Figure 1.
General scheme of the construction of cosmid X940.
Pseudomonas protegens Pf-5 (Pf-5), Pseudomonas stutzeri A1501 (A1501), kanamycin resistance gene (kan), ampicillin resistance gene (amp), BamHI (B), SalI (S), XbaI (X), MboI (M), AvaI (A), dephosphorylation (P-) are shown.
Figure 2.
PCR and Southern blot analyses of genomic DNA from the Pf-5 X940 strain carrying the nifH gene and the PST1302–PST1306 region.
PCR marker: 1 Kb Plus DNA Ladder (INVITROGEN). Southern blot marker: Dig marker III (Roche).
Figure 3.
Effect of the transformation of Pseudomonas Pf-5 with cosmid X940.
We analyzed the growth of Pf-5 and Pf-5 X940 in L medium with and without nitrogen under microaerobiotic (Erlenmeyers with lid) and aerobic (Erlenmayers sealed with parafilm) conditions by means of turbidity (a) and the relationship between the CFU (colony-forming units) at the beginning and at the end of the experiment (b). The statistical analysis was carried out with ANOVA followed by Tukey’s contrast test. The letters correspond to the treatments compared. The same letters correspond to non-significant differences. (a) and (b) showed significant differences with p<0.001.
Figure 4.
Adaptation of nitrogenase to new bacteria (a) nitrogenase activity, (b) nif gene expression and (c) ammonium production of recombinant Pseudomonas containing X940 cosmid.
Nitrogenase activity and extracellular ammonium concentration values are the mean±SEM of three independent measurements. RT-PCR and Real-time RT-PCR (nifA, nifH and nifB) studies of nif gene expression in Pf-5 X940 in the presence (+(NH4)2SO4) or absence (−(NH4)2SO4) of nitrogen. Transcript abundance for nifA, nifH and nifB genes was normalized to that for GAP-1 gene. Values represent media+SEM, n = 3 independent experiments.
Figure 5.
Effect of the inoculation with Pf-5 X940 on (a) Arabidopsis growth and (b) ammonium production.
We analyzed the productivity (rosette diameter) and measured the ammonium in solid (NH4+) of Arabidopsis plants inoculated with A1501, Pf-5 and Pf-5 X940 in the presence (+Ca(NO3)2) or absence (−Ca(NO3)2) of nitrogen in the substrate. The statistical analysis was carried out with two-way ANOVA in the case of ammonium measurements or one-way ANOVA in the case of rosette diameter, followed by Tukey’s multiple comparison tests in both cases. Comparisons were made between plants that received the same inoculation treatment in different conditions of nitrogen in the substrate (***p<0.001 **p<0.01 and *p<0.05). N.S: not significant.
Figure 6.
Effect of the inoculation with PF-5 X940 on alfalfa growth.
We analyzed the productivity (fresh weight) of plants inoculated with Pf-5 and Pf-5 X940 in the presence (+Ca(NO3)2) or absence (−Ca(NO3)2) of nitrogen in the substrate. The statistical analysis was carried out with ANOVA followed by Tukey’s contrast test. Comparisons were made between plants that received the same inoculation treatment in different conditions of nitrogen in the substrate (**p<0.01 and ***p<0.001).
Figure 7.
Effect of the inoculation with PF-5 X940 on tall fescue growth.
We analyzed the productivity (first leaf width) of plants inoculated with Pf-5 and Pf-5 X940 in the presence (+Ca(NO3)2) or absence (−Ca(NO3)2) of nitrogen in the substrate. The statistical analysis was carried out with ANOVA followed by Tukey’s contrast test. Comparisons were made between plants that received the same inoculation treatment in different conditions of nitrogen in the substrate (**p<0.01 and *p<0.05).
Figure 8.
Effect of the inoculation with PF-5 X940 on maize growth.
We analyzed the productivity (the area of the third leaf) of plants inoculated with Pf-5 and Pf-5 X940 in the presence (+NH4SO4) or absence (−NH4SO4) of nitrogen in the soil. Arrows indicate the presence of the fourth leaf. The statistical analysis was carried out with ANOVA followed by Tukey’s contrast test (***p<0.001).
Figure 9.
Persistence of bacteria in soil.
Sterile soil was inoculated with Pf-5 or Pf-5 X940. Persistence was expressed as a percentage of the number of colony forming units at time zero, that was taken as 1. The experiment was repeated three independent times. The statistical analysis was carried out with t-test (***p<0.001).
Table 1.
Quantification of ammonium and nitrate concentration in maize cultivation soil treated or not (uninoculated) with nitrogen (ammonium), wild-type strain (Pf-5) and recombinant bacterium (Pf-5 X940).