Achromobacter Species

JOURNAL OF BACTERIOLOGY, Mar., 1965 Copyright 0 1965 American Society for Microbiology Vol. 89, No. 3 Printed in U.S.A. Pyruvate Metabolism, Carbon Dioxide Assimilation, and Nitrogen Fixation by an Achromobacter Species I. R. HAMILTON,' R. H. BURRIS, P. W. WILSON, AND C. H. WANG Departments of Bacteriology and Biochemistry, University of Wisconsin, Madison, Wisconsin, and Radiation Center, Oregon State University, Corvallis, Oregon Received for publication 12 October 1964 ABSTRACT HAMILTON, I. R. (University of Wisconsin, Madison), R. H. BURRIS, P. W. WILSON, AND C. H. WANG. Pyruvate metabolism and carbon dioxide assimilation by an Achromobacter species. J. Bacteriol. 89:647-653. 1965.-Carbon dioxide fixation by washed whole cells of Achromobacter N4-B has been observed during anaerobic pyruvate metabolism with both nitrogen- and NH4+-grown cells. Labeled sodium bicarbonate- C'4 was assimilated into cells by a mechanism requiring pyruvate under conditions of nitrogen fixation, nitrogenase induction, and assimilation of NH4+. Of the assimilated radioactivity, 89% appeared in six amino acids and two ninhydrin-positive unknown compounds, with the distribution of the label essentially independent of the nitrogen nutritional state of the organism. Aspartic and glutamic acids were the most highly labeled, with lesser amounts in glycine, alanine, ornithine, arginine, and the unknowns. All of the radioactivity extracted from these cells by ethanol-boiling water appeared in a protein fraction precipitated by 20% trichloroacetic acid. Radiorespirometric experiments with individually labeled pyruvate substrates demonstrated the preferential decarboxylation of the C-1 of pyruvate by this organism in a flowing helium gas phase. This decarboxylation was almost completely inhibited by using flowing nitrogen in place of helium; the addition of 0.5% CO2 to the flowing nitrogen prevented inhibition and allowed 70% of the expected CO2 evolution. These results, coupled with those from growth experiments, indicate a carbon dioxide requirement for anaerobic growth and pyruvate metabolism, which appears to be coupled to the formation of protein precursors. While testing samples of water for nitrogenfixitig organisms, Jensen (1958) isolated a gramnegative rod that formed acid, but no gas, from carbohydrates, was Voges-Proskauer-negative, methyl red-positive, and fixed nitrogen anaerobically. Jensen observed, "The systematic position of this organism is difficult to establish with certainty. The key to the families of Eubacteriineae in Bergey's Manual (1948, p. 67) leads to family IX, Achromobacteriaceae, and within this family the only possible genus is Achromobacter Bergey et al., but it is not identical with any of the Achromobacter species described in the manual." Recent physiological and serological tests in several laboratories suggest that it probably will be placed in the genus Klebsiella, as defined by Edwards and Ewing (1962), but until there is a publication of these conclusions, l Present address: Department of Oral Biology, The University of Manitoba, Winnipeg, Manitoba, Canada. it appears less confusing to retain its oiiginal designation. Nitrogen-fixation studies with pyruvate as a substrate revealed what appears to be an obligate carbon dioxide requirement (Hamilton, Burris, and Wilson, 1964). Results from experiments in which this requirement was examined through use of labeled pyruvate and sodium bicarbonate are given in this report. MATERIALS AND METHODS Culture of organism. The organism used was Achromobacter N4-B, a nongummy strain selected from Jensen's original culture; the general conditions for its growth were given by Hamilton et al. (1964). The incorporation of C'4 from radioactive substrates into intact cells of Achromobacter N4-B was carried out in 60- to 125-ml suction flasks equipped for evacuation and gassing. Freshly harvested cells suspended in cold phosphate buffer (0.1 M, ph 7.5) were added to the flasks, which were immediately sealed, evacuated, and gassed five to 647

