Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72

We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp. BH72, which was isolated as an endophyte from Kallar grass. Nitrogenase is derepressed under microaerobic conditions at O₂ concentrations in the micromolar range. With increasing O₂ deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O₂. Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen-fixing conditions. The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies. Membrane deficiency in NifH/K⁻ mutants, a deletion mutant in the nifK gene and the character of NH₄⁺-grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen fixation. We propose the term 'diazosomes' for them. Development of intracytoplasmic membranes coincides with the appearance of a high-molecular-mass form of the iron protein of nitrogenase, which was detectable in membrane fractions. Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation. Development of diazosomes in nitrogen-fixing cells can be induced in pure cultures and in co-culture with a fungus isolated from the rhizosphere of Kallar grass.     

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.     

Nitrogenase activity in Alnus incana root nodules. Responses to O(2) and short-term N(2) deprivation

O(2) and host-microsymbiont interactions are key factors affecting the physiology of N(2)-fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O(2) concentration, and short-term N(2) deprivation, intact nodulated roots were exposed to various O(2) pressures (pO(2)) and Ar:O(2) in a continuous flow-through system. Nitrogenase activity (H(2) production) occurred at a maximal rate at 20% O(2). Exposure to short-term N(2) deprivation in Ar:O(2) carried out at either 17%, 21%, or 25% O(2) caused a decline in the nitrogenase activity at 21% and 25% O(2) by 12% and 25%, respectively. At 21% O(2), nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N(2) fixation. Respiration (net CO(2) evolution) decreased in response to the N(2) deprivation at all pO(2) values and did not recover during the time in Ar:O(2). Increasing the pO(2) from 21% to 25% and decreasing the pO(2) from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O(2) limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O(2) inhibition of nitrogenase at 25% O(2). These results are consistent with a fixed O(2) diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O(2) and N(2) deprivation.     

Global changes in protein composition of N2-fixing-Azoarcus sp. strain BH72 upon diazosome formation.

The strictly respiratory, diazotrophic bacterium Azoarcus sp. strain BH72 fixes nitrogen under microaerobic conditions. In empirically optimized batch cultures at nanomolar O2 concentrations in the presence of proline, cells can shift into a state of higher activity and respiratory efficiency of N2 fixation in which intracytoplasmic membrane stacks (diazosomes) related to N2 fixation are formed. Induction of intracytoplasmic membranes is most pronounced in coculture of Azoarcus sp. strain BH72 with an ascomycete originating from the same host plant, Kallar grass. To initiate studies on function of diazosomes and regulation of their formation, diazosome-containing bacteria were compared with respect to composition or total cellular and membrane proteins with diazosome-free cells fixing nitrogen under standard conditions. In two-dimensional protein gels, we detected striking differences in protein patterns upon diazosome formation: (i) 7.3% of major proteins disappeared, and only 73% of the total proteins of control cells were detectable, indicating that diazosome-containing cells have a more specialized metabolism; (ii) nine new proteins appeared and five proteins increased in concentration, designated DP1 to DP 15; and (iii) five new major membrane proteins (MP1 to MP6) were detected, indicating that membranes might have specialized functions. N-terminal amino acid sequence analysis of DP1 to DP4 allowed us to preliminarily identify DP4 as the glnB gene product P(II), an intracellular signal transmitter known to be involved in the regulation of nitrogen metabolism. According to its electrophoretic mobility, it might be uridylylated in diazosome-free cells but not in diazosome-containing cells, or it may represent a second, not identical P(II) protein. Oligonucleotides deduced from N-terminal sequences of DP1 and DP4 specifically hybridized to chromosomal DNA of Azoarcus sp. strain BH72 in Southern hybridizations.     

Effect of oxygen on batch and continuous cultures of a nitrogen-fixing Arthrobacter sp.

Growth, acetylene reduction, and respiration rate were studied in batch and continuous cultures of Arthrobacter fluorescents at different oxygen partial pressures. The optimum pO2 values for growth and acetylene reduction were 0.05 and 0.025 atm, respectively, but microorganisms can tolerate higher pO2 values. The growth of cultures provided with combined nitrogen was dependent on oxygen availability, and strict anaerobic conditions did not support growth. Acetylene reduction of a population grown in continuous culture and adapted to low pO2 (0.02 atm) was much more sensitive to oxygenation than that of a population adapted to high pO2 (0.4 atm). Their maximum nitrogenase activity, at their optimal pO2 values, were quite different. The respiratory activity of nitrogen-fixing cultures increased with increasing oxygen tensions until a pO2 of 0.2 atm. At higher pO2 values, the respiration rate began to decrease.     

