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The development of an effective biological (bio) agent detection capability based upon terahertz (THz) frequency absorption spectra will require insight into how the constituent cellular components contribute to the overall THz signature. In this work, the specific contribution of ribonucleic acid (RNA) to THz spectra is analyzed in detail. Previously, it has only been possible to simulate partial fragments of the RNA (or DNA) structures due to the excessive computational demands. For the first time, the molecular structure of the entire transfer RNA (tRNA) molecule of E. coli was simulated and the associated THz signature was derived theoretically. The tRNA that binds amino acid tyrosine (tRNAtyr) was studied. Here, the molecular structure was optimized using the potential energy minimization and molecular dynamical (MD) simulations. Solvation effects (water molecules) were also included explicitly in the MD simulations. To verify that realistic molecular signatures were simulated, a parallel experimental study of tRNAs of E. coli was also conducted. Two very similar molecules, valine and tyrosine tRNA were investigated experimentally. Samples were prepared in the form of water solutions with the concentrations in the range 0.01-1 mg/ml. A strong correlation of the measured THz signatures associated with valine tRNA and tyrosine tRNA was observed. These findings are consistent with the structural similarity of the two tRNAs. The calculated THz signature of the tyrosine tRNA of E. coli reproduces many features of our measured spectra, and, therefore, provides valuable new insights into bio-agent detection.
The experimental and computational study of bacterial thioredoxin, an E. coli protein, at THz frequencies is presented. The absorption spectrum of the entire protein in water was studied numerically in the terahertz range (0.1 – 2 THz). In our work, the initial X-ray molecular structure of thioredoxin was optimized using the molecular dynamical (MD) simulations at room temperature and atmospheric pressure. The effect of a liquid content of a bacterial cell was taken into account explicitly via the simulation of water molecules using the TIP3P water model. Using atomic trajectories from the room-temperature MD simulations, thioredoxin's THz vibrational spectrum and the absorption coefficient were calculated in a quasi harmonic approximation.
For our terahertz transmission measurements, we used solutions of thioredoxin in distilled water obtained from Sigma. The experimental and simulated signatures are correlated and dominant peaks are close in frequencies. The results of this study demonstrate that terahertz spectroscopy is a promising tool in generating spectral data for cellular components of bio agents such as bacterial cells and spores.
The distance distributions of the four types of bases A, C, G and T in the complete sequences of human genome are shown to have long-tail power-law but short-distance exponential behaviors. However, the random sequences with identical numbers of bases show only short-distance exponential behaviors. The DNA sequence of E. coli, which is much shorter than human's, is shown to exhibit essentially exponential behavior as its corresponding random sequence. Also, DNA sequence of human's with smaller C + G content is seen to enjoy longer power-law tail in its distributions of C and G. The coefficient in the exponential decaying function shows a linear dependent with the C + G content but the decay exponent in the power-law shows no such dependence.
In this paper, the asymptotical behavior of a chemostat model for E. coli and the virulent phage T4 is analyzed. The basic reproduction number R0 is proved to be a threshold which determines the outcome of the virulent phage T4. If R0 < 1, the virus dies out; if R0 > 1, the virus persists. Sufficient conditions for the Hopf bifurcation are also established. The theoretical results show that increasing the input of nutrient will result in an increase in the equilibrium population density of the virulent bacteriophage T4, but will have no effect on the equilibrium population density of E. coli. The results also show that increasing the input of nutrient or increasing the average lytic time for the infected E. coli can destabilize the interaction between E. coli and T4.
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Silver nanoparticles (AgNPs) have been synthesized by chemical reduction method using ascorbic acid as reducing agent. Silver nitrate (AgNO3) and sodium dodecyl sulfate (SDS) have been used as precursor and stabilizer, respectively. The prepared samples were characterized by UV-visible spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD). The antibacterial activity of prepared silver nanoparticles has been assessed by using the disc diffusion method against pathogenic, gram-negative bacterial strains including Escherichia coli and Pseudomonassp. To evaluate the potential antibacterial properties of AgNPs, the discs have been impregnated and dried with three different doses like 50, 100 and 150μl of 20μg/ml concentrated AgNPs solution and placed on the Petri-dishes. The antibiotic kanamycin (5μg) was used as control. In all the cases, a clear and distinct zone of inhibition is observed, which suggests that AgNPs can be used as effective growth inhibitors of various bacterial species and would be promising candidate for future development of antibacterial agents.