648 HAMILTON ET AL. J. BACTERIOL. seven times with the appropriate gas. After the equilibration of the flasks at 30 C for 30 min, the substrate (pyruvate) and the bicarbonate solutions (also equilibrated at 30 C) were added by a hypodermic syringe and needle through a side-arm serum cap to start the reaction. Reaction volumes ranged from 20 to 24 ml per flask. The reactions were stopped in individual flasks at hourly intervals by the addition of 1.5 ml of 6 N HC1 with a syringe. After the samples were inactivated, the cell suspensions were rinsed from the reaction vessels and titrated to ph 7.7 with 1.0 N NaOH. The cells were separated from the reaction mixture by centrifugation at 28,000 X g for 15 min and washed once in 10 ml of 0.1 M phosphate buffer (ph 7.5). The supernatant liquid fractions were pooled, made to 50 ml with distilled water, filtered through a 0.45-, Millipore filter, and frozen at -4 C until needed. The washed cells were suspended in 5.0 ml of 6 N HCI, and 0.2- to 0.4-ml samples were taken for dilution and plating in gelatin to give the total radioactivity incorporated into the cells. The remaining cell suspension was sealed under vacuum in a 25-ml glass tube and hydrolyzed by autoclaving at 121 C for 18 to 20 hr. Some samples were extracted with 10 ml of 80% ethanol three times, followed by two extractions with the same volume of boiling water. The autoclaved hydrolysates were used for anmino acid analysis, whereas the ethanol-extracted cells were used for the isolation of radioactive organic acids and labeled protein. The acid-hydrolyzed suspensions of cells were filtered and evaporated twice under vacuum to remove the HCl, brought to 5.0 ml with distilled water, and frozen. The ethanol-extracted solutions were also evaporated, dissolved in 5.0 ml of water, and frozen at -4 C. The separation of the labeled amino acids and organic acids was performed on Whatman no. 1 filter paper, according to the methods of Block, Durrum, and Zweig (1955), Palmer (1955), and Smith (1960). The solvent systems used most frequently for the separation of amino acids were butanol-acetic acid-water (4:1:5, v/v) in one direction, followed by phenol-water (4:1, v/v) in the other. The chromatograms were then cut into 2-cm strips in the phenol direction, connected in sequence, and passed through a Vanguard 880 automatic chromatogram scanner to locate the radioactivity. Radioactivity was also located by exposing the two-dimensional chromatograms to X-ray film for 17 to 20 days, followed by development. Quantitative radiometry. The total radioactivity added per flask ranged from 1.2 X 107 to 2.3 X 107 count/min, as measured by planchet counting with a Mylar end-window gas-flow detector. All samples for counting were plated with 1% gelatin in 0.01 N NaOH, such that 0.1 ml of the diluted radioactive sample was added to 1.0 ml of the gelatin. A portion (1 ml) of this was dried overnight at room temperature on an aluminum planchet (diameter, 3 cm). The radioactivity in the compounds isolated bv paper chromatography was quantitatively estimated by cutting the radioactive spots from the chromatogram strips and counting them in a Packard liquid scintillation counter, according to the method of Wang and Jones (1959). The pyruvate-carbon dioxide exchange reaction, which is known to occur during anaerobic pyruvate metabolism with cells and extracts of Achromobacter N4-B, was estimated by isolation of formic acid, which is a major product of pyruvate dissimilation under anaerobic conditions (Hamilton, 1962). The exchange of radioactive bicarbonate with the carboxyl of pyruvate leads to the formation of HC'4OOH following the cleavage of pyruvate in the phosphoroclastic reaction. Formic acid was isolated by column chromatography, and its radioactivity was measured; from these data the amount of radioactivity per mole of formate could be related to the pyruvate assimilated by the cells. The extent of the exchange reaction could thus be estimated without degrading the radioactive compounds isolated from the hydrolyzed cells. The experiments designed to determine the patterns of evolution of C'402 from the specifically labeled pyruvate C'4 substrates, under conditions approximating those made with the conventional Warburg respirometer, employed the techniques described by Wang et al. (1958). The one variable from the normal manometric methods was the use of a flowing-gas phase to carry evolved carbon dioxide to the trapping solution. Initial