Response of the endophytic diazotroph Gluconacetobacter diazotrophicus on solid media to changes in atmospheric partial O(2) pressure

Gluconacetobacter diazotrophicus is an N(2)-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O(2) pressures (pO(2)) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO(2) (5 to 60 kPa). Nitrogenase activity was measured by H(2) evolution in N(2)-O(2) and in Ar-O(2), and respiration rate was measured by CO(2) evolution in N(2)-O(2). To validate the use of H(2) production as an assay for nitrogenase activity, a non-N(2)-fixing (Nif(-)) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup(+)) activity (0.016 +/- 0.009 micromol of H(2) 10(10) cells(-1) h(-1)) when incubated in an atmosphere enriched in H(2). However, Hup(+) activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO(2) tested. However, when the assay atmospheric pO(2) was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO(2) for nitrogenase activity was 0 to 20 kPa above the pO(2) at which the bacteria had been grown. As atmospheric pO(2) was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO(2) was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO(2) from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O(2), 80% of nitrogenase activity was recovered within 10 min, indicating a "switch-off/switch-on" O(2) protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N(2) at a wide range of atmospheric pO(2) and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO(2).     

Hydrogen metabolism of Azospirillum brasilense in nitrogen-free medium

Production of H2 by Azospirillum brasilense under N2-fixing conditions was studied in continuous and batch cultures. Net H2 production was consistently observed only when the gas phase contained CO. Nitrogenase activity (C2H2 reduction) and H2 evolution (in the presence of 5% CO) showed a similar response to O2 and were highest at 0.75% dissolved O2. Uptake hydrogenase activity, ranging from 0.3 to 2.5 mumol H2/mg protein per hour was observed in batch cultures under N2. Such rates were more than sufficient to recycle nitrogenase-produced H2. Tritium-exchange assay showed that H2 uptake was higher under Ar than under N2. Uptake hydrogenase was strongly inhibited by CO and C2H2. Cyclic GMP inhibited both nitrogenase and uptake hydrogenase activities.     

Characterization of a nitrogen-fixing bacterial strain from the roots of Digitaria sanguinalis.

Rates of nitrogen fixation of 3 to 10 g of N2 fixed per hectare per day were associated with root systems of Digitaria sanguinalis. A Gram-negative motile aerobic bacterial strain that was capable of N2 fixation was isolated from a washed root sample of one of these plants. Optimal growth and N2 fixation occurred at a pH of about 6.5, a temperature of 30-37 degrees C, and at a pO2 of about 0.01 atm. Increased rates of N2 fixation resulted when this strain was grown in mixed cultures with aerobic or facultative bacteria. Observations of cellular and cultural morphology and results of biochemical and physiological studies indicate that the isolate may be related to the Azotobacteraceae but that it is not identical with any of the members of this family. The importance of N2 fixation by this isolate in nature is unknown.     

Characteristics of N2 fixation in Mo-limited batch and continuous cultures of Azotobacter vinelandii.

Steady-state chemostat cultures of Azotobacter vinelandii were established in a simple defined medium that had been chemically purified to minimize Mo and that contained no utilizable combined N source. Growth was dependent on N2 fixation, the limiting nutrient being the Mo contaminating the system. The Mo content of the organisms was at least 100-fold lower than that of Mo-sufficient cultures, and they lacked the characteristic g = 3.7 e.p.r. feature of the MoFe-protein of nitrogenase. A characteristic of nitrogenase activity in vivo in Mo-limited populations was a disproportionately low activity for acetylene reduction, which was 0.3 to 0.1 of that expected from the rate of N2 reduction. Acetylene was also a poor substrate in comparison with protons as a substrate for nitrogenase, and did not markedly inhibit H2 evolution, in contrast with Mo-sufficient populations. In batch cultures in similar medium or 'spent' chemostat medium inoculated with Mo-limited organisms, the addition of Mo elicited a biphasic increased growth response at concentrations as low as 2.5 nM, provided that sufficient Fe was supplied. In this system V did not substitute for Mo, and Mo-deficient cultures ceased growth at a 25-fold lower population density compared with cultures supplemented with Mo. Nitrogenase component proteins could not be unequivocally detected by visual inspection of fractionated crude extracts of Mo-limited organisms. 35SO42-pulse-labelling studies also showed that the rate of synthesis of the MoFe-protein component of nitrogenase was too low to be quantified. However, the Fe-protein of nitrogenase was apparently synthesized at high rates. The discussion includes an evaluation of the possibility that A. vinelandii possesses an Mo-independent N2-fixation system.     

