RESUMO
The tellurium oxyanion tellurate is toxic to living organisms even at low concentrations; however, its mechanism of toxicity is poorly understood. Here, we show that exposure of Escherichia coli K-12 to tellurate results in reduction to elemental tellurium (Te[0]) and the formation of intracellular reactive oxygen species (ROS). Toxicity assays performed with E. coli indicated that pre-oxidation of the intracellular thiol pools increases cellular resistance to tellurate-suggesting that intracellular thiols are important in tellurate toxicity. X-ray absorption spectroscopy experiments demonstrated that cysteine reduces tellurate to elemental tellurium. This redox reaction was found to generate superoxide anions. These results indicate that tellurate reduction to Te(0) by cysteine is a source of ROS in the cytoplasm of tellurate-exposed cells.
Assuntos
Cisteína/metabolismo , Escherichia coli K12/efeitos dos fármacos , Telúrio/farmacologia , Escherichia coli K12/citologia , Escherichia coli K12/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Telúrio/metabolismoRESUMO
Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants - presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring - influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.
Assuntos
Antineoplásicos/farmacologia , DNA Tumoral Circulante/química , Lignanas/farmacologia , RNA Neoplásico/química , Albumina Sérica Humana/química , Xantenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli K12/citologia , Escherichia coli K12/efeitos dos fármacos , Humanos , Lignanas/síntese química , Lignanas/química , Estrutura Molecular , Salmonella enterica/citologia , Salmonella enterica/efeitos dos fármacos , Staphylococcus aureus/citologia , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Xantenos/síntese química , Xantenos/químicaRESUMO
In Raman-activated cell ejection and sequencing (RACE-Seq), success rate and sequence coverage have generally been low for shotgun sequencing of individual post-RACE cells. Here we quantitatively evaluated the influence of cell lysis condition, nucleic acid amplification condition, and parameters of Raman measurement on RACE-Seq performance. Variations in laser energy input during Raman signal acquisition, but not duration of alkaline lysate lysis, temperature, or measurement under dry or aqueous conditions, are vital to the success of multiple displacement amplification (MDA). In fact, laser irradiation is reversely linked to MDA product quality. However, introduction of oils prior to MDA, by mitigating such negative effects of Raman irradiation, elevates genome coverage of post-RACE Escherichia coli cells from <20% to â¼50%, while greatly improving the success rate of RACE-Seq for soil microbiota. Our findings provide a practical solution for enhancing RACE-Seq performance and pinpoint protection of cells from laser irradiation as a priority in method development.
Assuntos
DNA Bacteriano/genética , Escherichia coli K12/genética , Análise de Sequência de DNA , Escherichia coli K12/citologia , Técnicas de Amplificação de Ácido Nucleico , Análise Espectral RamanRESUMO
We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a riboflavin-producing bacterial strain. We show that stimulation as well as inhibition of bacterial production can be performed through the liposomal membrane simply by passive diffusion of inducing or antibiotic compounds, respectively. The possibility to introduce as well as accumulate compounds in liposomal cultivation compartments represents great advantage over the current state of the art systems, emulsion droplets, and gel beads. Additionally, the encapsulation of bacteria and monitoring of individual lipid vesicles have been accomplished on a single microfluidic device. The presented system paves the way toward highly parallel microbial cultivation and monitoring as required in biotechnology, basic research, or drug discovery.
Assuntos
Escherichia coli K12/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip , Lipossomas Unilamelares/química , Emulsões , Escherichia coli K12/citologiaRESUMO
The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM.
