Organic Compounds' substrates is evident from the report of Silver (1960) that formate is metabolized, and the report

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JOURNAL OF BACTERIOLOGY, JUlY, 1965 Vol. 90, No. 1 Copyright 1965 American Society for Microbiology Printed in U.S.A. Permeability of Nitrobacter agilis to Organic Compounds' S. IDA2 AND M.

1 JOURNAL OF BACTERIOLOGY, JUlY, 1965 Vol. 90, No. 1 Copyright 1965 American Society for Microbiology Printed in U.S.A. Permeability of Nitrobacter agilis to Organic Compounds' S. IDA2 AND M. ALEXANDER Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York Received for publication 8 March 1965 ABSTRACT IDA, S. (Cornell University, Ithaca, N.Y.), AND M. ALEXANDER. Permeability of Nitrobacter agilis to organic compounds. J. Bacteriol. 90: None of a variety of inorganic ions or organic compounds served as a sole energy source for the growth of Nitrobacter agilis, and the test substrates were not oxidized by either intact cells or extracts of the obligate chemoautotroph. The organic substances did not serve as sole carbon sources for the bacterium in a synthetic medium, and they failed to enhance the rate of nitrite oxidation. The organism was permeable to acetate and a number of other simple carbon compounds, however, and exogenously supplied acetate was converted to a number of products. On the basis of these findings, possible reasons are examined for the inability of the chemoautotroph to use exogenous organic compounds as energy or carbon sources. Each of the two categories of nitrifying chemoautotrophs is restricted to a single source of energy to sustain growth, i.e., ammonium for Nitrosomonas and related bacteria and nitrite for Nitrobacter spp. Moreover, the sole source of carbon for growth and development of both microorganisms is carbon dioxide. Such an apparent restriction of this group of bacteria to a single energy and a single carbon source seems to be relatively unique even among the obligate chemoautotrophs, since strains of Thiobacillus are known to oxidize a number of inorganic sulfur compounds (Vishniac and Santer, 1957), and Ferrobacillus strains may likewise have the ability to grow heterotrophically under certain circumstances (Remsen and Lundgren, 1963). Nevertheless, the physiological basis of the apparent nutritional exclusiveness of the nitrogen autotrophs has never been examined in detail. The inability of the nitrifying chemoautotrophs to make use of organic energy or carbon sources may result from the impermeability of the cell to such substrates, the absence from the bacteria of the appropriate enzymes to metabolize those organic molecules that penetrate into the cell, or the inability of the organism to derive energy from, or couple phosphorylation with, the metabolism of those organic compounds that enter into it. The possibility that strains of Nitrobacter may be permeable to at least certain carbonaceous 2 1 Agronomy Paper No Present address: Research Institute for Food Sciences, Kyoto University, Kyoto, Japan. substrates is evident from the report of Silver (1960) that formate is metabolized, and the report of Zavarzin (1960) that the rate of reduction of methylene blue by intact cells of Nitrobacter winogradskyi is increased in the presence of pyruvate, benzaldehyde, xanthine, and hypoxanthine. It has already been shown that strains of Thiobacillus are permeable to glycerol, valine, and leucine (Vishniac and Trudinger, 1962). The present communication and the accompanying independent report of Delwiche and Finstein (1965) are designed to examine the physiological basis for the limitation of N. agilis to a single energy source and a single carbon source. MATERIALS AND METHODS Carbon and energy sources. Cultures of N. agilis were grown in the medium of Aleem and Alexander (1958), modified to contain other energy sources in place of nitrite. The cultures were incubated for 14 days at 30 C on a rotary shaker. The possible utilization of organic compounds as carbon sources for growth was determined with flasks containing 50 ml of a medium that initially received 40 ppm of nitrite-nitrogen. Half the flasks only received bicarbonate. The cultures were aerated with sterile air freed from CO2 by passage through a column of alkali. The organic carbon source and additional nitrite-nitrogen, 300,g/ml of each, were added after 2 days, and the nitrite levels were determined by the procedure of Morrill (1959). The possible oxidation of a variety of inorganic and organic substances was examined with suspensions or extracts of N. agilis. The cells were 151