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.     

Azorhizobium caulinodans respires with at least four terminal oxidases.

In culture, Azorhizobium caulinodans used at least four terminal oxidases, cytochrome aa3 (cytaa3), cytd, cyto, and a second a-type cytochrome, which together mediated general, respiratory electron (e-) transport to O2. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome genes were cloned, and three cytaa3 mutants, a cytd mutant, and a cytaa3, cytd double mutant were constructed by reverse genetics. These cytochrome oxidase mutants were tested for growth, oxidase activities, and N2 fixation properties both in culture and in symbiosis with the host plant Sesbania rostrata. The cytaa3 mutants grew normally, fixed N2 normally, and remained fully able to oxidize general respiratory e- donors (NADH, succinate) which utilize a cytc-dependent oxidase. By difference spectroscopy, a second, a-type cytochrome was detected in the cytaa3 mutants. This alternative a-type cytochrome (Amax = 610 nm) was also present in the wild type but was masked by bona fide cytaa3 (Amax = 605 nm). In late exponential-phase cultures, the cytaa3 mutants induced a new, membrane-bound, CO-binding cytc550, which also might serve as a cytc oxidase (a fifth terminal oxidase). The cloned Azorhizobium cytaa3 genes were strongly expressed during exponential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 40% lower N2 fixation rates in culture and in planta, but aerobic growth rates were wild type. The cytaa3, cytd double mutant showed 70% lower N2 fixation rates in planta. Pleiotropic cytc mutants were isolated by screening for strains unable to use N,N,N',N'-tetramethyl-p-phenylenediamine as a respiratory e- donor. These mutants synthesized no detectable cytc, excreted coproporphyrin, grew normally in aerobic minimal medium, grew poorly in rich medium, and fixed N2 poorly both in culture and in planta. Therefore, while aerobic growth was sustained by quinol oxidases alone, N2 fixation required cytc oxidase activities. Assuming that the terminal oxidases function as do their homologs in other bacteria, Azorhizobium respiration simultaneously employs both quinol and cytc oxidases. Because Azorhizobium terminal oxidase mutants were able to reformulate their terminal oxidase mix and grow more or less normally in aerobic culture, these terminal oxidases are somewhat degenerate. Its extensive terminal oxidase repertoire might allow Azorhizobium spp. to flourish in wide-ranging O2 environments.     

Role of the Azotobacter vinelandii nitrogenase-protective shethna protein in preventing oxygen-mediated cell death

Azotobacter vinelandii strains lacking the nitrogenase-protective Shethna protein lost viability upon carbon-substrate deprivation in the presence of oxygen. This viability loss was dependent upon the N(2)-fixing status of cultures (N(2)-fixing cells lost viability, while non-N(2)-fixing cells did not) and on the ambient O(2) level. Supra-atmosheric O(2) tensions (40% partial pressure) decreased the viable cell number of the mutant further, and the mutant had a slightly higher spontaneous mutation frequency than the wild type in the high-O(2) conditions. Iron starvation conditions, which resulted in fourfold-reduced superoxide dismutase levels, were also highly detrimental to the viability of the protective protein mutants, but these conditions did not affect the viability of the wild-type strain. Nitrogenase or other powerful reductants associated with N(2) fixation may be sources of damaging partially reduced oxygen species, and the production of such species are perhaps minimized by the Shethna protein.     

Role of Oxygen in the Limitation and Inhibition of Nitrogenase Activity and Respiration Rate in Individual Soybean Nodules