Assuntos
Membrana Externa Bacteriana , Fenômenos Biofísicos , Simulação de Dinâmica Molecular , Escherichia coli K12/citologia , Conformação Molecular , Difração de NêutronsRESUMO
Isothermal nucleic acid amplification (INAA) techniques such as loop-mediated isothermal amplification (LAMP) and isothermal multiple-self-matching-initiated amplification (IMSA) constitute simple and rapid approaches for the detection of pathogens. However, due to the employment of multiple primers, the detection of LAMP and IMSA products is easily influenced by high background signals from primer dimer-based nonspecific nucleic acid amplification (NSA) products. Moreover, time-consuming sample preparation steps are often required for the isolation of sufficiently pure nucleic acid prior to INAA. To address these drawbacks, hydrophobic magnetic ionic liquids (MILs) were used to rapidly preconcentrate DNA from complex biological samples followed by direct amplification by LAMP and IMSA. Careful control of the components within the isothermal buffer permitted direct addition of DNA-enriched MIL to the INAA reaction mixture, thereby circumventing tedious purification procedures that are ordinarily required prior to downstream DNA amplification. When added directly to INAA reactions, MIL solvents released metal ions that ultimately inhibited the primer dimer-mediated NSA, resulting in a flat or decreased baseline signal in no-template control samples and short threshold time for positive reactions. Using a MIL-based single droplet DNA extraction method, MIL-enhanced INAA reaction system, and a handheld 3D printed device for visual detection of the amplified product in customized tubes, we demonstrate the potential of the MIL-based approach for the onsite analysis of DNA from pathogens.
Assuntos
DNA/análise , Líquidos Iônicos/química , Técnicas de Amplificação de Ácido Nucleico , Células Cultivadas , Escherichia coli K12/citologia , Fenômenos Magnéticos , Plasmídeos/químicaRESUMO
Bacterial suspensions-a premier example of active fluids-show an unusual response to shear stresses. Instead of increasing the viscosity of the suspending fluid, the emergent collective motions of swimming bacteria can turn a suspension into a superfluid with zero apparent viscosity. Although the existence of active superfluids has been demonstrated in bulk rheological measurements, the microscopic origin and dynamics of such an exotic phase have not been experimentally probed. Here, using high-speed confocal rheometry, we study the dynamics of concentrated bacterial suspensions under simple planar shear. We find that bacterial superfluids under shear exhibit unusual symmetric shear bands, defying the conventional wisdom on shear banding of complex fluids, where the formation of steady shear bands necessarily breaks the symmetry of unsheared samples. We propose a simple hydrodynamic model based on the local stress balance and the ergodic sampling of nonequilibrium shear configurations, which quantitatively describes the observed symmetric shear-banding structure. The model also successfully predicts various interesting features of swarming vortices in stationary bacterial suspensions. Our study provides insights into the physical properties of collective swarming in active fluids and illustrates their profound influences on transport processes.
Assuntos
Escherichia coli K12/citologia , Escherichia coli K12/fisiologia , Modelos Biológicos , Resistência ao CisalhamentoRESUMO
In the last years, carbon-based nanomaterials have attracted considerable attention in a wide range of fields, particularly in biomedicine, owing to their remarkable photo-physical and chemical properties. In this study, we demonstrate that amine-terminated carbon dots (CDs-NH2) functionalized with ampicillin (AMP) offer a new perspective for antibacterial treatment. The amine-functionalized carbon dots were used as a carrier for immobilization and delivery of ampicillin (CDs-AMP) and as a visible light-triggered antibacterial material. Additionally, AMP immobilization on the CDs-NH2 surface improves its stability in solution as compared to free AMP. The AMP conjugated CDs platform combines the antibacterial function of AMP and conserves the intrinsic theranostic properties of CDs-NH2. Therefore, the AMP immobilized onto CDs-NH2 surface together with the generation of moderate quantities of reactive oxygen species under visible light illumination are very effective to inactivate the growth of Escherichia coli.
Assuntos
Aminas/farmacologia , Ampicilina/farmacologia , Antibacterianos/farmacologia , Carbono/farmacologia , Escherichia coli K12/efeitos dos fármacos , Luz , Pontos Quânticos/química , Adulto , Aminas/síntese química , Aminas/química , Ampicilina/química , Antibacterianos/síntese química , Antibacterianos/química , Carbono/química , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Escherichia coli K12/citologia , Feminino , Células HeLa , Humanos , Testes de Sensibilidade Microbiana , Fotoquimioterapia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Interactions between bacteria and colon cancer cells influence the transcription of the host cell. Yet is it undetermined whether the bacteria itself or the communication between the host and bacteria is responsible for the genomic changes in the eukaryotic cell. Now, we have investigated the genomic and epigenetic consequences of co-culturing colorectal carcinoma cells with membrane vesicles from pathogenic bacteria Vibrio cholerae and non-pathogenic commensal bacteria Escherichia coli. Our study reveals that membrane vesicles from pathogenic and commensal bacteria have a global impact on the gene expression of colon-carcinoma cells. The changes in gene expression correlate positively with both epigenetic changes and chromatin accessibility of promoters at transcription start sites of genes induced by both types of membrane vesicles. Moreover, we have demonstrated that membrane vesicles obtained only from V. cholerae induced the expression of genes associated with epithelial cell differentiation. Altogether, our study suggests that the observed genomic changes in host cells might be due to specific components of membrane vesicles and do not require communication by direct contact with the bacteria.
