Resequencing of E.coli for polyketide production
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Blake-Hedges, Jacquelyn↗
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Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation
Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.
59 BASIC BIOLOGICAL SCIENCES↗
The Gene Fitness Atlas: A Roadmap for Predicting Evolution
We developed a novel, high-throughput microfluidic device design containing “interaction zones” where progeny cell lines compete against each other allowing for accurate analysis of bacterial cell fitness. The goal of the project was to use the device for two applications: 1) gene knockout screening and 2) antibiotic resistance screening. The microfluidic platform was fabricated using photolithography and soft lithography in polydimethylsiloxane (PDMS). E.coli Keio mutants and fluorescent wildtype parent were chosen for the study. Cells were grown overnight and their loading into the devices and seeding in mother machines was optimized. For mutant screening, the least fit mutant and wildtype parent were cultured individually and then added to the microfluidic device. The mother machines which were seeded with mutant and wildtype were imaged through time lapse microscopy and the growth of cells was observed. For antibiotic screening, wildtype E.coli cells which were grown overnight were added to the device and washed with media containing the antibiotic ampicillin. The growth pattern in presence and absence of ampicillin was observed through time lapse microscopy. It was observed that over a period of four hours, both the mutant and the wildtype divided in the mother machine and pushed daughter cells out into the interaction zone. In case of the antibiotic screening experiment, the fluorescent wildtype divided both in the absence and presence of sublethal concentration of ampicillin. This study is a proof of concept demonstration of high- throughput single cell analysis of cells using a novel microfluidics device.
59 BASIC BIOLOGICAL SCIENCES↗
Spectro Analytical, Computational and In Vitro Biological Studies of Novel Substituted Quinolone Hydrazone and it’s Metal Complexes
Some novel transition metal [Cu (II), Ni (II) and Co (II)] complexes of nalidixic acid hydrazone have been prepared and characterized by employing spectro-analytical techniques viz: elemental analysis, {sup 1}H-NMR, Mass, UV–Vis, IR, TGA-DTA, SEM-EDX, ESR and Spectrophotometry studies. The HyperChem 7.5 software was used for geometry optimization of title compound in its molecular and ionic forms. Quantum mechanical parameters, contour maps of highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) and corresponding binding energy values were computed using semi empirical single point PM3 method. The stoichiometric equilibrium studies of metal complexes carried out spectrophotometrically using Job’s continuous variation and mole ratio methods inferred formation of 1:2 (ML{sub 2}) metal complexes in respective systems. The title compound and its metal complexes screened for antibacterial and antifungal properties, exemplified improved activity in metal complexes. The studies of nuclease activity for the cleavage of CT- DNA and MTT assay for in vitro cytotoxic properties involving metal complexes exhibited high activity. In addition, the DNA binding properties of Cu (II), Ni (II) and Co (II) complexes investigated by electronic absorption and fluorescence measurements revealed their good binding ability and commended agreement of K{sub b} values obtained from both the techniques. Molecular docking studies were also performed to find the binding affinity of synthesized compounds with DNA (PDB ID: 1N37) and “Thymidine phosphorylase from E.coli” (PDB ID: 4EAF) protein targets.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Escherichia coli single-stranded DNA binding protein SSB promotes AlkB-mediated DNA dealkylation repair
Highlights: • E. coli single stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA). • SSB is known to be involved in base excision repair and recombination repair but not in DNA dealkylation repair. • The alkyl-adduct positioned at the 5′ of ssDNA is more accessible to AlkB compared to the internally located alkyl-adducts. • The presence of SSB lowers the K{sub M} of AlkB-catalyzed reaction and promote repair of the longer substrate. • AlkB-mediated repair of methyl adducts from very short ssDNA is rather efficient and SSB-independent. Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N1-methyladenine (N{sup 1}meA) and N3-methylcytosine (N{sup 3}meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N{sup 1}meA and N{sup 3}meC from single-stranded DNA (ssDNA). It can also remove N{sup 1}meA and N{sup 3}meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N{sup 3}meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N{sup 3}meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N{sup 3}meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.
