Genetic Encoding of a Photocaged Histidine for Light-Control of Protein Activity

The use of light to control protein function is a critical tool in chemical biology. Here we describe the addition of a photocaged histidine to the genetic code. This unnatural amino acid becomes histidine upon exposure to light and allows for the optical control of enzymes that utilize active-site histidine residues. We demonstrate light-induced activation of a blue fluorescent protein and a chloramphenicol transferase. Further, we genetically encoded photocaged histidine in mammalian cells. We then used this approach in live cells for optical control of firefly luciferase and, Renilla luciferase. This tool should have utility in manipulating and controlling a wide range of biological processes.     

Hacking the Genetic Code of Mammalian Cells

A genetic shuttle : The highlighted article, which was recently published by Schultz, Geierstanger and co‐workers, describes a straightforward scheme for enlarging the genetic code of mammalian cells. An orthogonal tRNA/aminoacyl‐tRNA synthetase pair specific for a new amino acid can be evolved in E. coli and subsequently transferred into mammalian cells. The feasibility of this approach was demonstrated by adding a photocaged lysine derivative to the genetic repertoire of a human cell line.

     

Optochemical Control of Bacterial Gene Expression: Novel Photocaged Compounds for Different Promoter Systems

Photocaged compounds are applied for implementing precise, optochemical control of gene expression in bacteria. To broaden the scope of UV-light-responsive inducer molecules, six photocaged carbohydrates were synthesized and photochemically characterized, with the absorption exhibiting a red-shift. Their differing linkage through ether, carbonate, and carbamate bonds revealed that carbonate and carbamate bonds are convenient. Subsequently, those compounds were successfully applied in vivo for controlling gene expression in E. coli via blue light illumination. Furthermore, benzoate-based expression systems were subjected to light control by establishing a novel photocaged salicylic acid derivative. Besides its synthesis and in vitro characterization, we demonstrate the challenging choice of a suitable promoter system for light-controlled gene expression in E. coli. We illustrate various bottlenecks during both photocaged inducer synthesis and in vivo application and possibilities to overcome them. These findings pave the way towards novel caged inducer-dependent systems for wavelength-selective gene expression.     

Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro‐ and Eukaryotic Cells

A light‐activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site‐specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho‐nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro‐ and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.     

Synthetic Phosphoethanolamine Cellobiose Promotes Escherichia coli Biofilm Formation and Congo Red Binding

Urinary tract infections (UTIs) are caused by bacteria growing in complex, multicellular enclosed aggregates known as biofilms. Recently, a zwitterionic cellulose derivative produced in Escherichia coli (E. coli) was determined to play an important role in the formation and assembly of biofilms. In order to produce a minimal, yet structurally defined tool compound to probe the biology of the naturally occurring polymer, we have synthesized a zwitterionic phosphoethanolamine cellobiose (pEtN cellobiose) and evaluated its biofilm activity in the Gram-negative bacterium E. coli, a pathogen implicated in the pathogenesis of UTIs. The impact of synthetic pEtN cellobiose on biofilm formation was examined via colorimetric assays which revealed an increase in cellular adhesion to an abiotic substrate compared to untreated samples. Additionally, Congo red binding assays indicate that culturing E. coli in the presence of pEtN cellobiose enhances Congo Red binding to bacterial cells. These results reveal new opportunities to study the impact glycopolymers have on cellular adhesion in Gram-negative pathogens.     

Genetic Incorporation of ϵ-N-Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase

Post-translational modifications regulate protein structure and function. Lysine benzoylation is a newly discovered histone modification with unique physiological relevance. To construct proteins with this modification site-specifically, we generated orthogonal tRNA^Pyl-MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl-tRNA synthetase, allowing the genetic incorporation of ϵ-N-benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation.     

Developing antibacterial surgical adhesives: An enhancement of cyanoacrylate polymers

Surgical site infections (SSIs) and traumatic wounds have a significant risk of becoming contaminated by microbial pathogens of both endogenous and nosocomial origins, including Staphylococcus aureus and Enterococci sp.. One preventative approach is to protect wounds from infection by using a rapidly curing adhesive to seal the wound and prevent further contamination. Here, we demonstrate the covalent incorporation of an antimicrobial, quaternary ammonium chloride monomer (quat) into a 2-octyl cyanoacrylate (2oc) polymer adhesive. Copolymerization was confirmed via nuclear magnetic resonance spectroscopy. Cytotoxicity of the copolymer was assessed against: S. epidermidis and E. coli, and 3T3 mouse fibroblasts. The CA-Quat polymer was found to exhibit dose-dependent bacteriostatic and bactericidal effects against both E. coli and S. epidermidis, importantly without showing any demonstrable toxicity against mammalian 3T3 fibroblast cells. The described experiments provide promising data to suggest successful copolymerization, effective antibacterial properties, and remarkably low cytotoxic effects of the copolymer on mammalian cells.     

