RESUMO
The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.
Assuntos
Proteínas de Bactérias/genética , DNA Helicases/genética , Replicação do DNA/genética , Farmacorresistência Bacteriana/genética , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Geobacillus stearothermophilus/efeitos dos fármacos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/patogenicidade , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Tetraciclina/farmacologia , Transativadores/genéticaRESUMO
Novel methods and apparatus that employ the rapid heating characteristics of microwave irradiation to facilitate the aseptic transfer of nutrients, products, and other materials between microbially sensitive systems and the external environment are described. The microwave-sterilizable access port (MSAP) consists of a 600-W magnetron emitting at a frequency of 2.45 GHz, a sterilization chamber with inlet and outlet flow lines, and a specimen transfer interface. Energy is routed to the sterilization chamber via a coaxial transmission line where small quantities of water couple strongly with the incident radiation to produce a superheated vapor phase. The efficiency of energy transfer is enhanced through the use of microwave susceptors within the sterilization chamber. Mating surfaces are thermally sterilized through direct contact with the hot gas. Efficacy has been demonstrated using the thermophile Bacillus stearothermophilus.
Assuntos
Reatores Biológicos/normas , Contaminação de Equipamentos/prevenção & controle , Micro-Ondas , Esterilização/métodos , Reatores Biológicos/microbiologia , Geobacillus stearothermophilus/patogenicidade , Vapor , Esterilização/instrumentação , Temperatura , Fatores de TempoRESUMO
Se evaluó la presencia de residuos de antibióticos en 200 muestras de leche pasteurizada recolectadas en diez hospitales costaricenses. La detección de antibióticos se efectuó mediante las pruebas de inhibición del crecimiento del Bacillus stearothermophilus var. calidolactis (Delvo test P) y del Streptococcus thermophilus (T-101): (Valiotest 101). Los resultados señalan que el 13 por ciento (N=200) de las muestras presentaron residuos de antibióticos; no obstante, los porcentajes de positividad de las muestras variaron significativamente entre los hospitales evaluados, oscilando entre cero y 35 por ciento (N=20). El mayor número de muestras positivas correspondió a las industrias lácteas que suministran el 70 por ciento de la leche requerida en los centros hospitalarios incluídos en el estudio
Assuntos
Antibacterianos/uso terapêutico , Doenças Transmissíveis/patologia , Quimioterapia Combinada/uso terapêutico , Geobacillus stearothermophilus/patogenicidade , Hospitais/estatística & dados numéricosRESUMO
The pathogenicities of three proteases from Serratia marcescens, two proteases from Pseudomonas aeruginosa, and one thermolysin from Bacillus stearothermophilus were examined. All proteases tested caused acute liquefactive necrosis of the cornea and descemetocele formation in guinea pig eyes after intrastromal injection, with the exception of the 60-kilodalton protease from S. marcescens, which produced only an opaque lesion. When injected into guinea pig skin, the protease also enhanced vascular permeability, which was followed by edema formation. The permeability-enhancing activity of the proteases increased in parallel with the concentration of the enzymes. When tested in vitro for its effect on these bacterial proteases, chicken egg white ovomacroglobulin (ovoM) inhibited the enzymatic activity of all the proteases after a short incubation period at an enzyme/inhibitor ratio (molar) of 1:1 to 1:4 or at a lower concentration after a longer incubation period. Such treatment of the proteases with chicken egg white ovoM before injection intrastromally into the eyes or intradermally into the clipped flanks of guinea pigs protected the cornea from destruction or completely prevented the permeability reaction and edema formation. No inhibitory effects of plasma protease inhibitors against these bacterial proteases were noted. Since the proteases are critical in the pathogenic processes caused by the bacteria, these results suggest a beneficial effect of ovoM against bacterial infections.
