Control of respiration and growth yield in ammonium-assimilating cultures of Azotobacter vinelandii

1988

Biological dinitrogen fixation is largely restricted to conditions of low aeration and complete anaerobiosis, because the enzyme catalyzing the reduction of atmospheric dinitrogen to ammonia, nitrogenase, is extremely oxygen labile (18,22). Nevertheless, members of the Azotobacteraceae are capable of fixing dinitrogen while growing at high ambient oxygen concentrations. As originally proposed by Postgate (22), protection of nitrogenase against oxygen damage may involve two mechanisms in Azotobacter chroococcum and related species. The hypothesis of respiratory protection postulates that the unusually high activities of cellular oxygen consumption, characteristic of azotobacters, prevent the diffusion of oxygen into the cells and thus to nitrogenase. Whenever this steady state becomes disturbed by an increase in the dissolved oxygen concentration, conformational protection, i.e., switch-off of nitrogenase activity, protects nitrogenase proteins (3,28).Studies of the molecular basis of the switch-off effect indicated that a low-molecular-weight (two atoms of iron and two atoms of sulfur) protein associates with the iron-molybdenum and iron proteins of nitrogenase to form a threecomponent complex (25). Formation of the complex depends on the oxidation of the electron donor to nitrogenase, as well as on the reversible oxidation of nitrogenase (25,27,29).Following a switch-off, nitrogenase activity resumes either when dissolved oxygen decreases to about its original concentration or after adaptation of the cultures to the elevated oxygen concentration. The latter is accompanied by an enhanced formation of constituents of the respiratory chain, increasing the cellular respiratory activity (13). However, detailed investigations with chemostat cultures of Azotobacter vinelandii which were adapted to different oxygen concentrations revealed that, particularly at high ambient oxygen concentrations, the activity of oxygen consumption * Corresponding author. increased with increasing oxygen concentrations, but not linearly (1, 21). Thus, additional or even alternative mechanisms are required to provide maximal protection of nitrogenase against an oxygen-induced switch-off and, moreover, against irreversible damage. Protection against the latter may involve superoxide dismutase, the activity of which increased when the dissolved oxygen concentration was raised (7). The present investigation, focusing on the stability of nitrogenase activity under oxygen stress, demonstrates that the switch-off behavior depends entirely on the rate of supply of the energy and carbon source rather than on the rate of total oxygen consumption by the cells. MATERIALS AND METHODS

Section: Discussionmentioning

confidence: 91%

“…Total nitrogen was determined as described previously (2). Respiration activities were determined in situ on the basis of the gas flow rate through the culture and the difference between oxygen concentrations at the gas inlet and outlet (1).…”

mentioning

confidence: 99%

Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii

Kuhla

1988

“…Bühler et al (1,2) showed that ammonium-assimilating chemostat cultures increased the rate of cellular respiration when growing under steady-state conditions at increasing C/N ratios. The respiratory rate reached constancy when active nitrogenase proteins were expressed.…”

Section: Resultsmentioning

confidence: 99%

“…Respiratory activities of whole cells were measured polarographically (YSI 35; Yellow Springs Instrument Co., Yellow Springs, Ohio) after samples were suspended from chemostat cultures in air-saturated fresh medium. In situ respiratory activities were calculated on the basis of the rate of gas flow through the culture and the difference between oxygen concentrations at the gas inlet and outlet (1).…”