Although infected cell O2 concentration (Oi) is known to limit respiration and nitrogenase activity in legume nodules, techniques have not been available to measure both processes simultaneously in an individual legume nodule. Consequently, details of the relationship between nitrogenase activity and Oi are not fully appreciated. For the present study, a probe was designed that allowed open circuit measurements of H2 evolution (nitrogenase activity) and CO2 evolution (respiration rate) in a single attached soybean nodule while simultaneously monitoring fractional oxygenation of leghemoglobin (and thereby Oi) with a nodule oximeter. Compared to measurements of whole nodulated roots, use of the probe led to inhibition of nitrogenase activity in the single nodules. During oximetry measurements, total nitrogenase activity (TNA; peak H2 evolution in Ar/O2) in the single nodules was 16% of that in whole nodulated roots and 48% of nodulated root activity when Oi was not being measured simultaneously. This inhibition did not affect the nodules' ability to regulate Oi, because exposure to Ar/O2 (80:20, v/v) caused nitrogenase activity and respiration rate to decline, and this decline was linearly correlated with a concurrent decrease in Oi. When the nodules were subsequently exposed to a linear increase in external pO2 from 20 to 100% O2 at 2.7% O2/min, fractional leghemoglobin oxygenation first increased gradually and then more rapidly, reaching saturation at a pO2 between 76 and 100% O2. Plots of nitrogenase activity and respiration rate against Oi showed that rates increased with Oi up to a value of 57 nM, with half-maximal rates being attained at Oi values between 10 and 14 nM O2. The maximum nitrogenase activity achieved during the increase in pO2 (potential nitrogenase activity) was 30 to 57% of that measured in intact nodulated roots, showing that O2 limitation of nitrogenase activity could account for a significant proportion of the inhibition of TNA associated with the use of the probe. However, some factor(s) in addition to O2 must have limited the activity of single nodules at both subsaturating and saturating Oi. At Oi values greater than about 57 nM, nitrogenase activity and nodule respiration were inhibited, but, because this inhibition has been shown previously to be readily reversible when the Oi was lowered, it was not attributed to direct O2 inactivation of the nitrogenase protein. These results indicate that maximum nitrogenase activity in legume nodules is supported by a narrow range of Oi values. Possible biochemical mechanisms are discussed for both O2 limitation of nitrogenase activity at low Oi and inhibition of nitrogenase activity at high Oi.     

Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation.

Twenty Azorhizobium caulinodans vector insertion (Vi) mutants unable to catabolize nicotinate (Nic- phenotype) were identified and directly cloned as pVi plasmids. These pVi plasmids were used as DNA hybridization probes to isolate homologous wild-type sequences. From subsequent physical mapping experiments, the nic::Vi mutants defined four distinct loci. Two, possibly three, of these loci are physically linked. A. caulinodans nic loci II and III encode the structural genes for nicotinate catabolism; nic loci I and IV encode nicotinate-driven respiratory chain components. Recombinant lambda bacteriophages corresponding to three of these loci were subcloned in pRK293; resulting plasmids were used for complementation tests with resolved nic::IS50 derivatives of the nic::Vi mutants. When wild-type A. caulinodans was cultured in defined liquid medium under 3% O2, nicotinate catabolism stimulated N2 fixation 10-fold. In these exponentially growing cultures, the entire (300 microM) nicotinate supplement was exhausted within 10 h. While nic::Vi mutants retained the ability to fix some N2, they did so at rates only 10% of that of the wild type: nitrogenase activity by nic::Vi mutants was not stimulated by 300 microM added nicotinate. Higher-level (5 mM) nicotinate supplementation inhibited N2 fixation. Because 5 mM nicotinate repressed nitrogenase induction in all nic::Vi mutants as well, this repression was independent of nicotinate catabolism. During catabolism, nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase which drives a respiratory chain to O2. In A. caulinodans wild-type cultures, added 300 microM 6-OH-nicotinate stimulated N2 fixation twofold better than did added 300 microM nicotinate. Likewise, nic::Vi mutant 61302, defective in nicotinate hydroxylase, fixed N2 at wild-type levels when supplemented with 300 microM 6-OH-nicotinate. Therefore, nicotinate catabolism stimulates N2 fixation not by nicotinate hydroxylase-driven respiration but rather by some subsequent aspect(s) of nicotinate catabolism.     

