Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations

Abstract

Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 FM) in sucrose-linited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Avl) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting). When the cultures were grown at a constant dilution rate (D, representing the growth rate, ,u) of 0.15. h1, the cellular levels of both proteins were constant regardless of different dissolved oxygen concentrations. The same was true when the organisms were grown at D values above 0.15 h-1. At a lower growth rate (D = 0.09 h-1), however, and at lower oxygen concentrations cellular levels of both nitrogenase proteins were decreased. This means that catalytic activities of nitrogenase proteins were highest at low oxygen concentrations, but at higher oxygen concentrations they increased with growth rate. Under all conditions tested, however, the Av1:Av2 molar ratio was 1:(1.45 ± 0.12). Cellular levels of flavodoxin and FeS protein II were largely copstant as well. In order to estimate turnover of nitrogenase proteins in the absence of protein synthesis, chloramphenicol was added to cultures adapted to 3 and 216 ,uM oxygen, respectively.After 2 h of incubation, no significant decrease in the cellular levels of Avl and Av2 could be observed. Thbis suggests that oxygen has no significant effect on the breakdown of nitrogenase proteins.

Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

“…2A). This observation is consistent with previous analyses and supports the observation that nitrogenase activity is optimal under conditions of high Fe protein to MoFe protein ratios (13,14). Interestingly, the abundances of transcripts of nonessential nif genes (28) downstream of nifK increased by only 2-to 14-fold ( Fig.…”

“…Under N-fixing conditions, nitrogenase components make up nearly 10% of the total cellular proteins, the majority of which are structural proteins (14). The catalytic components necessary for FeMo-co biosynthesis are presumably necessary in much smaller amounts.…”

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations

“…This possibility can be excluded by previous findings showing that neither the cellular level nor the turnover of the nitrogenase polypeptides of A. vinelandii is affected by oxygen (7). Moreover, since it was shown that the cellular level of both nitrogenase polypeptides is largely independent of D, it was concluded that the D-dependent increase of the cellular nitrogenase activity is regulated by the supply of reducing equivalents and energy (7). The present investigation confirms the latter mechanism.…”

“…For example, it was shown with A. vinelandii growing at high dissolved oxygen concentrations that the respiratory activity does not significantly increase with an increase in the dissolved oxygen concentration (25). Moreover, cultures of A. vinelandii exhibited comparable cellular contents and nitrogenase activities in spite of considerable differences in cellular oxygen consumption (7,13). Finally, it has been demonstrated that oxygen enters the cells of A. vinelandii without impairing the function of nitrogenase (21).…”

“…In spite of this sensitivity, members of the diazotrophic azotobacters are able to grow under fully aerobic conditions. It was shown before that cells of Azotobacter vinelandii grown at different dissolved oxygen concentrations exhibited identical nitrogenase polypeptide contents and that ambient oxygen had no significant effect with respect to the turnover of these polypeptides (7). Protection of nitrogenase against oxygen damage has been proposed to occur in azotobacters on the basis of the following two mechanisms: (i) high respiratory activities that remove oxygen already at the surface of the cell and (ii) reversible conversion of the enzyme into a protected, yet-inactivated state (3,4,8,33).…”