Assuntos
Membrana Celular/metabolismo , Neoplasias do Colo/microbiologia , Neoplasias do Colo/patologia , Epigênese Genética , Escherichia coli K12/citologia , Transcrição Gênica , Vibrio cholerae/citologia , Linhagem Celular , Neoplasias do Colo/genética , HumanosRESUMO
The anode material is vital to improve the power generation of a microbial fuel cell (MFC). In this study, a carbon nanotube (CNT)-coated sponge with macro-porous structure, high surface area, and high conductivity was constructed as an anode to encapsulate Escherichia coli K12 (E. coli K12) cells. To achieve high power generation of the MFC, the optimal concentration of the CNT coating the sponge was found to be 30mgmL-1. At this concentration, a maximum power density of 787Wm-3 and a chemical oxygen demand (COD) removal of 80.9% were obtained with a long stable electricity generation process in batch mode. This indicates that the biofilm on the CNT (30mgmL-1)-coated sponge possessed excellent electroactivity and stability. Scanning electron microscope (SEM) images confirmed that the CNT-coated sponge provided a suitable microenvironment for E. coli K12 cells to maintain their attachment and colonization. Additionally, a CNT-dependent viability phenomenon of the E. coli K12 cells was discovered after electricity generation. This CNT-dependent viability of the E. coli K12 cells was stable and sustainable after storage at -20°C in a milk tube for one year.
Assuntos
Fontes de Energia Bioelétrica/microbiologia , Escherichia coli K12/citologia , Nanotubos de Carbono/química , Biofilmes/crescimento & desenvolvimento , Análise da Demanda Biológica de Oxigênio , Células Imobilizadas/química , Células Imobilizadas/citologia , Células Imobilizadas/fisiologia , Condutividade Elétrica , Eletricidade , Eletrodos , Escherichia coli K12/química , Escherichia coli K12/fisiologia , Viabilidade MicrobianaRESUMO
The Min system negatively regulates the position of the Z ring, which serves as a scaffold for the divisome that mediates bacterial cytokinesis. In Escherichia coli, this system consists of MinC, which antagonizes assembly of the tubulin homologue FtsZ. MinC is recruited to the membrane by MinD and induced by MinE to oscillate between the cell poles. MinC is a dimer with each monomer consisting of functionally distinct MinCN and MinCC domains, both of which contact FtsZ. According to one model, MinCC/MinD binding to the FtsZ tail positions MinCN at the junction of two GDP-containing subunits in the filament, leading to filament breakage. Others posit that MinC sequesters FtsZ-GDP monomers or that MinCN caps the minus end of FtsZ polymers and that MinCC interferes with lateral interactions between FtsZ filaments. Here, we isolated minC mutations that impair MinCN function and analyzed FtsZ mutants resistant to MinC/MinD. Surprisingly, we found mutations in both minC and ftsZ that differentiate inhibition by MinC from inhibition by MinC/MinD. Analysis of these mutations suggests that inhibition of the Z ring by MinC alone is due to sequestration, whereas inhibition by MinC/MinD is not. In conclusion, our genetic and biochemical data support the model that MinC/MinD fragments FtsZ filaments.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Escherichia coli K12/química , Escherichia coli K12/citologia , Escherichia coli K12/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Multimerização ProteicaRESUMO
The utility of engineering flocculation is wildly recognized in applied and environmental microbiology. We previously reported self-produced flocculation of Escherichia coli cells by overexpressing the native bcsB gene that encodes a component of the cellulose synthesis pathway. Further experiments clarified that the spontaneous E. coli flocs were proteinous, and elongation factor Ts (Tsf) was the main component. In this study, we demonstrated successful expression of a fusion protein consisting of Tsf and green fluorescence protein (GFP) on E. coli flocs. Interestingly, the percentage of Tsf-GFP in total floc protein reached approximately 15% (w/w). The proposed design of a fusion protein with Tsf enables displaying a recombinant target protein on the floc structure.
Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Técnicas Bacteriológicas , Bioengenharia , Escherichia coli K12/citologia , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Floculação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Alongamento de Peptídeos/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND: Bacterial small regulatory RNAs (sRNAs) play important roles in sensing environment changes through sRNA-target mRNA interactions. However, the current strategy for detecting sRNA-mRNA interactions usually combines bioinformatics prediction and experimental verification, which is hampered by low prediction accuracy and low-throughput. Additionally, among the 4736 sequenced bacterial genomes, only about 2164 sRNAs from 319 strains have been described. Furthermore, target mRNAs of only 157 sRNAs have been uncovered. Obviously, highly efficient methods were required to detect sRNA-mRNA interactions in the sequenced genomes. This study aimed to apply a modified CLASH (cross-linking, ligation and sequencing hybrids) method to detect RNA-RNA interactions in E. coli, a model bacterial organism. RESULTS: Statistically significant interactions were detected in 29 transcript pairs. To the best of our knowledge, 24 pairs were reported for the first time and were novel RNA interactions, including tRNA-tRNA, tRNA-ncRNA (non-coding RNA), tRNA-rRNA, rRNA-mRNA, rRNA-ncRNA, rRNA-rRNA, rRNA-IGT (intergenic transcript), and tRNA-IGT interactions. CONCLUSIONS: Discovery of novel RNA-RNA interactions in the present study demonstrates that RNA-RNA interactions might be far more complicated than ever expected. New methods may be required to help discover more novel RNA-RNA interactions. The present work describes a high-throughput protocol not only for discovering new RNA interactions, but also directly obtaining base-pairing sequences, which should be useful in assessing RNA structure and interactions.
Assuntos
Biologia Computacional/métodos , Escherichia coli K12/genética , RNA Bacteriano/metabolismo , Escherichia coli K12/citologia , Escherichia coli K12/efeitos da radiação , RNA Bacteriano/genética , Termodinâmica , Raios UltravioletaRESUMO
Two protocols that allow for the comparison of Raman spectra of planktonic cells and biofilm formed from these cells in their growth phase have been developed. Planktonic cells are washed and flash-frozen in <1 min to reduce the time for metabolic changes during processing, prior to freeze-drying. Biofilm is formed by standing cells in 50 µL indentations in aluminum foil in an atmosphere of saturated water vapor for 24-48 h. The results for Escherichia coli type K12 cells, which do not readily form biofilm, are compared to those for Staphylococcus epidermidis cells, which prolifically synthesize biofilm. For E. coli, the Raman spectra of the planktonic and biofilm samples are similar with the exception that the spectral signature of RNA, present in planktonic cells, could not be detected in biofilm. For S. epidermidis, major changes occur upon biofilm formation. In addition to the absence of the RNA features, new bands occur near 950 cm-1 and between 1350 and 1420 cm-1 that are associated with an increase in carbohydrate content. Unlike the case in E. coli biofilm, the intensity of G base ring modes is reduced in but A and T base ring signatures become more prominent. For S. epidermis in the biofilm's amide III region, there is evidence of an increase in the level of ß-sheet structure accompanied by a decrease in α-helical content. The presence of biofilm is confirmed by microscope-aided photography and, separately, by staining with methyl violet.
