Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids

Abstract

The relationship between the O 2 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 O 2 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 O 2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 M O 2 , a decline in the O 2 consumption rate was observed to 50 to 70% of the maximal O 2 consumption rate. A determination of the redox state of the cytochromes during an O 2 consumption experiment indicated that at O 2 concentrations above 5 M, 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 O 2 concentrations of >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (K m 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 O 2 .Nitrogen fixation is essentially an anaerobic process. The enzyme nitrogenase, which catalyzes the reduction of N 2 to NH 3 , consists of two O 2 -labile proteins (16,25). The physiological electron donors for nitrogenase, flavodoxin and ferredoxin, are auto-oxidizable (35). Aside from an anaerobic environment and a source of reducing equivalents, MgATP is necessary in vitro for nitrogenase activity. MgADP inhibits nitrogenase (30). This introduces a major problem inherent to aerobic nitrogen-fixing organisms, namely that O 2 is essential for ATP synthesis and probably also for electron transport to nitrogenase (14), and on the other hand, O 2 inhibits and inactivates nitrogenase. This paradox has been resolved in the symbiosis of Rhizobium or Bradyrhizobium species with legumes by the organogenesis of the root nodule. In the central zone of the root nodule a microaerobic environment is created. The microaerobic condition is maintained by a regulation of the influx of O 2 into the central tissue (21), which balances the O 2 uptake of the mitochondria and the bacteroids. Furthermore, a...

Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules

“…Previous in vitro analyses showed that the PBS is an acidic compartment (Bhandari & Nicholas ; Udvardi & Day ; Fougere & Le Rudulier ; Haaker et al . ) and in vivo data described in this manuscript, using LysoTrackers DND‐99 and DND‐189, confirm and reinforce that the PBS compartment is acidic. However, it remains to be determined whether pH variations occur in the PBS throughout symbiosis.…”

Rhizobium-Linked Nutritional and Phytochemical Changes Under Multitrophic Functional Contexts in Sustainable Food Systems

“…Endophytic rhizobia acquire carbon from host plants in the form of C 4 -dicarboxylates (fumarate, malate, and succinate), which can easily penetrate peribacteroid membranes of root nodules (Mitsch et al, 2018). Specifically, L-malate (C 4 H 6 O 5 ) is the key C 4 -dicarboxylate that supplies carbon to symbiotic rhizobia in root nodules (Haaker et al, 1996;Poole and Allaway, 2000;Mitsch et al, 2018). Rhizobial TCA cycle functions aerobically in free-living cells (Maier, 2004), and microaerobically involving anaplerotic enzymatic pathways in endophytic bacteroids (Dunn, 1998).…”

A Cytochrome cbb 3 (Cytochrome c) Terminal Oxidase in Azospirillum brasilense Sp7 Supports Microaerobic Growth

“…Cells were subsequently centrifuged and suspended in 3 ml of 25 mM TES [N-tris(hydroxymethyl)methyl)methyl-2-aminoethanesulfonic acid]-KOH-5 mM MgCl 2 buffer (pH 6.8) containing 10 g of RNase per ml, 10 g of DNase I per ml, and 1 mM phenylmethylsulfonyl fluoride. Membrane vesicles were prepared as described by Haaker et al (16).…”

“…The cytochrome cbb 3 cytochrome c oxidase, encoded by the fixNOQP operon in rhizobial species (18,23,32,38,50) or by a similar cco(cyt)NOQP operon in other bacteria (7,39,43,45), appears to be a cytochrome c terminal oxidase belonging to the heme-copper oxidase superfamily (14). In most rhizobial species this oxidase is essential for nitrogen-fixing endosymbiosis (18,32,50) and is characterized by an extremely high O 2 affinity (16,33). In the bacteria Magnetospirillum magnetoaceticum and Agrobacterium tumefaciens, and in Azorhizobium caulinodans growing nonsymbiotically, the cbb 3 -type cytochrome c terminal oxidase seems to be at least partially responsible for the microaerobic respiration (23,39,43).…”