Biochemical Characterization of the Lysine Acetylation of Tyrosyl‐tRNA Synthetase in Escherichia coli

Aminoacyl‐tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl‐tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic‐code‐expansion strategy to site‐specifically incorporate N^?‐acetyl‐l ‐lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E. coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5‐related N‐acetyltransferase family in E. coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl‐CoA or acetyl‐phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation.     

Effect of high shear stress on microbial viability

Escherichia coli and Saccharomyces cerevisiae suspensions were submitted to controlled shear stress. Above a threshold value shear stress induced a decrease in micro‐organism viability. The threshold of shear stress efficiency depended on the micro‐organisms, being between 1292 Pa and 2770 Pa for S cerevisiae, and about 1250 Pa for E coli. Above 1810 Pa, E coli cells were disrupted whereas the S cerevisiae cells remained intact. The higher the cellular concentration, the greater the rate of decrease in viability. Viability loss was influenced by the number of passages through the experimental shear stress device and by exposure time. © 2001 Society of Chemical Industry     

Synthesis and Evaluation of N‐Phenylpyrrolamides as DNA Gyrase B Inhibitors

ATP‐competitive inhibitors of DNA gyrase and topoisomerase IV are among the most interesting classes of antibacterial drugs that are unrepresented in the antibacterial pipeline. We developed 32 new N‐phenylpyrrolamides and evaluated them against DNA gyrase and topoisomerase IV from E. coli and Staphylococcus aureus. Antibacterial activities were studied against Gram‐positive and Gram‐negative bacterial strains. The most potent compound displayed an IC₅₀ of 47 nm against E. coli DNA gyrase, and a minimum inhibitory concentration (MIC) of 12.5 μm against the Gram‐positive Enterococcus faecalis. Some compounds displayed good antibacterial activities against an efflux‐pump‐deficient E. coli strain (MIC=6.25 μm ) and against wild‐type E. coli in the presence of efflux pump inhibitor PAβN (MIC=3.13 μm ). Here we describe new findings regarding the structure–activity relationships of N‐phenylpyrrolamide DNA gyrase B inhibitors and investigate the factors that are important for the antibacterial activity of this class of compounds.     

Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5-fluorocytosine toxicity in HT1080 cells

Improved prodrug-activating enzymes have the potential to increase the therapeutic efficacy of gene-directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site-saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants through Escherichia coli genetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitize E. coli and HT1080 fibrosarcoma cells to 5-FC. Several mutants identified in this study showed increased sensitization ability for both E. coli and HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.     

Capture of Uropathogenic E. coli by Using Synthetic Glycan Ligands Specific for the Pap‐Pilus

Biotinylated mono‐ and biantennary di‐/trisaccharides were synthesized to evaluate their ability to capture E. coli strains that express pilus types with different receptor specificities. The synthesized biotinylated di‐/trisaccharides contain Galα(1→4)Gal, Galα(1→4)GalNHAc, GalNHAcα(1→4)Gal, Galα(1→4)Galβ(1→4)Glc and GalNHAcα(1→4)Galβ(1→4)Glc as carbohydrate epitopes. These biotinylated oligosaccharides were immobilized on streptavidin‐coated magnetic beads, and incubated with different strains of live E. coli. Capturing ability was assessed by using a luciferase assay that detects bacterial ATP. The trisaccharides containing Galα(1→4)Galβ(1→4)Glc and the disaccharides containing Galα(1→4)Gal as the epitopes exhibited strong capturing ability for uropathogenic E. coli strains with the pap pilus genotype, including CFT073, J96 and J96 pilE. The same ligands failed to capture E. coli strains with fim, prs, or foc genotypes. Uropathogenic CFT073 was also captured moderately by biantennary disaccharides containing a GalNHAc moiety at the reducing end; however, other saccharides containing GalNHAc at the nonreducing end did not capture the CFT073 strain. These synthetic glycoconjugates could potentially be adapted as rapid diagnostic agents to differentiate between different E. coli pathovars.     

Photo-biohydrogen Production by Photosensitization with Biologically Precipitated Cadmium Sulfide in Hydrogen-Forming Recombinant Escherichia coli

An inorganic-biological hybrid system that integrates features of both stable and efficient semiconductors and selective and efficient enzymes is attractive for facilitating the conversion of solar energy to hydrogen. In this study, we aimed to develop a new photocatalytic hydrogen-production system based on Escherichia coli whole-cell genetically engineered as a biocatalysis for highly active hydrogen formation. The photocatalysis part was obtained by bacterial precipitation of cadmium sulfide (CdS), which is a visible-light-responsive semiconductor. The recombinant E. coli cells were sequentially subjected to CdS precipitation and heterologous [FeFe]-hydrogenase synthesis to yield a CdS@E. coli hybrid capable of light energy conversion and hydrogen formation in a single cell. The CdS@E. coli hybrid achieved photocatalytic hydrogen production with a sacrificial electron donor, thus demonstrating the feasibility of our system and expanding the current knowledge of photosensitization using a whole-cell biocatalyst with a bacterially precipitated semiconductor.     