60 APPLIED LIFE SCIENCES↗
Identification of novel Amurin-2 variants from the skin secretion of Rana amurensis, and the design of cationicity-enhanced analogues
Highlights: • Three novel Amurin-2 variants were identified from the skin secretion of Rana amurensis. • Cationic-enhanced analogues show broader antimicrobial activity spectrums. • The substitution by Lys at position 4 improves the antimicrobial selectivity on E. coli. • The cell lysis ability of Amurin-2 peptides is associated with their positive charge. Rana amurensis is important in Chinese medicine as its skin secretions contain abundant bioactive peptides. Here, we have identified the antimicrobial peptide Amurin-2 and three highly-conserved variants, Amurin-2a, Amurin-2b and Amurin-2c through a combination of molecular cloning and MS/MS fragmentation sequencing. Synthetic replicates of these peptides demonstrate potent antimicrobial activity against S. aureus, whilst some have activity against C.albicans and even resistant bacterial MRSA. Furthermore, two Lys-analogues (K{sup 4}-Amurin-2 and K{sup 11}-Amurin-2) were designed to improve the bioactive function and the antimicrobial activity of K{sup 4}-Amurin-2 against E.coli was enhanced distinctly. In addition, the two modified peptides also showed more potent activity against S. aureus, C. albicans and MRSA strains. Meanwhile, these peptides showed inhibitory effect on the cell viability of several cancer cells. As a result, these structural and functional studies of Amurin-2 variants and analogues could provide insights for future antimicrobial peptide design.
60 APPLIED LIFE SCIENCES↗
Pol I DNA polymerases stimulate DNA end-joining by Escherichia coli DNA ligase
Highlights: • Pol I DNA polymerases stimulate E. coli DNA ligase activity. • End-joining activity is enhanced by up to 600%, nick closure is slightly enhanced. • T4 ligase is not similarly effected. Klenow and Klentaq are the large fragment domains of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, respectively. Herein, we show that both polymerases can significantly stimulate complementary intermolecular end-joining ligations by E.coli DNA ligase when the polymerases are present at concentrations lower than that of the DNA substrates. In contrast, high polymerase concentrations relative to the DNA substrates inhibit the intermolecular ligation activity of DNA ligase. Neither polymerase was able to stimulate the DNA ligase from T4 bacteriophage. Additionally, nick-closure by E. coli DNA ligase (but not T4 ligase) is slightly stimulated by both polymerases, but only at about 10% of the magnitude seen for end-joining enhancement. The data represent one of the first observations of direct polymerase-ligase interactions in prokaryotes, and suggest that the polymerases stabilize the associated DNA ends during intermolecular ligation, and that such a complex can be taken advantage of by some, but not all, DNA ligases.
60 APPLIED LIFE SCIENCES↗
A potential substrate binding pocket of BdcA plays a critical role in NADPH recognition and biofilm dispersal
Highlights: • The crystal structure of BdcA in complex with NADPH is determined. • NADPH binding stabilizes a potential substrate binding pocket of BdcA. • The substrate binding pocket is conversely involved in BdcA recognizing NADPH. • Both NADPH and substrate binding are essential for BdcA inducing biofilm dispersal. Biofilm dispersal is characterized by the cell detachment from biofilms and expected to provide novel “anti-biofilm” approaches of prevention and treatment of biofilms in clinical and industrial settings. The E.coli protein BdcA has been identified as a biofilm dispersal factor and designed to be an important component in engineered applications to control biofilm formation. It belongs to short-chain dehydrogenase/reductase (SDR) family with the specific affinity to NADPH. Here, we show the structure of BdcA in complex with NADPH and confirm that NADPH binding is requisite for BdcA facilitating cell motility and increasing biofilm dispersal. Especially, we observe a potential substrate binding pocket surrounded by hydrophobic residues upon NADPH binding and present evidences that this pocket is essential for BdcA binding NADPH and exerting its biological functions. Our study provides the clues for illuminating the molecular mechanism of BdcA regulating biofilm dispersal and better utilizing BdcA to eliminate the biofilms.