Mapping of putative promoters within the entire genome of Escherichia coli (E. coli) by means of pattern-recognition software PlatProm revealed several thousand of sites having high probability to perform promoter function. Along with the expected promoters located upstream of coding sequences, PlatProm identified more than a thousand potential promoters for antisense transcription and several hundred very similar signals within coding sequences having the same direction with the genes. Since recently developed ChIP–chip technology also testified the presence of intragenic RNA polymerase binding sites, such distribution of putative promoters is likely to be a general biological phenomenon reflecting yet undiscovered regulatory events. Here, we provide experimental evidences that two internal promoters are recognized by bacterial RNA polymerase. One of them is located within the hns coding sequence and may initiate synthesis of RNA from the antisense strand. Another one is found within the overlapping genes htgA/yaaW and may control the production of a shortened mRNA or an RNA-product complementary to mRNA of yaaW. Both RNA-products can form secondary structures with free energies of folding close to those of small regulatory RNAs (sRNAs) of the same length. Folding propensity of known sRNAs was further compared with that of antisense RNAs (aRNAs), predicted in E. coli as well as in Salmonella typhimurium (S. typhimurium). Slightly lower stability observed for aRNAs assumes that their structural compactness may be less significant for biological function.
The performance of a nanoscale sensor is not limited by the sensitivity of the sensor itself but rather by the diffusion time required for target molecules to reach to the extremely small sensor surface. In this work, we developed a carbon nanotube device that performed the dual functions of concentrating and detecting microorganisms in a sample solution. The sensor surface area was increased by fabricating a carbon nanotube network device using thermal chemical vapor deposition and standard microfabrication techniques. The target Escherichia coli (E. coli) cells were concentrated at the sensor surface via dielectrophoretic concentration by the carbon nanotube network channels. After 10 min of collection, the chip was washed with ample amounts of a clean buffer solution, and only the E. coli cells that were bound to the antibodies remained on the sensor surface. The binding of E. coli to the CNT network device decreased the conductance, presumably due to an increase in the scattering at the sensor surface. The detection limit and the time required for microorganism detection was greatly improved by combining dielectrophoresis with the carbon nanotube devices.
Photodynamic inactivation of microorganisms known as antibacterial photodynamic therapy (APDT) is one of the most promising and innovative approaches for the destruction of pathogenic microorganisms. Among the photosensitizers (PSs), compounds based on cationic porphyrins/metalloporphyrins are most successfully used to inactivate microorganisms. Series of meso-substituted cationic pyridylporphyrins and metalloporphyrins with various peripheral groups in the third and fourth positions of the pyrrole ring have been synthesized in Armenia. The aim of this work was to determine and test the most effective cationic porphyrins and metalloporphyrins with high photoactivity against Gram negative and Gram positive microorganisms. It was shown that the synthesized cationic pyridylporphyrins/metalloporphyrins exhibit a high degree of phototoxicity towards both types of bacteria, including the methicillin-resistant S. aureus strain. Zinc complexes of porphyrins are more phototoxic than metal-free porphyrin analogs. The effectiveness of these Zn–metalloporphyrins on bacteria is consistent with the level of singlet oxygen generation. It was found that the high antibacterial activity of the studied cationic porphyrins/metalloporphyrins depends on four factors: the presence in the porphyrin macrocycle of a positive charge (+4), a central metal atom (Zn2+) and hydrophobic peripheral functional groups as well as high values of quantum yields of singlet oxygen. The results indicate that meso-substituted cationic pyridylporphyrins/metalloporphyrins can find wider application in photoinactivation of bacteria than anionic or neutral PSs usually used in APDT.
Bacterial resistance is today a matter of great medical concern, so it is urgent to investigate alternatives to alleviate it. Photodynamic inactivation (PDI) is a method that has been endorsed to inactivate different pathogens, including bacteria, fungi and viruses. PDI is achieved by using a photosensitizer (PS) molecule which generates reactive oxygen species under visible or UV radiation. We use visible light and UV-A radiation to excite four commercial PSs (methylene blue, rose bengal, riboflavin and curcumin), and nanoparticles synthesized in our laboratory. Despite these PSs having been thoroughly studied in the past by other research groups, in order to compare their effects in an appropriate way, we matched the number of photons they absorb. We found that methylene blue leads to the major inactivation of Escherichia coli. Furthermore, we evaluated the production of singlet oxygen and hydroxyl radicals in the photoinactivation process.