experiments with sodium bicarbonate-c'4 showed that no appreciable carbon dioxide is retained at ph 7.5 under the experimental conditions; virtually all of the carbon dioxide evolved from the radioactive substrates is swept from solution by the flowing gas and then is trapped in the hyamine hydroxide. This use of a flowing-gas phase provided the first suggestion of the significance of carbon dioxide in the pyruvate metabolism of the organism. RESULTS Carbon dioxide fixation during anaerobic pyruvate dissimilation. Fermentation studies with intact cells of Achromobacter N4-B incubated with pyruvate under nitrogen-fixing conditions demonstrated that a small portion of the pyruvate initially added was not recovered from the reaction filtrates as products (Hamilton et al., 1963). The fermentation balances lacked sufficient oxidized products, which led to the conclusion that pyruvate or a product of pyruvate metabolism was being incorporated into the cells during the incubation. This conclusion was confirmed by exposing both ammonia-grown and nitrogen-grown cells to pyruvate-3-c'4 under anaerobic conditions. As shown in Fig. 1, a small portion of the total

VOL. 89, 1965 METABOLISM OF ACHROMOBACTER SP. 649 A 1 in experiment 2, no N2 was fixed, as nitrogenase %0 x NH+4-GROWN was being induced; in experiment 3, NH4+ was 12 \ 14 2 120 the source of nitrogen. z \ c / That assimilation of C14 from radioactive XJ \ /bicarbonate does occur is shown in Fig. 2 (ex- 0 8- \ /80 z periment 1), where the total amount of C'4 o incorporatedintocellswas determinedfrom a PYRUVATE o sample of the reaction mixture. Pyruvate ap- 0 t / \ / 40 F parently is involved, since the incorporation of 8 / ENDOG- \ z / a C14 was higher in its presence thani in its absence. The incorporation was also prol)ortional to the 1-0 0 dissimilation of pyruvate, with a sharp break > B a at 2 hr as pyruvate neared complete utilization. I NzGROWN Nitrogen fixation, as measured by the net gain o 12 120 U in total combined nitrogen, continued until 4 -Jcl4 c _ ~~~~~0 all the pyruvate was utilized. _J \_a The cells, after separation from the reaction 880 mixture and subsequent washing, were extracted with 80% ethanol and boiling water; this treatzy 0 PYRUVATE ment produced an extract which contained 25% F 4- / \/ '40 of the total radioactivity incorporated by the z/endog- / intact cells. When this extract was subjected O 1 2 3 4 TI ME (HRS) \ FIG. 1. Incorporation of pyruvate-3-c14 into ZPYRUVATE 50z -JI washed whole cells of Achromobacter N4-B. Experi- 4 I fj ment A: Cells were grown in medium containing 200 0 I- I ~~~~~~~c./14404,ug of NH4+-N per ml in an atmosphere of hydrogen. 2,2-0 40 Reaction mixture contained SO Amoles of ammonium X phosphate per milliliter. Flask contained 2.85 mg cc w 0 of total cell nitrogen. Experiment B: Cells were U \304 grown on N2 in nitrogen-free medium. Cell concen- -z> tration was 3.52 mg of total cell nitrogen. Each flask -/\ contained 386,umoles of unlabeled pyruvate in addi- -J / tion to pyruvate-s-c'4 (negligible concentration), 0 / \c 3.75 X 104 count/min per flask. Final volume was 10 ' / \ ml at 30 C. F / 0 0~~~~~~~~~~~~~~1 1 added radioactivity was incorporated into both E types of cells. The calculated oxidation value of the assimilated product was in the range of a co +3 to +5 (0 = 2, H = 1) by considering the z TOTAL N carbon not accounted for in relation to the defi- ;A ciency in the oxidized product. This oxidation R 120/ range includes a number of the 4-carbon tri- / carboxylic acid cycle intermediates, which can a be formed by carbon dioxide fixation with pyru- xc 60/ vate, followed by assimilation into cellular z components. To test this possibility, cells of Achromobacter N4-B were incubated with high specific activity bicarbonate-c'4 and unlabeled TI M E (H RS) sodium pyruvate. The cells from this reaction FIG. 2. Incorporation of C'4 into intact washed were isolated at hourly intervals and hydrolyzed, cells of Achromobacter N4-B incubated in sodium as described in Materials and Methods. Three pyruvate and radioactive bicarbonate under optimal types of experiments were made: in experiment nitrogen-fixing conditions (experiment 1). For 1, N2 was fixed by already induced nitrogenase; conditions, see Table 1.