60 APPLIED LIFE SCIENCES↗
Resistance of biofilm-covered mortars to microbiologically influenced deterioration simulated by sulfuric acid exposure
Following the reported success of biofilm applications on metal surfaces to inhibit microbiologically influenced corrosion, effectiveness and sustainability of E. coli DH5α biofilm on mortar surface to prevent microbiologically influenced concrete deterioration (MICD) are investigated. Experiments simulating microbial attack were carried out by exposing incrementally biofilm-covered mortar specimens to sulfuric acid solutions with pH ranging from 3 to 6. Results showed that calcium concentration in control reactors without biofilm was 23–47% higher than the reactors with biofilm-covered mortar. Formation of amorphous silica gel as an indication of early stages of acid attack was observed only on the control mortar specimens without biofilm. During acidification, the biofilm continued to grow and its thickness almost doubled from ∼ 30 μm before acidification to ∼ 60 μm after acidification. These results demonstrated that E. coli DH5α biofilm was able to provide a protective and sustainable barrier on mortar surfaces against medium to strong sulfuric acid attack. -- Highlights: •Effectiveness of E.coli DH5α biofilm to prevent MICD was studied. •Conditions that lead to MICD were simulated by chemical acidification. •Biofilm-covered mortar specimens were exposed to sulfuric acid solutions. •The presence of biofilm helped reduce the chemically-induced mortar deterioration. •Biofilm remained alive and continued to grow during the acidification process.
36 MATERIALS SCIENCE↗
TiO{sub 2}-based photocatalytic disinfection of microbes in aqueous media: A review
The TiO{sub 2} based photocatalyst has great potential for the disinfection/inactivation of harmful pathogens (such as E.coli in aqueous media) along with its well-known usefulness on various chemical pollutants. The disinfection property of TiO{sub 2} is primarily attributed to surface generation of reactive oxygen species (ROS) as well as free metal ions formation. Furthermore, its disinfection capacity and overall performance can be significantly improved through modifications of the TiO{sub 2} material. In this review, we provide a brief survey on the effect of various TiO{sub 2} materials in the disinfection of a wide range of environmentally harmful microbial pathogens (e.g., bacteria, fungi, algae, and viruses) in aqueous media. The influencing factors (such as reactor design, water chemistry, and TiO{sub 2} modifications) of such processes are discussed along with the mechanisms of such disinfection. It is believed that the combined application of disinfection and decontamination will greatly enhance the utilization of TiO{sub 2} photocatalyst as a potential alternative to conventional methods of water purification. - Highlights: • The advent of industrialization jeopardized the quality of drinking water. • TiO{sub 2} photocatalysis holds promise both in the degradation of pollutants and for disinfection. • The applicability of TiO{sub 2}-based decontamination is explored for microbial disinfection. • Here we provide a comprehensive review on titania-based photocatalysts for disinfection.
54 ENVIRONMENTAL SCIENCES↗
ZnO/CuO nanostructures anchored over Ni/Cu tubular films via pulse electrodeposition for photocatalytic and antibacterial applications
In this work, we report the fabrication of Ni and Cu tubular substrates and the synthesis of ZnO/CuO nanocomposite on them through the process of pulse electrodeposition. The systematic variation in CuO incorporation in the ZnO matrix and the processing technique were noticed to affect the structural, optical, photocatalytic, and anti-bacterial properties, which are well in accordance with the Field Emission-Scanning Electron Microscope, X-ray Diffraction, Fourier transform Infrared Spectroscopy and UV-Differential reflectance spectroscopy results. The remediation capabilities of the photocatalytic substrates were assessed through the degradation of methylene blue (MB) dye under solar irradiation. Optimized CuO incorporation within the ZnO nanorods resulted in the degradation of a 20 ppm of MB dye solution within 40 min and a higher concentration of 50 ppm within 95 min. The Ni and Cu electroformed tubes as substrates provided not only a reusable supporting frame but also a large surface-area for the growth of ZnO/CuO nanocomposite. The current study also dealt with the anti-bacterial efficacy of the above-mentioned substrates against E.coli. Hence, the Ni and Cu tubular thin film substrates with nanorods of ZnO/CuO composite were explored for the removal of organic as well as biological contaminants from waste water.