Assuntos
Biofilmes , Escherichia coli K12/fisiologia , Plâncton/fisiologia , Staphylococcus epidermidis/fisiologia , Métodos Analíticos de Preparação de Amostras , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Carboidratos/biossíntese , Carboidratos/isolamento & purificação , Escherichia coli K12/química , Escherichia coli K12/citologia , Escherichia coli K12/crescimento & desenvolvimento , Liofilização , Microtecnologia , Plâncton/crescimento & desenvolvimento , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , RNA Bacteriano/biossíntese , RNA Bacteriano/isolamento & purificação , Reprodutibilidade dos Testes , Análise Espectral Raman , Staphylococcus epidermidis/química , Staphylococcus epidermidis/citologia , Staphylococcus epidermidis/crescimento & desenvolvimentoRESUMO
Perturbation of cellular processes is a prevailing approach to understanding biology. To better understand the complicated biology that defines bacterial shape, a sensitive, high-content platform was developed to detect multiple morphological defect phenotypes using microscopy. We examined morphological phenotypes across the Escherichia coli K-12 deletion (Keio) collection at the mid-exponential growth phase, revealing 111 deletions perturbing shape. Interestingly, 64% of these were uncharacterized mutants, illustrating the complex nature of shape maintenance and regulation in bacteria. To understand the roles these genes play in defining morphology, 53 mutants with knockouts resulting in abnormal cell shape were crossed with the Keio collection in high throughput, generating 1,373 synthetic lethal interactions across 1.7 million double deletion mutants. This analysis yielded a highly populated interaction network spanning and linking multiple phenotypes, with a preponderance of interactions involved in transport, oxidation-reduction, and metabolic processes.IMPORTANCE Genetic perturbations of cellular functions are a prevailing approach to understanding cell systems, which are increasingly being practiced in very high throughput. Here, we report a high-content microscopy platform tailored to bacteria, which probes the impact of genetic mutation on cell morphology. This has particular utility in revealing elusive and subtle morphological phenotypes associated with blocks in nonessential cellular functions. We report 111 nonessential mutations impacting E. coli morphology, with nearly half of those genes being poorly annotated or uncharacterized. Further, these genes appear to be tightly linked to transport or redox processes within the cell. The screening platform is simple and low cost and is broadly applicable to any bacterial genomic library or chemical collection. Indeed, this is a powerful tool in understanding the biology behind bacterial shape.
Assuntos
Escherichia coli K12/citologia , Escherichia coli K12/genética , Genes Bacterianos , Deleção de Genes , Redes Reguladoras de Genes , Testes Genéticos , MicroscopiaRESUMO
For electrochemical regulations of the intracellular metabolisms, lipophilic electron mediators with cell membrane permeability have been conventionally used. We have recently developed amphiphilic, cell-membrane permeable polymer composed of hydrophilic 2-methacryloyloxyethyl phosphorylcholine and hydrophobic redox-active units as a new category of electron mediator. The advantage of the redox active polymer is that we can obtain appropriate molecules in a synthetic bottom-up manner. Here we report that the rate of the extracellular electron transfer (EET) through the redox active polymer can be regulated by sophisticated molecular design of the polymers. It was also shown that the cellular metabolism of yeast Saccharomyces cerevisiae was regulated by using the polymer with the highest EET rate.
Assuntos
Desenho de Fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Fosforilcolina/análogos & derivados , Ácidos Polimetacrílicos/química , Ácidos Polimetacrílicos/farmacologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Escherichia coli K12/citologia , Fosforilcolina/química , Fosforilcolina/farmacologia , Saccharomyces cerevisiae/citologiaRESUMO
The asymmetric outer membrane of Gram-negative bacteria is formed of the inner leaflet with phospholipids and the outer leaflet with lipopolysaccharides (LPS). Outer membrane protein F (OmpF) is a trimeric porin responsible for the passive transport of small molecules across the outer membrane of Escherichia coli. Here, we report the impact of different levels of heterogeneity in LPS environments on the structure and dynamics of OmpF using all-atom molecular dynamics simulations. The simulations provide insight into the flexibility and dynamics of LPS components that are highly dependent on local environments, with lipid A being the most rigid and O-antigen being the most flexible. Increased flexibility of O-antigen polysaccharides is observed in heterogeneous LPS systems, where the adjacent O-antigen repeating units are weakly interacting and thus more dynamic, compared to homogeneous LPS systems in which LPS interacts strongly with each other with limited overall flexibility due to dense packing. The model systems were validated by comparing molecular-level details of interactions between OmpF surface residues and LPS core sugars with experimental data, establishing the importance of LPS core oligosaccharides in shielding OmpF surface epitopes recognized by monoclonal antibodies. There are LPS environmental influences on the movement of bulk ions (K(+) and Cl(-)), but the ion selectivity of OmpF is mainly affected by bulk ion concentration.