Hydrogels for water filters: Characterization and regeneration

Acrylic acid was crosslinked with N,N′‐methylenebisacrylamide followed by a reaction with hexamethylenetetramine (HMTA) to form a new hydrogel, Gel ( 2 ). Water absorbance rate and retention of Gel ( 2 ) were characterized. At the same time, factors affecting absorbance rate such as pH, temperature, and ions concentration were studied. The rate decreased with decreasing pH and increasing ions concentration, whereas increased with raising temperature. The effect of the hydrogel on bacterial (Staphylococcus aureus and Escherichia coli) viability and growth rate was determined. Gel ( 2 ) has achieved a 5 log reduction on both E. coli and S. aureus in 2 h while no cells were detected after 3 h in case of S. aureus and 4 h in case of E. coli. Lifetime and regeneration of Gel ( 2 ) using both stirred flask and column (a model for a water filter) methods were determined. Identifying the lifetime using columns showed that the gel stays active up to 5 and 7 runs for both E. coli and S. aureus respectively. In addition, it was recycled successfully up to 10 times, using column and stirred flask methods, against both types of bacteria. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ?X174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC₅₀ 200–600 μM ), and demonstrated antimicrobial activity against E. coli (MIC 31–125 μg mL^?1). Cationic antimicrobial peptides at a concentration of 100 μg mL^?1 containing Arg‐Trp sequences also showed 30–60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.     

Effect of addition of amino acids on microfiltration behavior of a microorganism slurry

The effect of the addition of amino acids on the microfiltration behavior of Escherichia coli (E coli) and Corynebacterium glutamicum (C glutamicum) slurry was examined using a dead‐end microfilter. It was found that the average specific filtration resistance, α_av, of a slurry of microorganisms increased markedly by adding amino acids. Amino acid concentration ranged from 110 to 876 mol m^?3. However the same concentration of ammonium chloride did not increase α_av. The cells were found to disperse in the presence of lysine, and this caused an increase in α_av. In the case of kaolin slurry, α_av was not affected by adding amino acids. Copyright © 2004 Society of Chemical Industry     

A sandwich-type assay based on quantum dot/aptamer bioconjugates for analysis of E. Coli O157:H7 in microtiter plate format

An optical assay based on CdSe/ZnS quantum dots (QD)/NH₂-Apt bioconjugates for the pathogen detection was presented. QDs with carboxyl functional groups and 72-mer aptamer (against E. coli outer membrane proteins) were used as probes and sensing element. E. coli O157:H7 was selected as a model pathogen and 96-well plate assay in the sandwich hybridization format was constructed. Poly-L-lysine-coated 96-well plate surfaces were used as support material where thiol functionalized aptamers were immobilized by 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide (sulfo-SMCC). After incubation with the bacteria, CdSe/ZnS QDs/aptamer bioconjugates were added. The fluorescence signals were followed before and after addition of bioconjugates. Probe concentrations, incubation time with E. coli O157:H7 were also optimized. The bioassay could detect the pathogen down to 10² CFU/mL with high selectivity. The detection system was successfully employed in samples, in the presence of interfering compounds.     

Expanding the Genetic Code for a Dinitrophenyl Hapten

Haptens, such as dinitrophenyl (DNP) are small molecules that induce strong immune responses when attached to proteins or peptides and, as such, have been exploited for diverse applications. We engineered a Methanosarcina barkeri pyrrolysyl‐tRNA synthetase (mbPylRS) to genetically encode a DNP‐containing unnatural amino acid, N⁶‐(2‐(2,4‐dinitrophenyl)acetyl)lysine (DnpK). Although this moiety was unstable in Escherichia coli, we found that its stability was enhanced in mammalian HEK 293T cells and was able to induce selective interactions with anti‐DNP antibodies. The capability of genetically introducing DNP into proteins is expected to find broad applications in biosensing, immunology, and therapeutics.     

Effect of Media on Biofilter Performance Following Ozonation of Secondary Treated Municipal Wastewater Effluent: Sand vs. GAC

Ozone has been shown to be effective in the transformation of several chemicals of emerging concern that escape the wastewater treatment process, but there is concern whether toxic transformation products are formed. Two parallel biofilter columns with granular activated carbon (GAC) and filter sand following a pilot-scale ozone unit to treat secondary treated municipal wastewater were studied. Results show reduced wastewater genotoxicity following ozonation and further reduction following biofiltration. The BAC biofilter outperformed the sand biofilter in terms of reduction in both organics and genotoxicity. Biofilter performance correlated with biological indicators (dissolved oxygen reduction and effluent E. coli counts) but not with ATP bioactivity measurements. Limited bacterial (E. coli) regrowth was observed in treated effluent from both biofilters.