RESUMO
BACKGROUND: The tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics. METHODS: We utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs. RESULTS: TolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. coli nor does it reduce MIC values of antibiotics erythromycin and fusidic acid. CONCLUSIONS: TolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target. GENERAL SIGNIFICANCE: TolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Fenômenos Biofísicos , Cobalto/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Ressonância de Plasmônio de SuperfícieRESUMO
The objectives of this study were to develop a high-density chromosome bin map of homoeologous group 7 in hexaploid wheat (Triticum aestivum L.), to identify gene distribution in these chromosomes, and to perform comparative studies of wheat with rice and barley. We mapped 2148 loci from 919 EST clones onto group 7 chromosomes of wheat. In the majority of cases the numbers of loci were significantly lower in the centromeric regions and tended to increase in the distal regions. The level of duplicated loci in this group was 24% with most of these loci being localized toward the distal regions. One hundred nineteen EST probes that hybridized to three fragments and mapped to the three group 7 chromosomes were designated landmark probes and were used to construct a consensus homoeologous group 7 map. An additional 49 probes that mapped to 7AS, 7DS, and the ancestral translocated segment involving 7BS also were designated landmarks. Landmark probe orders and comparative maps of wheat, rice, and barley were produced on the basis of corresponding rice BAC/PAC and genetic markers that mapped on chromosomes 6 and 8 of rice. Identification of landmark ESTs and development of consensus maps may provide a framework of conserved coding regions predating the evolution of wheat genomes.
Assuntos
Mapeamento Cromossômico , Cromossomos de Plantas/genética , Etiquetas de Sequências Expressas , Genes de Plantas , Triticum/genética , Deleção de Genes , Duplicação Gênica , Marcadores Genéticos , Genoma de Planta , Hordeum/genética , Oryza/genética , Alinhamento de SequênciaRESUMO
Optimal steady-state performance of any biofilm reactor requires a fully developed and mature biofilm. During fixed-film reactor startup phase, biofilm is in process of development and accordingly, process performance is difficult to quantify. Environmental, cellular and surface factors greatly influence the process of biofilm formation during reactor startup. Improved knowledge of nutritional, toxicological and environmental requirements of wastewater degrading microorganisms has helped define optimal microbial growth conditions. In case of anaerobic fixed film reactors the startup is hindered by low microbial growth rates, strict environmental requirements and limited ability of methanogens to adhere and form fixed biofilms. These obstacles could be overcome by proper support media selection and formulation of appropriate inoculation procedures and startup strategies.