ABSTRACT
Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.
Quatorze linhagens de bactérias fixadoras de nitrogênio foram isoladas de diferentes espécies de plantas, incluindo cassava, milho e cana-de-açúcar, usando condições seletivas desprovidas de nitrogênio. A capacidade de fixar nitrogênio foi verificada por ensaio de redução de acetileno. Todas as linhagens fixadoras de nitrogênio testadas apresentaram hibridização positiva com sonda de gene nifH derivada de Azospirillum brasilense. As linhagens foram caracterizadas por RAPD, ARDRA e sequenciamento do gene 16S rDNA. As análises de RAPD revelaram 8 genótipos, as 6 linhagens restantes foram agrupadas em 3 grupos de RAPD, sugerindo uma origem clonal. ARDRA e seqüências de 16S rDNA foram alocadas em 13 grupos conhecidos de bactérias fixadoras de nitrogênio, incluindo organismos dos gêneros Azospirillum, Herbaspirillum, Pseudomonas e Enterobacteriaceae. Duas linhagens foram classificadas como Stenotrophomonas ssp. Os resultados da identificação molecular baseados em sequencias de 16S rDNA corroboram com dados obtidos em testes morfológicos e bioquímicos.
Subject(s)
Azospirillum brasilense/isolation & purification , Hybridization, Genetic , In Vitro Techniques , Nitrogen Fixation , Plant Structures , Random Amplified Polymorphic DNA Technique , Classification , Genotype , Methods , MethodsABSTRACT
In order to evaluate the response of Beijerinckia derxii ICB-10 to different environmental factors, growth curves and specific nitrogenase activity were studied. Tested conditions were as follows: a) media with different pH values (2.5, 2.8, 4.2 and 5.7); b) medium supplemented with 230 æM aluminium sulphate; c) media with two different potassium phosphate concentrations (50 mM and 100 mM); d) shaken or still cultures; e) medium supplemented with 40 mM sodium thiosulphate. Growth curves and specific nitrogenase activity at pH 4.2 were closely similar to those for cultures at pH 5.7 (standard condition), whereas no growth occurred at pH 2.5. Changes in growth curves and/or specific nitrogenase activity were observed under the following conditions: I) pH 2.8 (decrease in initial CFU number, reduction of maximum specific growth rate, reduced number of generations and stimulation of nitrogenase activity), II) presence of aluminium (early death phase), III) 50 mM PO4(3-) (reduction of maximum specific growth rate), IV) 100 mM PO4(3-) (reduction of both number of generations and maximum specific growth rate as well as early death phase), V) low O2 availability (increasing nitrogenase activity), and VI) presence of thiosulphate (reduction of maximum specific growth rate; early death phase and high stimulation of nitrogenase activity). The data obtained showed the high variability of the cell growth response to environmental factors. Nitrogenase activity was always preserved even when population growth was affected
Subject(s)
Gram-Negative Aerobic Bacteria/growth & development , Gram-Negative Aerobic Bacteria/isolation & purification , Clinical Enzyme Tests , In Vitro Techniques , Nitrogen Fixation , Nitrogenase , Environment , Genetic Variation , MethodsABSTRACT
The interactions between the nitrogen-fixing microorganism "Beijerinckia derxii" with two non-diazotrophic bacteria, either "Escherichia coli" or a faculty sulphur-oxidizing chemolitroph, were studied in mixed cultures. Direct and indirect contact between "B. derxii" and "E. coli" were tested. "B. derxii" increased CFU numbers and/or maintained the viability of the non-diazotrophic bacteria, but neither growth nor nitrogenase activity of the nitrogen-fixing bacterium were affected by either partner