650 HAMILTON ET AL. J. BACTERIOL. to paper chromatography with solvent systems separating both organic acids and amino acids, all of the radioactivity remained at the origin; no organic acids contained radioactivity. If the extracts were treated with 20% trichloroacetic acid, all of the radioactivity in the samples was precipitated; no label appeared in the trichloroacetic acid-soluble fraction. Acid hydrolysates of these cells subjected to paper chromatography yielded eight ninhydrin - positive compounds containing 89% of the incorporated radioactivity. Of the eight compounds, six were identified as amino acids, whereas two other TABLE 1. Expt J. 2c 3d ninhydrin-positive spots with little radioactivity were not identified. The six amino acids were aspartic acid, glutamic acid, ornithine, arginine, glycine, and alanine. An unknown acid (unknown 1) moved near aspartic acid (RF of 0.24 in butanol-acetic acid-water and 0.22 in phenol-water), and a second unknown acid (2) moved close to glutamic acid (RF of 0.33 and 0.23 in butanol and phenol, respectively). Only traces of radioactivity could be detected at other locations on the chromatograms. As the amount of hydrolysate added to the chromatograms was known, and efficiency of counting was determined Fraction of NaHC1403 incorporated into amino acids of the proteins of Achromobacter N4B I Amino acid Alanine Arginine Aspartic Glutamic Glycine Ornithine Asparagine Unknown (1) Unknown (2) Alanine Arginine Aspartic Glutamic Glycine Ornithine Asparagine Unknown (1) Unknown (2) Alanine Arginine Aspartic Glutamic Glycine Ornithine Asparagine Unknown (1) Unknown (2) 1 b 0.02 0.10 0.00 0.04 0.03 0.25 0.07 0.12 0.02 0.04 0.02 0.22 0.20 1.41 0.43 0.42 Time of harvest (hr) 2 0.29 0.72 0.31 0.25 0.04 0.34 0.08 0.10 0.50 0.32 0.09 0.60 0.42 2.02 1.40 0.90 0.30 0.81 0.37 3 0.14 0.32 0.72 0.30 0.43 0.08 0.22 0.19 0.87 0.72 0.34 0.37 0.07 0.33 0.88 3.12 2.52 1.00 0.40 0.84 0.40 4 0.12 0.43 0.73 0.51 0.09 0.14 0.24 0.44 0.92 0.74 0.21 0.16 0.39 0.31 0.58 1.20 3.78 2.70 1.30 0.62 0.65 0.50 Total label per mg of cell N 2.4 3.4 11.3 Pyruvate added per mg of cell N a Cells (16.2 mg of nitrogen) grown on N2 incubated with 924 jumoles of pyruvate, 20 pmoles of sodium bicarbonate (radioactivity of 1.31 X 107 count/min) in 0.1 M phosphate buffer (ph 7.5); total volume, 20 ml. Gas phase: N2. bvalues represent the fraction of the total radioactivity incorporated into cells per milligram of total nitrogen X 103. c Cells (12.95 mg of nitrogen) grown on NH4+ incubated with 960,umoles of pyruvate, 24,umoles of sodium bicarbonate (radioactivity of 1.29 X 107 count/min), and 32,ug of ammonium phosphate in 0.1 M phosphate buffer (ph 7.5) in total volume of 24 ml. Ammonia nitrogen completely utilized in 90-min preincubation period before addition of substrates. Gas phase: N2. d Cells (5.7 mg of nitrogen) grown on ammonium phosphate incubated with 1,075jumoles of pyruvate, 1,200 Amoles of ammonium phosphate, 24,moles of sodium bicarbonate (radioactivity of 2.3 X 107 counts/min) in 0.1 M phosphate buffer (ph 7.5) in total volume of 24 ml; atmosphere was H2. pmotes 57 74 189