Assuntos
Lipopolissacarídeos/metabolismo , Simulação de Dinâmica Molecular , Porinas/química , Porinas/metabolismo , Membrana Celular/metabolismo , Escherichia coli K12/citologia , Escherichia coli K12/metabolismo , Lipopolissacarídeos/química , Permeabilidade , Porosidade , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Especificidade por Substrato , Propriedades de SuperfícieRESUMO
Genotoxic/antigenotoxic, mutagenic/antimutagenic and cytotoxic effects of monoterpenes camphor, eucalyptol and thujone were determined in bacteria and mammalian cells using alkaline comet assay, Escherichia coli K12 reversion test and MTT assay, respectively. When applied in low doses (up to 200 µM in bacterial assay and 50 µM in comet assay) monoterpenes protected repair proficient E. coli and Vero cells against UV-induced mutagenesis and 4NQO-induced DNA strand breaks, respectively. Antimutagenic response was not detected in nucleotide excision repair (NER) deficient bacteria. When monoterpenes were applied in higher doses, a weak mutagenic effect was found in mismatch repair (MMR) and NER deficient E. coli strains, while induction of DNA strand breaks was evident in human fetal lung fibroblasts MRC-5, colorectal carcinoma HT-29 and HCT 116 cells, as well as in Vero cells. Moreover, the involvement of NER, MMR and RecBCD pathways in repair of DNA lesions induced by monoterpenes was demonstrated in E. coli. Camphor, eucalyptol and thujone were cytotoxic to MRC-5, HT-29 and HCT 116 cells. The most susceptible cell line was HCT 116, with IC50 values of 4.5 mM for camphor, 4 mM for eucalyptol and 1 mM for thujone. Observed effects of monoterpenes are consistent with hormesis response, characterized by a low dose beneficial effect and a high dose adverse effect of a stressor agent, and provide a basis for further study of both chemopreventive and chemotherapeutic potential of camphor, eucalyptol and thujone.
Assuntos
Citotoxinas/farmacologia , Citotoxinas/toxicidade , Escherichia coli K12/citologia , Mutagênicos/farmacologia , Mutagênicos/toxicidade , Animais , Monoterpenos Bicíclicos , Cânfora/farmacologia , Cânfora/toxicidade , Cicloexanóis/farmacologia , Cicloexanóis/toxicidade , Eucaliptol , Humanos , Monoterpenos/farmacologia , Monoterpenos/toxicidadeRESUMO
Fluorescence microscopy has revolutionized in vivo cellular biology. Through the specific labeling of a protein of interest with a fluorescent protein, one is able to study movement and colocalization, and even count individual proteins in a live cell. Different algorithms exist to quantify the total intensity and position of a fluorescent focus. Although these algorithms have been rigorously studied for in vitro conditions, which are greatly different than the in-homogenous and variable cellular environments, their exact limits and applicability in the context of a live cell have not been thoroughly and systematically evaluated. In this study, we quantitatively characterize the influence of different background subtraction algorithms on several focus analysis algorithms. We use, to our knowledge, a novel approach to assess the sensitivity of the focus analysis algorithms to background removal, in which simulated and experimental data are combined to maintain full control over the sensitivity of a focus within a realistic background of cellular fluorescence. We demonstrate that the choice of algorithm and the corresponding error are dependent on both the brightness of the focus, and the cellular context. Expectedly, focus intensity estimation and localization accuracy suffer in all algorithms at low focus to background ratios, with the bacteroidal background subtraction in combination with the median excess algorithm, and the region of interest background subtraction in combination with a two-dimensional Gaussian fit algorithm, performing the best. We furthermore show that the choice of background subtraction algorithm is dependent on the expression level of the protein under investigation, and that the localization error is dependent on the distance of a focus from the bacterial edge and pole. Our results establish a set of guidelines for what signals can be analyzed to give a targeted spatial and intensity accuracy within a bacterial cell.
Assuntos
Escherichia coli K12/citologia , Espaço Intracelular/metabolismo , Microscopia de FluorescênciaRESUMO
The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes.