The respiratory system and diazotrophic activity of Acetobacter diazotrophicus PAL5

Abstract

The characteristics of the respiratory system of Acetobacter diazotrophicus PAL5 were investigated. Increasing aeration (from 0.5 to 4.0 liters of air min(-1) liter of medium(-1)) had a strong positive effect on growth and on the diazotrophic activity of cultures. Cells obtained from well-aerated and diazotrophically active cultures possessed a highly active, membrane-bound electron transport system with dehydrogenases for NADH, glucose, and acetaldehyde as the main electron donors. Ethanol, succinate, and gluconate were also oxidized but to only a minor extent. Terminal cytochrome c oxidase-type activity was poor as measured by reduced N, N,N,N'-tetramethyl-p-phenylenediamine, but quinol oxidase-type activity, as measured by 2,3,5,6-tetrachloro-1,4-benzenediol, was high. Spectral and high-pressure liquid chromatography analysis of membranes revealed the presence of cytochrome ba as a putative oxidase in cells obtained from diazotrophically active cultures. Cells were also rich in c-type cytochromes; four bands of high molecular mass (i.e., 67, 56, 52, and 45 kDa) were revealed by a peroxidase activity stain in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KCN inhibition curves of respiratory oxidase activities were biphasic, with a highly resistant component. Treatment of membranes with 0.2% Triton X-100 solubilized c-type cytochromes and resulted in a preparation that was significantly more sensitive to cyanide. Repression of diazotrophic activity in well-aerated cultures by 40 mM (NH(4))(2)SO(4) caused a significant decrease of the respiratory activities. It is noteworthy that the levels of glucose dehydrogenase and putative oxidase ba decreased 6. 8- and 10-fold, respectively. In these cells, a bd-type cytochrome seems to be the major terminal oxidase. Thus, it would seem that glucose dehydrogenase and cytochrome ba are key components of the respiratory system of A. diazotrophicus during aerobic diazotrophy.

FIG. 1

Growth properties and diazotrophic activity of A. diazotrophicus on LGIP medium with increased aereation (liters of air min⁻¹ liters of medium⁻¹): ▿, 0.5; □, 1.0; ◊, 1.5; ▵, 2.5; ○, 4.0. The medium contained 1.0 mM (NH₄)₂SO₄ as the nitrogen starting dose; cultures were performed at 30°C in a fermentor with a working volume of 1.0 liter, agitated to 320 rpm. Cultures were initiated with 20 ml of inoculum from a 24-h shaker culture (200 rpm). At the times noted, samples were withdrawn to determine culture growth at 560 nm (A), culture oxygen demand (B), nitrogenase activity as measured by the acetylene reduction assay (C), medium pH (D), concentration of ammonium in the medium (E), and dissolved-oxygen concentration in the medium (F). For comparison, growth profiles obtained in LPGIP medium containing 40 mM (NH₄)₂SO₄ and with an air flow of 4.0 liters of air min⁻¹ are displayed (●) in each of the sets. Details of culture and assay methods are described in Materials and Methods.

FIG. 2

Nitrogenase activities of A. diazotrophicus in a fermentor (1.0-liter working volume) at increased values of oxygen transfer coefficient (K_La). Values for K_La (see Materials and Methods) were determined under each of the aeration conditions shown in Fig. 1 and plotted against the recorded maximal values of nitrogenase activity (as shown in Fig. 1C).

FIG. 3

Low-temperature (77 K) spectra of membranes of A. diazotrophicus PAL5 grown aerobically in LGIP medium supplemented with 1.0 mM (A to C) or 40 mM (D to E) (NH₄)₂SO₄. (A and D) reduced-minus-oxidized spectra. Difference spectra were generated by adding sodium dithionite (spectra a), NADH (spectra b), and glucose (spectra c) to sample cuvettes and ammonium persulfate to reference cuvettes. (B and E) Reduced plus CO minus reduced difference spectra. Membranes in both cuvettes were reduced by dithionite and CO gas bubbled through sample cuvettes. (C and F) Reduced plus 1.0 mM KCN minus oxidized spectra. Sample cuvettes were reduced with dithionite in the presence of 1.0 mM KCN. Reference cuvettes were oxidized with ammonium persulfate. All samples contained 2.5 mg of membrane protein ml⁻¹. Cells were collected at early stationary phase once they reached OD₅₆₀ = 2.5 (low NH⁺₄) and 6 to 8 (high NH⁺₄).

FIG. 4

c-type cytochromes associated with membranes of A. diazotrophicus PAL5 grown aerobically in LGIP medium supplemented with 1.0 mM (A) or 40 mM (B) (NH₄)₂SO₄. Membranes (50 mg of protein) were extracted with 5.0 ml of 0.2% Triton X-100 in 50 mM potassium phosphate (pH 6.0) for 2 h at 4°C. Membrane residues were sedimented at 140,000 × g for 1 h. SDS-PAGE and peroxidase stain of heme C-containing proteins of whole membranes (lanes a₁ and a₂), membrane residues after 0.2% Triton X-100 treatment (lanes b), and supernatant after 0.2% Triton X-100 treatment (lanes c). Dithionite-reduced minus persulfate-oxidized spectra at 77 K were obtained from the same samples with spectra a to c as above. The protein contents for spectra a, b, and c were 5.0, 4.0, and 1.0 mg ml⁻¹, respectively. The protein contents in SDS-PAGE of samples in panel A were 80, 160, 940, and 1,000 μg for samples a₁, a₂, b, and c, respectively; those in panel B were: 500 μg for sample a₁ and 1,000 μg for samples a₂, b, and c. SDS treatment removes noncovalently bound hemes. Hence, a peroxidase stain on SDS-gels specifically reveals c-type cytochromes.

FIG. 5

Reverse-phase HPLC chromatograms of the membrane-bound hemes from A. diazotrophicus PAL5 grown aerobically in LGIP medium supplemented with 1.0 mM (A) or 40 mM (B) (NH₄)₂SO₄. The system was calibrated with the following standards: hemes B and O extracted from membranes of E. coli, hemes B and A extracted from submitochondrial particles from bovine heart, and photoheme IX obtained from Sigma Chemical Co. The retention times for the standards and hemes of A. diazotrophicus were as follows: heme B, 28.5 min; heme A, 31.4 min; heme O, 34 min. The relative amounts of hemes B and A were estimated to be 1.0:0.26 (A) and 1.0:0.042 (B).

FIG. 6

Cyanide inhibition of oxidase respiratory activities of A. diazotrophicus PAL5 grown aerobically in LGIP medium supplemented with 1.0 mM (A) or 40 mM (B) (NH₄)₂SO₄. Oxidase activities for NADH (○), glucose (▵), and acetaldehyde (□) in membranes were titrated with KCN. Alternatively, the KCN titration was performed on (▴) glucose oxidase of membrane residues obtained after treatment with 0.2% Triton X-100 (see Results and Fig. 4). The membrane protein used, assay conditions, and oxidase specific activities were similar to those shown in Table 2.

FIG. 7

Postulated composition and organization of the aerobic respiratory system of A. diazotrophicus PAL5. Cytochrome a₁ (also called ba) putative oxidase is preferentially expressed in N₂-fixing cells, while cytochrome bd putative oxidase is conspicuous in cells grown with excess NH₄⁺. Respiratory activities, notably glucose oxidase, are higher in cells displaying diazotrophic activity. Cyt, cytochrome.