N2 Fixation by Streptomyces thermoautotrophicus Involves a Molybdenum-Dinitrogenase and a Manganese-Superoxide Oxidoreductase That Couple N2Reduction to the Oxidation of Superoxide Produced from O2by a Molybdenum-CO Dehydrogenase

Abstract

N 2 fixation by Streptomyces thermoautotrophicus follows the equation N 2 ؉ 4 -12MgATP ؉ 8H؉ ؉ 8e ؊ 3 2NH 3 ؉ H 2 ؉ 4 -12MgADP ؉ 4 -12P i and exhibits features which are not obvious in the diazotrophic bacteria studied so far. The reaction is coupled to the oxidation of carbon monoxide (CO) by a molybdenum-containing CO dehydrogenase that transfers the electrons derived from CO oxidation to O 2 , thereby producing superoxide anion radicals (O 2 . Streptomyces thermoautotrophicus UBT1 occurs in natural enrichments in the covering soil of burning charcoal piles (1, 2). The bacterium is characterized by the utilization of gases as sources of energy, carbon and nitrogen (gasotrophy). S. thermoautotrophicus grows with CO or H 2 plus CO 2 under aerobic, chemolithoautotrophic, and thermophilic conditions (3). It is a free living dinitrogen fixer (4). Although CO and H 2 are known as inhibitors of nitrogenase activity, S. thermoautotrophicus is able to fix N 2 with CO or H 2 plus CO 2 as growth substrates as well as in the presence of CO plus ethine (4). The N 2 -fixing system of S. thermoautotrophicus is expressed constitutively (5). Intact bacteria reduced ethine to ethene (6) at a negligible level of less than 0.001% of the activity of Azotobacter vinelandii (7). We were, therefore, interested in the analysis of N 2 fixation by S. thermoautotrophicus with respect to the unusual components, structure, and reactivity of the nitrogenase system. EXPERIMENTAL PROCEDURESBacterial Strain and Growth Conditions-S. thermoautotrophicus UBT1 (DSM 41605, ATCC 49746) was employed throughout this study (3). Bacteria were grown chemolithoautotrophically with CO as a sole source of carbon and energy in mineral medium supplied with trace elements (8) and 1.5 g of NH 4 Cl/liter (8) at 65°C. Fermentors of 70 liters total volume were flushed with a gas mixture composed of (v/v) 78% air, 13% CO, and 9% CO 2 at a flow rate of 3 liters/min. Bacteria were harvested by centrifugation and stored at Ϫ20°C until use.Preparation of Subcellular Fractions-Crude extracts were prepared by passing bacterial suspensions (200 g of wet weight in 200 ml of 50 mM potassium phosphate buffer, pH 7.5, containing 0.5 mM phenylmethylsulfonyl fluoride and few crystals of DNase I) about 20 times through a French pressure cell under anoxic or oxic conditions. Solutions were made anoxic by evacuation, sparging with N 2 , and the addition of sodium dithionite (100 mg/liter). Cytoplasmic fractions were obtained from crude extracts by ultracentrifugation.Purification of Nitrogenase Proteins-Cytoplasmic fractions were loaded onto a DEAE-52 cellulose column (height, 10.6 cm; diameter, 6.2 cm; bed volume, 250 ml) and eluted with 250 ml of phosphate buffer (50 mM, pH 7.5), followed by a linear gradient of 0 to 1 M NaCl in phosphate buffer. Fractions were analyzed for nitrogenase activity. The St2

Role of Diazotrophic Bacteria in Biological Nitrogen Fixation and Plant Growth Improvement

“…However, it was shown that the alternative systems may be important for N 2 -fixation in certain environments, such as termite gut. Only recently a completely novel nitrogenase system was found in Streptomyces thermoautotrophicus (Ribbe et al, 1997). The nitrogenase reductase in this system is a manganese superoxide dismutase that shows no homology to nifH.…”

“…The nitrogenase reductase in this system is a manganese superoxide dismutase that shows no homology to nifH. Interestingly this Mo nitrogenase is not O 2 -sensitive and its energy requirements are significantly lower than the classical nitrogenases (Ribbe et al, 1997).…”

CO-Sensing Mechanisms

“…e.g., references 7 and 72). In contrast, expression of the monofunctional CODH enzymes can readily be associated with environmental CO: the anaerobic Carboxydothermus hydrogenoformans (Ni-containing CODH) was obtained from a volcanic vent (121), while the aerobic Streptomyces thermoautotrophicus (Mo-containing CODH) was isolated from soil covering mounds of burning charcoal (110).…”

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

“…The longest-known and best-studied nitrogenase is the Mo nitrogenase, which occurs in all N 2 -fixing organisms with the exception of some CO-oxidizing bacteria (178). The Mo nitrogenase is encoded by the structural genes nifHDK.…”