36 MATERIALS SCIENCE↗
Microbial community response and SDS-PAGE reveal possible mechanism of waste activated sludge acidification enhanced by microaeration coupled thermophilic pretreatment
Aiming to strengthen the performance of waste activated sludge digestion, pretreatment is a prerequisite for keeping operations within an industrially acceptable time- frame. The performance of microaeration coupled with thermophilic (MT) pretreatment on waste activated sludge solubilization and acidification was investigated. The results showed that the maximum soluble organics concentrations, including soluble proteins and carbohydrates, reached 2290 mg COD/L in 24 h when the ventilation rate and temperature were 0.05 vvm and 70 °C. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) test on pure-culture (E.coli) and scanning electron microscopy analysis indicated that MT pretreatment effectively destroyed microbial cell wall and resulted in an increase in soluble proteins. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed that MT pretreatment reduced the diversity of the bacterial community during pretreatment. Along with the dissolution of a large quantity of organic matter, microbial species such as Lactococcus and Methyloversatilis bloomed at the end of acidification period, which contributed to SCFAs production. Finally, this study revealed that MT pretreatment facilitated the WAS hydrolysis efficiency and enhanced SCFAs (3149 mg COD/L with fermentation for 96 h) production, especially for acetic acid (50%) accumulation, which provides a new perspective for the application and recovery of sludge resources.
59 BASIC BIOLOGICAL SCIENCES↗
Development of an Efficient Enzyme Production and Structure-Based Discovery Platform for BACE1 Inhibitors
BACE1 (Beta-site Amyloid Precursor Protein (APP) Cleaving Enzyme 1) is a promising therapeutic target for Alzheimer’s Disease (AD). However, efficient expression, purification, and crystallization systems are not well described or detailed in the literature nor are approaches for treatment of enzyme kinetic data for potent inhibitors well described. As such, we developed a platform for expression and purification of BACE1, including protein refolding from E.coli inclusion bodies, in addition to optimizing a reproducible crystallization procedure of BACE1 bound with inhibitors. We also report a detailed approach to the proper analysis of enzyme kinetic data for compounds that exhibit either rapid-equilibrium or tight-binding mechanisms. Our methods allow for the purification of ~15 mg of BACE1 enzyme from 1 L of culture which is higher than reported yields in the current literature. To evaluate the data analysis approach developed here, a well-known potent inhibitor and two of its derivatives were tested, analyzed, and compared. The inhibitory constants (K i ) obtained from the kinetic studies are in agreement with dissociation constants (K d ) that were also determined using isothermal titration calorimetry (ITC) experiments. The X-ray structures of these three compounds in complex with BACE1 were readily obtained and provide important insight into the structure and thermodynamics of the BACE1-inhibitor interactions.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Antimicrobial polymers - The antibacterial effect of photoactivated nano titanium dioxide polymer composites
To obtain a polymer with antimicrobial properties for medical and sanitary applications nanoscale titanium dioxide (TiO{sub 2}) particles have been incorporated into a medical grade polypropylene (PP) matrix with various filler contents (0 wt %, 2 wt %, 10 wt % and 15 wt %). The standard application of TiO{sub 2} for antimicrobial efficacy is to deposit a thin TiO{sub 2} coating on the surface. In contrast to the common way of applying a coating, TiO{sub 2} particles were applied into the bulk polymer. With this design we want to ensure antimicrobial properties even after application of impact effects that could lead to surface defects. The filler material (Aeroxide® TiO{sub 2} P25, Evonik) was applied via melt compounding and the compounding parameters were optimized with respect to nanoscale titanium dioxide. In a next step the effect of UV-irradiation on the compounds concerning their photocatalytic activity, which is related to the titanium dioxide amount, was investigated. The photocatalytic effect of TiO{sub 2}-PP-composites was analyzed by contact angle measurement, by methylene blue testing and by evaluation of inactivation potential for Escherichia coli (E.coli) bacteria. The dependence of antimicrobial activity on the filler content was evaluated, and on the basis of different titanium dioxide fractions adequate amounts of additives within the compounds were discussed. Specimens displayed a higher photocatalytic and also antimicrobial activity and lower contact angles with increasing titania content. The results suggest that the presence of titania embedded in the PP matrix leads to a surface change and a photocatalytic effect with bacteria killing result.