An immunoassay has been developed for the detection of bacterium Escherichia coli (E. coli) through β-Galactosidase (β-Gal). For developing an immunoassay on piezoelectric quartz crystal a polystyrene nanoparticle (PSNP) surface was developed by anchoring synthesized PSNPs (60 ± 5 nm) via self-assembled monolayer of p-aminothiophenol patterned on gold-coated quartz crystal. In order to make immunoassay specific to β-Gal, antibody specific to β-Gal (anti-β-Bal) was captured through physical attraction. On exposing this immunoassay to the β-Gal antigen (3.03 ng/ml), a strong detecting signal was observed, with simultaneous detachment of antibody from immunoassay as antigen–antibody complex. No appreciable change in the frequency shift, Δf, of quartz crystal was observed upon exposure to nonspecific Brucella antigen, confirming the specific behavior of immunoassay.
Natural products span broad activities and applications; however, their access and production are often limited by native cellular sources. As a result, the heterologous production of a siderophore termed yersiniabactin (Ybt) was completed using the surrogate host Escherichia coli. Post-production and purification steps are complicated by the complex nature of most media used for cell growth, prompting the development in this work of an aqueous two-phase pre-purification system capable of rapidly and simply enhancing the concentration of the target Ybt compound.
Non-point source microbial pollution damages the overall health of marine ecosystems. Beyond their direct effects, contaminating microbes can introduce antimicrobial resistance and virulence genes into resident species by horizontal gene transfer, a process that may be enhanced by co-pollutants such as heavy metals. In this work, a number of heavy metal- and antimicrobial-resistant (AMR) strains of E. coli were isolated from water and sediment samples collected in the Causeway Bay, Hong Kong (22.2833 N, 114.1847 E) and their complete genomes obtained by hybrid assembly using both Illumina MiSeq and Oxford Nanopore MinION sequencing platforms. Genomic analysis showed an abundance of plasmids and mobile elements bearing heavy metal- and AMR genes, such as the mer operon and tetR-tetA efflux system, with these features frequently co-located. In addition, NCBIBLAST–using isolates collected from in-land soil, freshwater, and animal faeces in Hong Kong, as well as the NCBI database – showed that these genes, while common amongst human and animal-derived Enterobacteriaceae, could also be shown present in a number of strains of obligate halophilic Vibrio spp.. Previous water chemistry analysis at this location showed high levels of heavy metal contamination (98 ppm Pb, 0.12 ppm Hg, and 0.4 ppm Cd), which has been shown to promote AMR transfer by selecting for co- resistance. Further isolation and characterisation of marine bacterial species from Hong Kong coastal waters will help to determine the extent of this process.
Photodynamic inactivation of microorganisms known as antibacterial photodynamic therapy (APDT) is one of the most promising and innovative approaches for the destruction of pathogenic microorganisms. Among the photosensitizers (PSs), compounds based on cationic porphyrins/metalloporphyrins are most successfully used to inactivate microorganisms. Series of meso-substituted cationic pyridylporphyrins and metalloporphyrins with various peripheral groups in the third and fourth positions of the pyrrole ring have been synthesized in Armenia. The aim of this work was to determine and test the most effective cationic porphyrins and metalloporphyrins with high photoactivity against Gram negative and Gram positive microorganisms. It was shown that the synthesized cationic pyridylporphyrins/metalloporphyrins exhibit a high degree of phototoxicity towards both types of bacteria, including the methicillin-resistant S. aureus strain. Zinc complexes of porphyrins are more phototoxic than metal-free porphyrin analogs. The effectiveness of these Zn–metalloporphyrins on bacteria is consistent with the level of singlet oxygen generation. It was found that the high antibacterial activity of the studied cationic porphyrins/metalloporphyrins depends on four factors: the presence in the porphyrin macrocycle of a positive charge (+4), a central metal atom (Zn2+) and hydrophobic peripheral functional groups as well as high values of quantum yields of singlet oxygen. The results indicate that meso-substituted cationic pyridylporphyrins/metalloporphyrins can find wider application in photoinactivation of bacteria than anionic or neutral PSs usually used in APDT.
Bacterial resistance is today a matter of great medical concern, so it is urgent to investigate alternatives to alleviate it. Photodynamic inactivation (PDI) is a method that has been endorsed to inactivate different pathogens, including bacteria, fungi and viruses. PDI is achieved by using a photosensitizer (PS) molecule which generates reactive oxygen species under visible or UV radiation. We use visible light and UV-A radiation to excite four commercial PSs (methylene blue, rose bengal, riboflavin and curcumin), and nanoparticles synthesized in our laboratory. Despite these PSs having been thoroughly studied in the past by other research groups, in order to compare their effects in an appropriate way, we matched the number of photons they absorb. We found that methylene blue leads to the major inactivation of Escherichia coli. Furthermore, we evaluated the production of singlet oxygen and hydroxyl radicals in the photoinactivation process.
