Rhodopseudomonas palustris
cells grown on limiting nitrogen produced four- to eightfold higher
nitrogenase
specific activity relative to cells sparged with N2.
The high activity of N-limited cells was the result of
overproduction
of the nitrogenase proteins. This was shown by four independent
techniques:
(i) titration of the Mo-Fe protein in cell-free extracts with Fe
protein
from Azotobacter vinelandii; (ii) direct detection of the
subunits
of Mo-Fe protein by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis;
(iii) monitoring of the electron paramagnetic resonance spectrum of
Mo-Fe
protein in whole cells; and (iv) immunological assay of the Fe protein
level with an antiserum against the homologous protein of Rhodospirillum
rubrum. The derepressed level of nitrogenase found in N2-grown
cells was not due to an increased turnover of nitrogenase. The apparent
half-lives of nitrogenase in N2-grown and
N-limited
cells were 58 and 98 h, respectively, but were too long to account for
the difference in enzyme level. Half-lives were determined by measuring
nitrogenase after repression of de novo synthesis by ammonia and
subsequent
release of nitrogenase switch-off by methionine sulfoximine.
Observations
were extended to R. rubrum, Rhodopseudomonos capsulata,
and
Rhodomicrobium vannielii and indicated that overproduction of
nitrogenase
under nitrogen limitation is not an exceptional property of
R. palustris,
but rather a general property of phototrophic bacteria.