36 MATERIALS SCIENCE↗
Roles of individual radicals generated by a submerged dielectric barrier discharge plasma reactor during Escherichia coli O157:H7 inactivation
A submerged dielectric barrier discharge plasma reactor (underwater DBD) has been used on Escherichia coli O157:H7 (ATCC 35150). Plasma treatment was carried out using clean dry air gas to investigate the individual effects of the radicals produced by underwater DBD on an E. coli O157:H7 suspension (8.0 log CFU/ml). E. coli O157:H7 was reduced by 6.0 log CFU/ml for 2 min of underwater DBD plasma treatment. Optical Emission Spectra (OES) shows that OH and NO (α, β) radicals, generated by underwater DBD along with ozone gas. E. coli O157:H7 were reduced by 2.3 log CFU/ml for 10 min of underwater DBD plasma treatment with the terephthalic acid (TA) OH radical scavenger solution, which is significantly lower (3.7 log CFU/ml) than the result obtained without using the OH radical scavenger. A maximum of 1.5 ppm of ozone gas was produced during the discharge of underwater DBD, and the obtained reduction difference in E.coli O157:H7 in presence and in absence of ozone gas was 1.68 log CFU/ml. The remainder of the 0.62 log CFU/ml reduction might be due to the effect of the NO (α, β) radicals or due to the combined effect of all the radicals produced by underwater DBD. A small amount of hydrogen peroxide was also generated but does not play any role in E. coli O157:H7 inactivation.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Application of VitaVallis dressing for infected wounds
Today there is a growing demand for safe and efficient antimicrobial dressings for infected wound treatment. The antimicrobial sorption material for VitaVallis dressings was produced by one-stage oxidation of aluminum nanopowder in water in the presence of fibrous acetylcellulose matrix. Scanning electron microscopy revealed that the material is made up of fibers of diameter 1.5–3.0 µm with adhered agglomerated alumina nanosheets. An antimicrobial study revealed a high inhibitory effect of VitaVallis against the growth of gram-negative (E.coli, P. aeruginosa) and gram-positive (S. aureus) strains. The antimicrobial activity of the dressing against microbial pathogens on the wound surface was demonstrated in in vivo experiments on male rats. The dressing was also tested on volunteer patients. The testing showed reduction of the wound healing period, accelerated cleaning of the infected wound and enhanced tissue regeneration in the wound. The results demonstrate that the VitaVallis dressing can be used for the treatment of deep infected wounds.
36 MATERIALS SCIENCE↗
Preparation of silver-chitosan nanocomposites and coating on bandage for antibacterial wound dressing application
Bandage is a medical device that is essential for wound dressing. To improve the performance of the bandage, it has been coated by silver-chitosan nanocomposites (Ag/Chit) with pad-dry-cure method. The nanocomposites were performed by chemical reduction method at room temperature using glucose as reducing agent, sodium hydroxide (NaOH) as accelerator reagent, silver nitrate (AgNO{sub 3}) as metal precursor and chitosan as stabilizing agent. Localized surface plasmon resonance (LSPR) absorption band of silver nanoparticles was investigated using UV-Vis spectrophotometer. The bandage coated Ag/Chit nanocomposites (B-Ag/Chit) were characterized by fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscope (SEM). In addition, antibacterial activity of the bandage toward Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli) were also studied. The formation of silver nanoparticles was confirmed by the appearance of LSPR absorption peak at 412.2 – 423.2 nm. Coating of nanocomposite cause increasing rigidity of bandage and decreasing on crystallinity. The bandages of B-Ag/Chit demonstrated good activity against both Gram positive (S. aureus) and Gram negative (E.Coli). Thus the bandages have a potential to be used for antibacterial wound dressing application.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium
Not Available
47 OTHER INSTRUMENTATION↗