Figure 1.
R. etli nodB and nifA mutation constructs.
(A). The deletion constructs of nodB and nifA (left panel) were made by cloning flanking regions of nodB and nifA into a suicide vector harboring the sacB counter-selective marker. The resulting plasmids were then introduced into R. etli and double cross-over events were selected. The mutations generated by this method are permanent. The insertional mutants (right panel) were constructed by cloning internal fragments of nodB and nifA into a suicide vector. The resulting plasmids were then introduced into R. etli and single cross-over events were selected. The reversion to wild type may occur if the strains undergo a second cross-over event. CmR: chloramphenicol-resistant gene; TcR: tetracycline-resistant gene; KmR: kanamycin-resistant gene. (B). Growth of R. etli and its derivative mutants. The cultures were grown in PY medium at 28°C. At the time points indicated, OD600 was measured. Data are mean and s.d. of three independent experiments.
Figure 2.
Nodule formation by wild type R. etli and its derivative nodB mutants.
(A). The number of nodules on Phaseolus vulgaris formed 30 days post-inoculation by wild type (empty circles), ΔnodB (empty squares), ::nodB (empty triangles), 1∶1 mixed inoculation of wild type and ΔnodB (filled squares), and mixed inoculation of wild type and ::nodB (filled triangles). (B). Average biomass of the nodules. Data are the combination of six individual experiments. Statistical analysis was performed using the Student’s t-test comparing nodule formation to that of wild type. NS: no significance; ***: p<0.0001; NA: not applicable.
Figure 3.
The nodB mutant competitiveness in nodulation.
(A). Nodule occupancy. Nodules formed by 1∶1 mixed inoculation of wild type and ΔnodB (left panel) or ::nodB (right panel) were harvested 30-day post-inoculation, surface-sterilized, crushed, and sequentially stabbed on PY agar containing Sm+Rif, Sm+Cm, Sm+Km. After incubation at 28°C for 72 hrs, plates were inspected for the identity of strains. Nodule occupancy is based on 90 nodules isolated from 3 plants. Data are mean and s.d. of four independent experiments. Statistical analysis was performed using the Student’s t-test comparing to that of wild type. ***: p<0.0001. (B). Competitive index. For nodules containing mixed strains, CFU of wild type (SmR, RifR), ΔnodB (SmR, CmR), ::nodB (SmR, KmR), nodB revertants (SmR, KmS) was determined by plating onto PY medium plates containing Sm only, Sm+Rif, Sm+Cm, or Sm+Km. Competitive index was calculated as the output ratio of mutant to wild type. The data are combinations of four independent experiments. Statistical analysis was performed using the Student’s t-test comparing competitive index to that of ::nodB revertant. ***: p<0.0001.
Figure 4.
The ::nodB reversion rate in vitro and in planta.
(A). The reversion in vitro. ::nodB mutants were grown in PY medium in the absence of kanamycin (squares) at 28°C and were subcultured to fresh medium every other day or grown in rhizosphere of Phaseolus vulgaris planted in autoclaved Vermiculite (triangles). Samples were withdrawn at the time points indicated and colony formation units (CFU) of ::nodB (SmR, KmR) and nodB revertants (SmR, KmS) were determined by plating onto PY medium plates containing streptomycin (Sm) only and streptomycin plus kanamycin (Km). The reversion rate was calculated by the percentage of nodB revertants relative to total SmR cell number. Data are mean and s.d. of three independent experiments. (B). The reversion in nodules. Nodules formed by ::nodB single inoculation (squares) or by::nodB-wild type mixed inoculation (triangles). Nodules formed by mixed inoculations contained either only nodB (empty triangles) or a mixture of nodB and wild type strains (filled triangles). Nodules were harvested 30-day post-inoculation, surface-sterilized, crushed, and resuspended in PY medium. Colony forming units (CFU) of ::nodB (SmR, KmR), nodB revertants (SmR, KmS), and wild type [SmR, Rifampin (RifR)] were determined by plating onto PY medium plates containing Sm only, Sm+Rif, or Sm+Km. The reversion rate was calculated by the percentage of nodB revertants (KmS) to total number (SmR, RifS) of cells. Statistical analyses were performed using the Student’s t-test comparing to reversion rate of ::nodB mutants ex planta. NS: no significance; **: p<0.005.
