RESUMO
AIMS: The aim of this work was to clarify the mechanism of monounsaturated fatty acid (MUFA) synthesis in Bradyrhizobium TAL1000 and the effect of high temperature on this process. METHODS AND RESULTS: Bradyrhizobium TAL1000 was exposed to a high growth temperature and heat shock, and fatty acid composition and synthesis were tested. To determine the presence of a possible desaturase, a gene was identify and overexpressed in Escherichia coli. The desaturase expression was detected by RT-PCR and Western blotting. In B. TAL1000, an aerobic mechanism for MUFA synthesis was detected. Desaturation was decreased by high growth temperature and by heat shock. Two hours of exposure to 37°C were required for the change in MUFA levels. A potential ∆9 desaturase gene was identified and successfully expressed in E. coli. A high growth temperature and not heat shock reduced transcript and protein desaturase levels in rhizobial strain. CONCLUSIONS: In B. TAL1000, the anaerobic MUFA biosynthetic pathway is supplemented by an aerobic mechanism mediated by desaturase and is down-regulated by temperature to maintain membrane fluidity under stressful conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This knowledge will be useful for developing strategies to improve a sustainable practice of this bacterium under stress and to enhance the bioprocess for the inoculants' manufacture.
Assuntos
Arachis/microbiologia , Bradyrhizobium/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos/biossíntese , Temperatura , Aerobiose , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos Dessaturases/genética , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Fluidez de Membrana , Dados de Sequência Molecular , NodulaçãoRESUMO
We investigated whole blood coagulation by thrombelastography (TEG) employing activation with minute amounts of tissue factor (TF). Continuous raw data captured were transformed into novel parameters, such as the maximum velocity (MaxVel) and the time to maximum velocity (t,MaxVel) of whole blood clot formation. The courses of the whole blood clot development were very similar to thrombin generation curves reported in plasma. In this assay healthy women (n = 30) showed an earlier onset and an increased coagulation velocity compared to healthy men (n = 30). In patients with severe hemophilia, and persons undergoing thromboprophylaxis, distinctly abnormal coagulation profiles were observed with a decrease in the MaxVel, as well as a prolonged t,MaxVel. Changes appeared to be dependent on the nature and severity of the hemostatic deficit. Preliminary studies in patients substituted with recombinant factor VIIa demonstrated a marked change in the coagulation profile, in which the MaxVel and t,MaxVel shifted towards normal in a dose-dependent way. Data suggest that the whole blood coagulation TEG profile, following activation with minute amounts of TF, may reflect the hemostatic potential in patients suspected of impaired hemostasis.