VoL. 89, 1965 METABOLISM OF ACHROMOBACTER SP. 65-1 for the paper strips, the fraction of the total incorporated radioactivity in each of the amino acids per milligram of total cell nitrogen could be calculated. The data in Table 1 reveal that differences in the distribution of the C'4 in the six identified amino acids were essentially independent of the state of nitrogen nutrition of the cells. The total label incorporated was proportional to the pyruvate-cell ratio, and this may have influenced the final distribution to some extent. For example, in experiment 1, with the lowest value of this ratio, the pyruvate had practically disappeared by the end of the 3rd hr. During the next hour, the C14 label in glutamic acid and arginine increased, that in aspartic acid remained constant, and glycine lost over one-half of its label. In general, the highest fraction of the label was found in aspartic and glutamic acids. When NH4+ was the source of nitrogen, asparagine was labeled (probably in the amide group), and the label in unknown 2 disappeared. Calculation of the extent of the exchange reaction between carbon dioxide and pyruvate indicated that it accounted for about 25 to 30% of the total C'4 incorporated when N2 was fixed, and only 11% when NH4+ was the source of nitrogen. Radiorespirometry with labeled pyruvate. Washed intact cells of Achromobacter N4-B have been shown to decarboxylate pyruvate under nitrogenfixing conditions by conventional manometric techniques (Hamilton, Burris, and Wilson, 1964). To determine which pyruvate carbon was being decarboxylated and to study the effects of varying the gaseous atmosphere, the ph, and the cell concentration, radiorespirometric experiments similar to those of Wang et al. (1958) were undertaken. For these experiments, radioactive potassium pyruvate, labeled in the C-1, C-2, or C-3 position, was employed with 13.6 or 50 mm unlabeled potassium pyruvate as carrier substrate. Cells incubated with unlabeled pyruvate in 0.1 M phosphate buffer (ph 7.5) will decarboxylate 5 to 15% of the pyruvate, as measured in a nonflowing nitrogen atmosphere by manometry. When, however, intact cells of Achromobacter N4-B were incubated in an identical fashion in a radiorespirometer, with radioactive pyruvate and a flowing N2 gas phase, less than 1% of the C-1, C-2, or C-3 of the pyruvate was decarboxylated (Fig. 3a). The addition of 0.54 mm thiamine to the growth medium or varying the ph of the reaction mixture did not stimulate decarboxylation under these conditions, as it does in nonflowing nitrogen atmospheres. In the flowing-gas experiments, little pyruvate was utilized during the incuba- - cu 0-0 TIME (HRS) FIG. 3. Effect of gas phase on decarboxylation of sodium pyruvate substrates specifically labeled with C14 when incubated with washed whole cells of Achromobacter N4-B at ph 7.5. Cells, at concentration of 58.0 mg (dry weight) of cells, incubated with 150 pmoles of potassium pyruvate in a total volume of 11 ml at 80 C. The gas flow rate was 60 cc/min. Cells were grown in nitrogen-free medium with N2. Substrates: 0, pyruvate-l-c"4 (1.10 X 106 count/min); A, pyruvate-2-c'4 (1.04 X 106 count/ min); *, pyruvate-s-c'4 (1.07 X 106 count/min). Gas phase: A = nitrogen, B = nitrogen + 0.5% carbon dioxide, C = helium. tions. The addition of 2 $M thiamine pyrophosphate and 20 AM MgCl2 to the reactions carried out in the respirometer stimulated C-1 decarboxylation to 3% (C-2 and C-3 were negligible), which was only one fifth the level observed in manometric experiments. As conditions for the manometric and radiorespirometric experiments were the same, except for the flowing nitrogen atmosphere, the nonevolution of CO2 apparently was the result of N2 inhibition of pyruvate metabolism. This conclusion was supported by experiments in which flowing nitrogen inhibited the growth of Achromobacter N4-B incubated in a nitrogen..

