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Changes in oxygen tension are a part of the environmental conditions in the habitat of the soil bacterium Bacillus subtilis. Oxygen is the major electron acceptor for aerobic growth. In the absence of oxygen B. subtilis develops alternative strategies for survival. Two general principles of anaerobic catabolism are known. Reducing equivalents (NADH and reduced flavin adenine dinucleotide) produced mainly by glycolysis and the Krebs cycle during the consumption of energy substrates like glucose are oxidized by membrane-localized electron transport chains. During these respiratory processes a transmembrane proton gradient is generated and used as the energy supply for ATP synthesis and transport of molecules through the cytoplasmic membrane. In the absence of external electron acceptors, the reducing equivalents are reoxidized by endogenous electron acceptors without proton gradient formation. This redox-balanced dismutation of the substrate is coupled directly on the substrate level to energy conservation.

The energetic yields of these fermentation processes are much lower than those of anaerobic respiration. However, anaerobic respiratory electron transport requires the presence and efficient utilization of alternative terminal electron acceptors (1). Recently, the utilization of nitrate as an alternative anaerobic electron acceptor was described for B. subtilis and genes for the respiratory nitrate reductase system (narGHJI) were cloned (2-4). A large variety of bacilli have been identified as respiring nitrate (10, 14). For Bacillus licheniformis, denitrification from nitrate via nitrite, NO, and N₂O to N₂ was shown (9). Bacillus macerans performs ammonification with the production of ammonia from nitrate (10). For the gram-positive model bacterium B. subtilis, anaerobic nitrate utilization has been demonstrated without elucidation of the biochemical fate of the reduced nitrate. Induction of anaerobic nitrate respiration in B. subtilis is dependent on the regulator Fnr (2). The currently known location of potential Fnr binding sites and the functional analysis of other anaerobically induced genes (6) indicate the limitation of Fnr function in B. subtilis to the regulation of nitrate respiration. Moreover, in contrast to Fnr function in enterobacteria, the B. subtilis fnr gene is under the control of a second regulatory component, the multifunctional ResDE system (6, 8, 13). Besides its recently discovered importance for anaerobic metabolism, the resDE locus is involved in the regulation of various other central cellular functions, such as sporulation, carbon source utilization, the phosphate regulon, heme A synthesis, and formation of the cytochrome bf complex (5, 13).

With this study we identified B. subtilis as an ammonifying bacterium converting nitrate via nitrite into ammonia. A dissimilatory nitrite reductase activity which required resDE but not fnr for its formation under anaerobic growth conditions was identified and measured.

Anaerobic conversion of nitrate via nitrite into ammonia by B. subtilis. B. subtilis JH642 (pheA1 trpC2) (Bacillus Genetic Stock Center) and THB1 (pheA1 trpC2 narGH::Tet Tet^r) (4) were grown anaerobically at 37°C on Luria-Bertani medium (7) supplemented with 20 mM K₃PO₄ (pH 7), 2 mM (NH₄)₂SO₄, 1 mM L-glutamic acid, 1 mM L-tryptophan, 0.8 mM L-phenylalanine, 0.005% (wt/vol) ammonium iron(III) citrate, 1 mM glucose, and, when indicated, 10 mM nitrite or nitrate (4, 6). The bacteria were incubated in completely filled flasks with rubber stoppers and with shaking at 180 rpm in an incubation shaker to minimize aggregation of the bacteria. Inoculation was performed aerobically at a 1:100 ratio of aerobically grown overnight culture and prewarmed medium. Anaerobic conditions were achieved after a short time through consumption of residual oxygen by the inoculated bacteria (11). Nitrate and ammonia concentrations in the growth media were monitored with standard enzymatic test systems (Boehringer, Mannheim, Germany). The ammonia concentration of the employed medium was subtracted from the measured values. Nitrite concentrations were measured by the diazotation procedure (12).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Anaerobic growth of B. subtilis JH642 (wild type) (A), THB1 (nitrate reductase-deficient mutant) (B), THB2 (fnr mutant) (C), and MH5081 (resDE mutant) (D) with 10 mM nitrate () or 10 mM nitrite () as a terminal electron acceptor and fermentation in the absence of external electron acceptors (). Growth was monitored by determination of the optical density at 578 nm (OD578nm) at the indicated time points. Values reported are the averages from at least 10 independent experiments performed in triplicate.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Changing concentrations of nitrate (), nitrite ( and ), and ammonia ( and ) during anaerobic growth of B. subtilis JH642 (wild type) (A), THB1 (nitrate reductase-deficient mutant) (B), THB2 (fnr mutant) (C), and MH5081 (resDE mutant) (D). The concentrations given were determined at the indicated time points in the media of B. subtilis cultures grown with 10 mM nitrate (closed symbols) or 10 mM nitrite (opened symbols). Values reported are the averages from at least 10 independent experiments performed in triplicate.