The development of an effective biological (bio) agent detection capability based upon terahertz (THz) frequency absorption spectra will require insight into how the constituent cellular components contribute to the overall THz signature. In this work, the specific contribution of ribonucleic acid (RNA) to THz spectra is analyzed in detail. Previously, it has only been possible to simulate partial fragments of the RNA (or DNA) structures due to the excessive computational demands. For the first time, the molecular structure of the entire transfer RNA (tRNA) molecule of E. coli was simulated and the associated THz signature was derived theoretically. The tRNA that binds amino acid tyrosine (tRNAtyr) was studied. Here, the molecular structure was optimized using the potential energy minimization and molecular dynamical (MD) simulations. Solvation effects (water molecules) were also included explicitly in the MD simulations. To verify that realistic molecular signatures were simulated, a parallel experimental study of tRNAs of E. Coli was also conducted. Two very similar molecules, valine and tyrosine tRNA were investigated experimentally. Samples were prepared in the form of water solutions with the concentrations in the range 0.01-1 mg/ml. A strong correlation of the measured THz signatures associated with valine tRNA and tyrosine tRNA was observed. These findings are consistent with the structural similarity of the two tRNAs. The calculated THz signature of the tyrosine tRNA of E.coli reproduces many features of our measured spectra, and, therefore, provides valuable new insights into bio-agent detection.
The experimental and computational study of bacterial thioredoxin, an E. coli protein, at THz frequencies is presented. The absorption spectrum of the entire protein in water was studied numerically in the terahertz range (0.1 – 2 THz). In our work, the initial X-ray molecular structure of thioredoxin was optimized using the molecular dynamical (MD) simulations at room temperature and atmospheric pressure. The effect of a liquid content of a bacterial cell was taken into account explicitly via the simulation of water molecules using the TIP3P water model. Using atomic trajectories from the room-temperature MD simulations, thioredoxin's THz vibrational spectrum and the absorption coefficient were calculated in a quasi harmonic approximation.
For our terahertz transmission measurements, we used solutions of thioredoxin in distilled water obtained from Sigma. The experimental and simulated signatures are correlated and dominant peaks are close in frequencies. The results of this study demonstrate that terahertz spectroscopy is a promising tool in generating spectral data for cellular components of bio agents such as bacterial cells and spores.
The purpose of the present study was to asses the thermal inactivation of E. coli and coliform during the kneading step, in hot water, in the making process of Oaxaca cheese – a fresh pasta filata Mexican cheese. A three-strain cocktail of E. coli isolated from industrial Oaxaca cheeses was used for the assay. Three batches of pasteurised and, then, inoculated milk were processed into Oaxaca cheese following a traditional open-vat process until before the kneading step. At this point, the process was halted and a simulated kneading step at 55 °C for 15 min was carried out. Counts of E. coli and coliform were performed on the curd after 0, 5, 10 and 15 min of the begining of the sumulated kneading. The data of these counts were analysed using linear regression, and the correspondent D55°C values were calculated. The heating taking place during the kneading can reduce considerably the population of E. coli and coliform of the curd. However, it did not eliminate entirely the risk of foodborne illness in Oaxaca cheese.
Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.
Enteroccocus faecalis, a gram-positive bacterium ubiquitous in the environment and normal inhabitant of the human gastrointestinal tract, has emerged as a major health problem exacerbated by its ability at surviving adverse conditions, including prolonged stay on environmental surfaces. Here its tolerance to detergents that are routine household chemicals was investigated and compared to that of E. coli. A clinical isolate of E. faecalis with plasmid-mediated antibiotics resistance grown in the presence of increasing concentrations of sodium N-lauroylsarcosinate (sarkosyl) and sodium dodecyl sulphate (SDS) exhibited a sarkosyl minimum inhibitory concentration (MIC) of 0.1% (10% for E. coli) and a SDS MIC of 0.05% (6% for E. coli). A mutant with accrued sarkosyl resistance (MIC = 0.6%) was isolated after growth in the presence of 0.06% sarkosyl. While there was no cross-resistance to SDS, other mutant characteristics gave it advantages over the original strain, emphasizing the potential emergence of E. faecalis with increasing tolerance to common household detergents.