Section: Methodsmentioning

confidence: 99%

Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii

Linkerhägner

1995

When Azotobacter vinelandii, growing diazotrophically in chemostat culture, was subjected to sudden increases in the ambient oxygen concentration (oxygen stress), nitrogenase activity was switched off and cellular ATP pools decreased at rates depending on the stress level. Following a fast decrease, the ATP pool approached a lower level. When the stress was released, these effects were reversed. The reversible decrease of the ATP pool upon oxygen stress could also be observed with cultures assimilating ammonium and, at the same time, fixing dinitrogen because of growth at a high C/N ratio but not with cultures growing only at the expense of ammonium. When strains OP and UW136 of A. vinelandii were subjected to long-term increases in ambient oxygen, the sizes of cellular ATP pools eventually started to increase to the level before stress and diazotrophic growth resumed. The cytochrome d-deficient mutant MK5 of A. vinelandii, however, impaired in aerotolerant diazotrophic growth, was unable to recover from stress on the basis of its ATP pool. The results suggest that adaptation to higher ambient oxygen depends on increased ATP synthesis requiring increased electron flow through the entire respiratory chain, which is possible only in combination with the more active, yet possibly uncoupled, branch terminated by cytochrome d. It is proposed that the decrease of the cellular ATP level under oxygen stress resulted from the increased energy and electron donor requirement of nitrogenase in reacting with oxygen.

“…Respiratory activities were estimated on the basis of the gas flow rate through the culture and the difference between oxygen concentrations at the gas inlet and outlet (3). Essentially the same method was used to determine specific rates of hydrogen evolution.…”

mentioning

confidence: 99%

Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation

Linkerhägner

1995

The presumed beneficial effect of hydrogenase on growth of diazotrophic bacteria was reinvestigated with carbon-limited chemostat cultures of the hydrogenase-deficient mutant hoxKG of Azotobacter vinelandii and its parent. The results revealed that hydrogen recycling was too low to benefit the cellular energy metabolism or activities of nitrogenase and respiration.Reduction of dinitrogen to ammonia by nitrogenase is accompanied by the production of hydrogen (17). Since organisms which express an uptake hydrogenase are able to recycle hydrogen, it has been proposed that recycling of hydrogen benefits diazotrophic growth (5, 19). Yates and colleagues showed that hydrogenase-negative mutants of Azotobacter chroococcum were outcompeted by the wild type, when growing diazotrophically in sucrose-limited mixed populations (1,20). These results have become widely accepted as evidence for the beneficial effect of hydrogen recycling on carbon-limited growth of diazotrophs (2, 19). However, results obtained with mixed populations may be questioned because it is very likely that the wild type utilized hydrogen produced not only by itself but also by the mutant. In fact, the present communication shows that the amount of hydrogen produced by glucose-limited hydrogenase-positive Azotobacter vinelandii is too low to benefit diazotrophic growth.Glucose-limited cultures of mutant hoxKG of A. vinelandii and its parent strain (15) were grown with 15 mM glucose in a pH-controlled chemostat (6, 13). Dissolved oxygen concentrations (DOC) of 135 and 11.3 M were measured and kept constant as described previously (13). In order to prevent washing out of the culture, the dilution rate (D) was not increased above 0.25 h Ϫ1 (6, 13). Since in carbon-limited chemostat cultures the ratio of protein to biomass is constant (6), steady-state biomass levels of the cultures were estimated on the basis of protein (10). At both DOC, the mutant and its parent exhibited the same dependence of protein formation on D (Fig. 1). Both strains consumed all of the carbon and energy source, glucose. Consequently, yield coefficients (Y, grams of biomass formed per mole of substrate consumed) were identical as well. The maintenance coefficients and the theoretical maximum Y were extrapolated from plots of 1/Y versus 1/D (12) (Fig. 2). The theoretical maximum Y of both strains were 20 g/mol, and maintenance processes required 1.0 and 16 mmol of glucose per g of protein and h at 11.3 and 135 M DOC, respectively. The identical maintenance coefficient and Y values may be considered sufficient to infer lack of any significant differences in the efficiency of the energy metabolism of both strains. Nevertheless, in the following, we examined the suggestion that hydrogenase benefits diazotrophs by giving rise to extra ATP formation or to protection of nitrogenase against oxygen (1,20).Adenine nucleotide levels were determined according to published methods (9,11,18). The different parameters of the energy status of both strains as compiled in Table 1 do not reveal any ...