B. subtilis contains a dissimilatory nitrite reductase. To determine whether the conversion of nitrite into ammonia sustains anaerobic growth, B. subtilis JH642 was incubated anaerobically on medium containing nitrite. B. subtilis efficiently grew anaerobically with nitrite as an electron acceptor (Fig. 1A). To exclude any residual or side activity of the nitrate reducing system, the dissimilatory-nitrate-reductase-deficient mutant THB1 was incubated anaerobically with nitrite. As shown in Fig. 1B, THB1 grew efficiently with nitrite as an electron acceptor, demonstrating the presence of a dissimilatory nitrite reductase in B. subtilis. Almost all supplied nitrite was converted into ammonia by THB1 (Fig. 2B). In control experiments, the mutant THB1 failed to grow efficiently on nitrate as an electron acceptor (Fig. 1B) and consequently no nitrate conversion was detected (Fig. 2B). Observed residual growth in the absence of external terminal electron acceptors was on the level of fermentative growth (Fig. 1B).

An oxygen-sensitive benzyl viologen-dependent nitrite reductase activity is present in cell extracts prepared from B. subtilis. To measure the newly discovered enzymatic activity directly in B. subtilis cell extracts, an in vitro test system had to be established. For this purpose, B. subtilis cultures grown anaerobically in the presence of 10 mM nitrate and nitrite were used at the end of the exponential growth phase. Harvesting, washing, and disruption of cells were performed under strictly anaerobic conditions. Cells were harvested and washed with 10 mM Tris (pH 7.4) containing 1 mg of MgCl₂·6H₂O per ml. Cells were anaerobically disrupted with a French press (20,000 lb/in²). To obtain cell extracts, the suspensions were filtered through a 0.2-µm-pore-size sterile filter. Dissimilatory nitrite reductase activity was measured spectroscopically by the nitrite-dependent oxidation of reduced benzyl viologen in anoxic cuvettes containing 50 mM Tris (pH 7.4), 2 mM benzyl viologen reduced with 0.2 µM sodium dithionite, 1 mM KNO₂, and 5 to 50 µl of cell extract (containing 2 mg of protein per ml) at room temperature. Reaction mixtures were incubated in the absence of external electron acceptors to determine the endogenous rate of benzyl viologen oxidation. No obvious background activities were observed. Reactions were started by the addition of nitrite. Enzymatic oxidation of benzyl viologen was monitored spectroscopically at a wavelength of 578 nm. The extinction coefficient of benzyl viologen was used to determine the amount of reduced nitrite. One unit of activity corresponded to the reduction of 1 µmol of nitrite per min. Important for successful enzyme activity measurements was the strictly anaerobic handling during extract preparation. The nitrite reductase activity of approximately 5.6 U/mg of protein in cell extracts prepared from B. subtilis THB1 (Table 1) was reduced to approximately 2.9 U/mg of protein after 15 min of aerobic treatment and further to approximately 0.7 U/mg of protein after 90 min of exposure to aerobic conditions.

The regulatory system resDE but not fnr is required for induction of dissimilatory nitrite reductase activity. The two-component system encoded by resDE is responsible for induction of respiratory nitrate reductase partially via transcriptional activation of fnr (8, 13). To determine the influence of resDE and fnr on dissimilatory nitrite reductase formation, the resDE mutant MH5081 (pheA1 trpC2 resDE::pRC22 Tet^r) (13) and the fnr mutant THB2 (pheA1 trpC2 fnr::spec Spec^r) were incubated anaerobically in the presence of nitrite.