Figure 5.
Nodule formation by wild type R. etli and its derivative nifA mutants.
(A). The number of nodules on Phaseolus vulgaris formed 30 days post-inoculation by wild type (empty circles), ΔnifA (empty squares), ::nifA (empty triangles), 1∶1 mixed inoculation of wild type and ΔnifA (filled squares), and mixed inoculation of wild type and ::nifA (filled triangles). (B). Average biomass of the nodules. The data are the combination of 6 individual experiments. Statistical analyses were performed using the Student’s t-test acomparing to nodule formation in the wild type strain. NS: no significance; ***: p<0.0001.
Figure 6.
Competitiveness of nifA mutants in nodulation.
(A). Nodule occupancy. Nodules formed by 1∶1 mixed inoculation of wild type and ΔnifA (left panel) or ::nifA (right panel) were harvested 30-day post-inoculation, surface-sterilized, crushed, and sequentially stabbed on PY agar containing Sm+Rif, Sm+Tc, Sm+Km. After incubation at 28°C for 72 hrs, plates were inspected for the identity of strains. Nodule occupancy is based on 90 nodules isolated from 3 plants. Data are mean and s.d. of four independent experiments. Statistical analyses were performed using the Student’s t-test comparing colonization to that of wild type. ***: p<0.0001. (B). Competitive index. For nodules containing mixed strains, CFU of wild type (SmR, RifR), ΔnifA (SmR, TcR), ::nifA (SmR, KmR), nifArevertants (SmR, KmS) was determined by plating on PY medium plates containing Sm only, Sm+Rif, Sm+Tc, or Sm+Km. Competitive index was calculated as the output ratio of mutant to wild type. The data are a combination of four independent experiments. Statistical analyses was performed using the Student’s t-test comparing competitive index to that of ::nifA revertant. ***: p<0.0001.
Figure 7.
The ::nifA reversion rate in vitro and in planta.
(A). The reversion in vitro. ::nifA strains were grown in PY medium in the absence of kanamycin (squares) at 28°C and were subcultured to fresh medium every other day or grown in rhizosphere of Phaseolus vulgaris planted in autoclaved Vermiculite (triangles). Samples were withdrawn at the time points indicated and colony forming units (CFU) of ::nifA (SmR, KmR) and nifA revertants (SmR, KmS) were determined by plating on PY medium plates containing streptomycin (Sm) only and streptomycin plus kanamycin (Km). The reversion rate was calculated as percentage of nifA revertants (KmS) to total number of cells (SmR). Data are the mean and s.d. of three independent experiments. (B). The reversion in nodules. Nodules formed by ::nifA single inoculation (squares), or ::nifA-wild type mixed inoculations (triangles). Of the mixed inoculations, nodules either contained only nifA (empty triangles) or a mixture of mutants with wild type strains (filled triangles). Nodules were harvested 30-day post-inoculation, surface-sterilized, crushed, and resuspended in PY medium. CFU of ::nifA (SmR, KmR), nifA revertants (SmR, KmS), and wild type (SmR, RifR) were determined by plating on PY medium plates containing Sm only, Sm+Rif, or Sm+Km. The reversion rate was calculated as percentage of nifA revertants (KmS) in total number of cells (SmR, RifS) cells. Statistical analyses were performed using the Student’s t-test comparing to reversion rate of ::nifA mutants ex planta. NS: no significance.
Figure 8.
Nitrogenase activity in nodules.
Nodules were formed by inoculation of a 1∶1 ratio of wild type to either ΔnifA or ::nifA mutants. Nodules were harvested 30-days post-inoculation and acetylene reduction activity was determined using a HP 6890 Series gas chromatograph system (see Methods). Nitrogenase activity is expressed as % of acetylene production per gram of nodule dry weight. Data shows mean and s.d. of four independent experiments. Statistical analysis was performed using the Student’s t-test comparing nitrogenase activity to levels seen in nodules formed by a single inoculation of wild type. NS: no significance; ***: p<0.0001.