652 HAMILTON ET AL. J. BACTERIOL. free medium compared with cells in the same medium in a nonflowing gas phase (Hamilton, Burris, and Wilson, 1964). When the previously described radiorespirometric experiments were carried out with helium as the carrier gas, the expected decarboxylation occurred, with 15.8% of the pyruvate-1-c'4 being decarboxylated at ph 7.5 (Fig. 3e) and 16.2%ho at ph 6.4. Less than 4% of the label of pyruvate-2-c'4 and pyruvate-3-cg4 was evolved as C'402 during these experiments. The predominant cleavage of the pyruvate-1-c'4 would be expected if the phosphoroclastic reaction functions in anaerobic pyruvate degradation (Lipmann, 1946). The absence of an effect by an inert gas on the system further implies the specific inhibition of pyruvate metabolism by flowing N2. The nitrogen inhibition of the pyruvate system in Achromobacter N4-B was also reversed by the addition of 0.5% carbon dioxide to the N2- flowing gas phase. As illustrated in Fig. 3b, a total of 12% of the C-1 of pyruvate was decarboxylated in an atmosphere of N2 + 0.5% C02, which represents 70% of the expected carbon dioxide evolution. Attempts were made to reverse the N2 inhibition during the course of these experiments by changing the carrier gas to N2 + 0.5% carbon dioxide after 3 hr with flowing N2. This slightly stimulated the decarboxylation of the pyruvate- 1-C4, but had no effect on the labeled C-2 and C-3 substrates. This partial reversal of the N2 inhibition could also be shown by changing from N2 to helium. The reduced evolution of C402 from the pyruvate-1-c"4 was noted if the cells were preincubated in a flowing N2 atmosphere for 2 hr, followed by changing to helium at the time of substrate addition. When cells grown in NH4+ were incubated under N2 with specifically labeled pyruvate substrates, no decarboxylation was noted at ph 7.5 or 6.9 with any of the substrates. The same was also observed with helium as the carrier gas. The fact that nitrogen-grown cells are inhibited in a flowing-nitrogen system, but not in flowing helium, indicates that differences exist between the pyruvate-metabolizing systems in the two types of cells. The reason for this difference has not been determined. DISCUSSION In contrast to the active pyruvate decarboxylating systems in other nitrogen fixers (Clostridiunm. pasteurianumn, Bacillus polymyxa, and Aerobacter aerogenes), that in Achromobacter N4-B is weak, as is its hydrogenase activity (Hamilton, Burris, and Wilson, 1964). During nitrogen fixation with intact cell suspensions of this organism, small quantities of carbon dioxide and hydrogen are formed from pyruvate, but the yield is low, nonequimolar, and characterized by the reduced evolution of hydrogen in the presence of N2, in comparison with that in helium (Hamilton, 1963). It has been shown that cells fixing N2 reassimilate carbon dioxide when the pyruvate becomes exhausted, and that succinic and isocitric acids appear as products of pyruvate metabolism (Hamilton, 1963). These observations point to the operation of a carbon dioxide-fixing system which may be essential for formation of amino acids. The importance of carbon dioxide fixation is evident in the labeling of these amino acids. It appears that with a 3-carbon substrate, such as pyruvate, carbon dioxide fixation is required to produce intermediates of the tricarboxylic acid cycle that are necessary for the formation of protein precursors. The evidence from the growth studies with mannitol as the carbon source in a flowing N2 atmosphere also suggests this function for carbon dioxide fixation. Possibly, under anaerobic conditions, this is the only mechanism of entry to the citr'ic acid cycle. The reversal of N2 inhibition of pyyruvate decarboxylation by carbon dioxide in the radiorespirometer further indicates the importance of carbon dioxide to Achroitobacter N4-B. Although the requirement of C-1 units for the biosynthesis of 4-carbon dicarboxylic acids, which in turn act as precursors for amino acids, explains partially the reversal of N2 inhibition, the complete reversal of N2 inhibition by helium suggests that factors other than the C-1 requirement may be involved in the observed growth responses. Hamilton et al. (1964) demonstrated that carbon dioxide reversed nitrogen inhibition of growth in a 2%y, mannitol, miileral salts, nitrogen-free medium, but found that the growth that occurred at several carbon dioxide tensions, or with added bicarbonate, was never equal to that of cells grown in a closed sy-stem. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grants AI 01417-09 anid Al 00848 from the National Institute of Allergy and Infectious Diseases, and grant GB-483 from the National Science Foundation. One of the authors (IRH) was a predoctoral research trainee on National Institutes of General MIedical Sciences training grant 2G-686-MTC. LITERATURE CITED BLOCK, R. J., L. DLRRUM, AND G. ZWIEG. 1955. A manual of paper chromatography and paper

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