderophore, requires adenylation of 2,3-dihydroxybenzoic acid (2,3-DHB) by the cytoplasmic enzyme EntE. The DHB-AMP product is then transferred to the active site of holo-EntB subsequent to formation of an EntE–EntB complex. Here we investigate the binding of 2,3-DHB to EntE and how DHB binding affects EntE–EntB interaction. We overexpressed and purified recombinant forms of EntE and EntB with N-terminal hexahistidine tags (H6-EntE and H6-EntB). Isothermal titration calorimetry showed that 2,3-DHB binds to H6-EntE with a 1:1 stoichiometry and a Kd of 7.4 μM. Fluorescence spectra revealed enhanced 2,3-DHB emission at 440 nm (λex = 280 nm) when bound to H6-EntE due to fluorescence resonance energy transfer (FRET) between EntE intrinsic fluorophore donors and bound 2,3-DHB acceptor. A FRET signal was not observed when H6-EntE was mixed with either 2,5-dihydroxybenzoic acid or 3,5-dihydroxybenzoic acid. The H6-EntE–2,3-DHB FRET signal was quenched by H6-EntB in a concentration-dependent manner. From these data, we were able to determine the EC50 of EntE–EntB interaction to be approximately 1.5 μM. We also found by fluorescence and CD measurements that H6-EntB can bind 2,3-DHB, resulting in conformational changes in the protein. Additional alterations in H6-EntB near-UV and far-UV CD spectra were observed upon mixture with H6-EntE and 2,3-DHB, suggesting that further conformational rearrangements occur in EntB upon interaction with substrate-loaded EntE. We also found that H6-EntB as a bait protein pulled down a higher concentration of chromosomally expressed EntE in the presence of exogenous 2,3-DHB. Taken together, our results show that binding of 2,3-DHB to EntE and EntB primes these proteins for efficient complexation, thus facilitating direct channeling of the siderophore precursor 2,3-DHB-AMP

Identification of a Surface Glutamine Residue (Q64) of Escherichia coli EntA Required for Interaction with EntE

The enterobactin biosynthetic enzyme EntA forms a complex with EntE, the next enzyme in the pathway, to enhance activation of the enterobactin precursor 2,3-dihydroxybenzoate. Here we used phage display to identify an EntE-interacting region on the surface of EntA. Upon panning immobilized EntE with a random peptide phage library, we recovered 47 unique EntE-binding dodecamer peptide sequences that aligned to a region of the EntA primary sequence corresponding to helix α4. In order to further investigate this region, we mutagenized EntA Q64, a hydrogen-bonding residue found on the surface-exposed face α4. Far-UV circular dichroism, thermal denaturation experiments, and enzymatic assays showed that mutation of EntA residue Gln 64 to alanine (Q64A) had no deleterious effect on EntA structure or function. By following near-UV CD spectral changes, we found that the spectrum of wild-type EntA was altered in the presence of EntE, indicative of conformational changes in EntA aromatic chromophores upon formation of the EntA-EntE complex. However, EntE did not affect the CD spectrum of EntA variant Q64A, demonstrating that this variant did not interact with EntE in a manner similar to wild-type EntA. Analytical ultracentrifugation of wild-type and variant EntA proteins showed that EntA Q64A was predominantly dimeric at 20 μM, unlike wild-type EntA which was predominantly tetrameric. Taken together, our findings establish that EntA α4 is required for efficient formation of the EntA-EntE as well as for EntA oligomerization