Root-Zone-Specific Oxygen Tolerance of Azospirillum spp. and Diazotrophic Rods Closely Associated with Kallar Grass

The effect of oxygen on N₂-dependent growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) was studied. To enable precise comparison, bacteria were grown in dissolved-oxygen-controlled batch and continuous cultures. Steady states were obtained from about 1 to 30 μM O₂, some of them being carbon limited. All strains needed a minimum amount of oxygen for N₂-dependent growth. Nitrogen contents between 10 and 13% of cell dry weight were observed. The response of steady-state cultures to increasing O₂ concentrations suggested that carbon limitation shifted to internal nitrogen limitation when N₂ fixation became so low that the bacteria could no longer meet their requirements for fixed nitrogen. For Azospirillum lipoferum Rp5, increase of the dilution rate resulted in decreased N₂ fixation in steady-state cultures with internal nitrogen limitation. Oxygen tolerance was found to be strain specific in A. lipoferum with strain Sp59b as a reference organism. Oxygen tolerance of strains from Kallar grass was found to be root zone specific. A. halopraeferens Au 4 and A. lipoferum Rp5, predominating on the rhizoplane of Kallar grass, and strains H6a2 and BH72, predominating in the endorhizosphere, differed in their oxygen tolerance profiles. Strains H6a2 and BH72 still grew and fixed nitrogen in steady-state cultures at O₂ concentrations exceeding those which absolutely inhibited nitrogen fixation of both Azospirillum strains. It is proposed that root-zone-specific oxygen tolerance reflects an adaptation of the isolates to the microenvironments provided by the host plant.     

N2 Fixation and the Respiratory Costs of Nodules, Nitrogenase Activity, and Nodule Growth and Maintenance in Fiskeby Soyabean

The respiratory effluxes of nodules and of roots of FiskebyV soyabean (Glycine max (L.) Merr.), grown in a controlled environment,were measured at intervals in air and 3% O₂ from shortly afterthe onset of N₂ fixation until plant senescence. The respiratoryburdens linked with nitrogenase plus ammonia metabolism, andnodule growth and maintenance, were calculated from gas exchangedata and related to the concurrent rates of N₂ fixation. The specific respiration rates of nodules increased to a maximumof 21 mg CO₂ g^–1 h^–1 at the time pods began development:the equivalent maximum for roots was c. 4.5 mg CO₂ g^–1h^–1. Maximum nodule and root respiration rates per plantwere attained about 25 d later at the time N₂ fixation peakedat 15 mg N d^–1 plant^–1. The relationship between nodule respiration and N₂ fixationindicated an average respiratory cost of 13.2 mg CO₂ mg^–1N until the last few days of plant development Separation ofnodule respiration into the two components: nitrogenase (+ NH₃metabolism) respiration and nodule growth and maintenance respiration,indicated that the latter efflux accounted for c. 20% of nodulerespiration while N₂ fixation was increasing and new noduletissue was being formed. When nodule growth ceased and N₂ fixationdeclined, this component of respiration also declined. The respiratorycost of nitrogenase activity plus the associated metabolismof NH₃ varied between 11 mg CO₂ mg^–1 N during vegetativeand early reproductive growth, to 12.5 mg CO₂ mg^–1 N duringthe later stages of pod development. Key words: N2 fixation, Respiration, Nodules, Nitrogenase     

Dependence of oxygen-tolerant nitrogenase activity on divalent cations in Azotobacter vinelandii.

Nitrogenase activity of washed Azotobacter vinelandii cells was enhanced by the addition of Ca2+ and Mg2+, and the enhancement increased with the O2 concentration. In assays provided with a level of O2 that was initially supraoptimal and inhibitory to nitrogenase activity, the addition of Ca2+ or Mg2+ affected both the maximum respiration rate (Vmax) of the cells and the apparent affinity [KS(O2)] of cell respiration for O2. Changes in these parameters correlated with changes in nitrogenase activity. Aeration-dependent increases in Vmax and KS(O2) were inhibited by rifampin and chloramphenicol and were also observed in ammonium-grown cultures.     

Factors affecting growth and nitrogen fixation of Spirillum lipoferum.

Spirillum lipoferum grows vigorously on malate, succinate, lactate, or pyruvate, moderately on galactose or acetate, and poorly on glucose or citrate. It reduces 15N2. Acetylene reduction rates decrease rapidly when the pH of the culture rises above 7.8. The organism is highly aerobic and had doubling times as low as 2 h when grown on NH4+. However, S. lipoferum reduces N2 well only under microaerophilic conditions. The optimal pO2 for acetylene reduction by stagnant cultures was 0.006 to 0.02 atm depending upon the cell density; aerated cultures grew well at dissolved O2 concentration corresponding to a pO2 of about 0.008 atm. Shaking S. lipoferum with air temporarily inactivates its nitrogenase; reactivation is inhibited by chloramphenicol. The organism assimilated 20 to 24 mg of N/g of organic acid oxidized during growth. The strains studied can be placed in two groups based upon their morphology and physiological characteristics.