RESUMO
The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.
Assuntos
Azospirillum brasilense/metabolismo , Citometria de Fluxo/métodos , Herbaspirillum/metabolismo , Hidroxibutiratos/metabolismo , Raízes de Plantas/microbiologia , Poliésteres/metabolismo , Microscopia de FluorescênciaRESUMO
The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.
Assuntos
Azospirillum brasilense/metabolismo , Citometria de Fluxo/métodos , Herbaspirillum/metabolismo , Hidroxibutiratos/metabolismo , Raízes de Plantas/microbiologia , Poliésteres/metabolismo , Microscopia de FluorescênciaRESUMO
Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.