RESUMEN
The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium. Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere. Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions. The DF57-P2 reporter strain responded to phosphate limitation, and the luxAB gene fusion was controlled by a promoter containing regulatory sequences characteristic of members of the phosphate (Pho) regulon. DF57 generally had higher metabolic activity in a gnotobiotic rhizosphere than in the corresponding bulk soil. Within the rhizosphere the distribution of metabolic activity along the root differed between the rhizosphere soil and the rhizoplane, suggesting that growth conditions may differ between these two habitats. The DF57-P2 reporter strain encountered phosphate limitation in a gnotobiotic rhizosphere but not in a natural rhizosphere. This difference in phosphate availability seemed to be due to the indigenous microbial population, as DF57-P2 did not report phosphate limitation when established in the rhizosphere of plants in sterilized soil amended with indigenous microorganisms.
Asunto(s)
Genes Bacterianos , Hordeum/microbiología , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Genes Reporteros , Luciferasas/genética , Luminiscencia , Datos de Secuencia Molecular , Fosfatos/metabolismo , SimbiosisRESUMEN
The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.
RESUMEN
Changes in culturability and outer membrane protein profiles were investigated in Pseudomonas fluorescens DF57 and Pseudomonas putida DF14 during starvation for carbon, nitrogen, and phosphorus. P. fluorescens DF57 remained fully culturable for 4 days in all starvation regimes. The cell mass increased during starvation for nitrogen and phosphorus, indicating the accumulation of storage compounds, whereas it decreased slightly in carbon-starved cells. P. putida DF14 lost culturability during phosphorus starvation, and the mass of phosphate-starved cells did not increase. Analysis of additional P. fluorescens and P. putida strains, however, showed that the ability to preserve culturability during phosphorus starvation was not species but strain dependent. In DF57, an outer membrane protein of 55 kDa appeared during starvation for phosphorus, while another protein of 63 kDa was seen during all starvation conditions. DF14 induced two outer membrane proteins of 28 and 29 kDa during starvation for carbon and nitrogen, but no phosphorus-specific starvation protein could be detected. Therefore, starvation-induced outer membrane proteins do not seem to be conserved among the fluorescent pseudomonads and a unique starvation response might be found in individual strains.