Prophylactic antibiotics for staged bone augmentation in implant dentistry

Background: The objective of the study was to assess the effect of prophylactic antibiotics on the outcome of bone augmentation and subsequent dental implant placement by combining the recommended quality assessment methods for systematic reviews and primary studies. Materials and methods: This is a complex systematic review in which systematic reviews as well as primary studies are scrutinised. A search of Medline (OVID), The Cochrane Library (Wiley) and EMBASE, PubMed and Health technology assessment (HTA) organisations as-well as a complementary hand-search was carried out. Selected primary studies were assessed using GRADE. Each study was reviewed by three authors independently. Results: Abstract screening yielded six potential systematic reviews allocated for full-text inspection. A total of ten primary studies were read in full-text. No relevant systematic reviews regarding the topic of this article were found. The quality assessment resulted in two primary studies with a moderate risk of bias. Of the two studies with a moderate risk of bias, one compared a single dose of clindamycin 600 mg preoperatively with the same preoperative dose followed by four doses of 300 mg every 6 h. The second study compared a single dose prophylaxis of two different types of antibiotic compounds. Conclusion: In conclusion, the scientific evidence regarding the use of antibiotic prophylaxis for reducing the risk of infection in conjunction with bone augmentation procedures during dental implant placement is very limited. The infection rate as compared to nonusage of prophylactic antibiotics, selection of the most suitable compound, and the optimal duration of prophylactic treatment is still unknown.publishedVersio

Intractable Hepatic Hydrothorax: A Successful Outcome following CPAP Treatment

Hepatic hydrothorax is an uncommon complication in patients with end-stage liver disease. It may result in dyspnoea, hypoxia and infection, and carries a poor prognosis. Initial treatment is based on a sodium-free diet together with diuretics. In case of recurrent hydrothorax, a transjugular intrahepatic portosystemic shunt (TIPS) or liver transplant should be considered. Here we describe an 80-year-old woman with decompensated liver cirrhosis related to NASH who presented with refractory hepatic hydrothorax. Treatment with CPAP resulted in a marked improvement in her pleural effusion

Coordinated Rearrangements between Cytoplasmic and Periplasmic Domains of the Membrane Protein Complex ExbB-ExbD of Escherichia coli

SummaryGram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240 kDa protein-detergent complex, measured by light scattering and by native gels. Quantitative Coomassie staining revealed a stoichiometry of ExbB4-ExbD2. Negative stain electron microscopy and 2D analysis showed particles of ∼10 nm diameter in multiple structural states. Nanogold labeling identified the position of the ExbD periplasmic domain. Random conical tilt was used to reconstruct the particles in three structural states followed by sorting of the single particles and refinement of each state. The different states are interpreted by coordinated structural rearrangements between the cytoplasmic domain and the periplasmic domain, concordant with in vivo predictions

Climate change and potential distribution of potato (Solanum tuberosum) crop cultivation in Pakistan using Maxent

The impacts of climate change are projected to become more intense and frequent. One of the indirect impacts of climate change is food insecurity. Agriculture in Pakistan, measured fourth best in the world, is already experiencing visible adverse impacts of climate change. Among many other food sources, potato crop remains one of the food security crops for developing nations. Potatoes are widely cultivated in Pakistan. To assess the impact of climate change on potato crop in Pakistan, it is imperative to analyze its distribution under future climate change scenarios using Species Distribution Models (SDMs). Maximum Entropy Model is used in this study to predict the spatial distribution of Potato in 2070 using two CMIP5 models for two climate change scenarios (RCP 4.5 and RCP 8.5). 19 Bioclimatic variables are incorporated along with other contributing variables like soil type, elevation and irrigation. The results indicate slight decrease in the suitable area for potato growth in RCP 4.5 and drastic decrease in suitable area in RCP 8.5 for both models. The performance evaluation of the model is based on AUC. AUC value of 0.85 suggests the fitness of the model and thus, it is applicable to predict the suitable climate for potato production in Pakistan. Sustainable potato cultivation is needed to increase productivity in developing countries while promoting better resource management and optimization

Trace and major element contents, microbial communities, and enzymatic activities of urban soils of Marrakech city along an anthropization gradient

Due to their close proximity with the population, urban soils are extensively affected by human activities that release considerable technogenic inputs resulting in an overall soil degradation and leading to an increase of water-extractable fraction of trace elements. This work aimed to determine the influence of anthropization on trace and major element concentrations and to assess how it might also affect soil biochemical and microbiological parameters in an urban area of Marrakech city, Morocco.info:eu-repo/semantics/publishedVersio