2 152 IDA ANI) ALEXANDER J. BACTERIOL. washed well and suspended in 0.05 M K2HPO4. The extracts were prepared by use of a 30-min exposure in a 10-kc Raytheon magnetorestrictive sonic oscillator, the residual cells and cellular debris being removed by a 30-min centrifugation at 4,000 X g. Substrate oxidation was measured manometrically at 30 C, with each Warburg flask containing 25,umoles of substrate, 50,moles of phosphate (ph 8.0), and, in the center compartment, 0.2 ml of 20% KOH. Permeability to organic compounds. N. agilis cells were obtained from cultures grown in 10-liter serum bottles containing 8.0 liters of the following medium: KNO2, 2.0 g; K2HPO4, 1.5 g; KHCO3, 12.0 g; MgSO4-7H20, 1.5 g; NaCl, 1.5 g; FeSO4V 7H20, 40 mg; 1.0 ml of Arnon's (1938) A-4 solution containing molybdenum; deionized, distilled water, 1.0 liter. The phosphate, iron, and bicarbonate salts were sterilized separately, and the resulting medium showed no evidence of turbidity. Air sterilized by passage through a column of cotton was provided continuously. The inoculum consisted of 600 ml of an active culture not more than 8 days old. After the nitrite initially added to the medium was oxidized to nitrate, additional increments of nitrite were supplied according to the method of Aleem and Alexander (1958). After 6 to 7 days of growth at 30 C, the yield was 50 to 60 mg of dry cells per liter of medium. The cells were collected by centrifugation; the paste was washed twice with distilled water, and suitable dilutions of the microbial suspension were prepared. A 5-ml amount of the cell suspension was incubated at 25 C with 1.0,uc of the carrier-free labeled compound. At regular intervals, 0.5-ml samples were taken from the reaction mixture and immediately cooled by mixing with 1.5 ml of ice water. The cells were removed from suspension by passage through a Millipore filter (mean pore size, 0.45 Iu), and the bacteria were washed three times with ice water. The radioactivity associated with the cells was determined directly on the Millipore filter with a Baird-Atomic scaler, model 123 (Baird- Atomic, Inc., Cambridge, Mass.). The radioactivity was corrected for background, and the data are expressed on a dry-weight basis. Acetate incorporation. The reaction mixture was incubated at 25 C for 20 min, at which time the cell suspension was cooled, and the cells were collected by centrifugation and immediately washed twice with water. The bacterial pellet was extracted with boiling 80% ethyl alcohol, as described by Bassham and Calvin (1957). The alcohol extracts were dried in a flash evaporator at temperatures below 40 C, and the dry material was extracted three times with petroleum ether (30 to 60 C boiling range) to yield a fraction regarded as the lipid component. The residue after ether extraction was taken up in water and passed through a column (6 by 1 cm) of Dowex 50W-X8 (H+). The resulting effluent was passed through a column (6 by 1 cm) of Dowex 1-X1O (formate). The amino acid-containing fraction was eluted from the first column with 50 ml of 1.0 N NH40H, and the organic acid-containing fraction was eluted from the Dowex 1-X1O column with 50 ml of 4.0 N formic acid. These two solutions and the neutral fraction that passed through both the resins were dried at temperatures not exceeding 40 C, and the residue was dissolved in a small amount of water. The amino acid-containing fraction was resolved by use of paper chromatography with a phenol-water (80:20, v/v) solvent system. The amino acids were located with a ninhydrin spray and identified by cochromatography with authentic compounds. The location of the radioactive peaks on the chromatograms was determined with a Baird-Atomic ratemeter, model 432A. Substrates. The following compounds (all from Calbiochem) were used: acetate-i-c14, 12.2 mc/ mmole; acetate-2-c14, 20.5 mc/mmole; glycine- 1),2-C14, 20.4 mc/mmole; hypoxanthine-8-c'4, 11.2 mc/mmole; glycerol-2-c14, 7.0 mc/mmole; pyruvate-i-c'4, 4.0 mc/mmole; xanthine-8-ci4, 4.12 mc/mmole; L-a-alanine-l-C14, 9.1 mc/mmole; methanol-c'4, 10.0 mc/mmole; formate-c14, 3.4 mc/mmole. Culture purity. The cultures were examined repeatedly for contamination by use of nutrient broth, nutrient broth supplemented with 1.0% yeast extract, Czapek broth, Thioglycolate broth, and lactose broth (all from Difco). After the media were inoculated with samples of the N. agilis cultures, the culture vessels were incubated for 2 weeks and examined for signs of microbial contamination. RESULTS Carbon and energy sources. The population of N. agilis did not increase, as determined microscopically or by plating on silica gel, in nitritefree media containing the following as potential energy sources: thiosulfate, sulfite, phosphite, hypophosphite, ferrous iron, divalent manganese, arsenite, oxalate, succinate, acetate, malate, glyceraldehyde, formaldehyde, or urea. However, the number of bacterial cells increased from 5.4 x 104 on the day of inoculation to 7.7 X 107 and 9.4 X 104 per milliliter after 14 days of incubation in media provided with nitrite and formate, respectively. The increase in number of cells in the formate medium was small and within the range of experimental error, however. Similarly, there was no detectable oxygen utilization in a 2-hr period by cell suspensions or extracts provided with any of these substrates except nitrite and formate. The addition of bicarbonate to the respirometer flasks did not alter the results. Although the oxygen uptake by intact cells or extracts in the presence of formate was small, the results were consistent and reproducible. Despite its inability to oxidize these organic compounds, N. agilis might conceivably be able

3 VOL. 90, 1965 PERMEABILITY OF N. AGILIS 153 TABLE 1. Nitrite content of Nitrobacter agilis cultur es provided with bicarbonate and organic carbon Organic carbon source Nitrite (N, pg/ml) 5 days 8 days None Formate Oxalate Citrate Malate Succinate Formaldehyde Glyceraldehyde Urea Acetate Ethyl alcohol Methanol Fructose Pyruvate to use organic substances as sole carbon sources for growth. To test this possibility, the organism was inoculated into a nitrite-salts medium that was aerated with CO2-free air. In the absence of bicarbonate, there was no nitrite disappearance, and the concentrations in all flasks remained at 320 to 330 jig of nitrite-nitrogen per ml. By contrast, the bacterium grew well if the medium contained bicarbonate, and the nitrite was metabolized (Table 1). However, even in this bicarbonate-fortified medium, the rate of nitrite oxidation was not stimulated by the addition of any of the organic compounds. Thus, these organic substances are apparently unable to serve as sole carbon sources for growth and nitrite oxidation. The rate of nitrite oxidation by cell-free preparations was likewise not enhanced by formate, oxalate, acetate, malate, succinate, pyruvate, citrate, glyceraldehyde, urea, or fructose. The cell extracts were incubated in Warburg flasks with 25,pmoles of nitrite, 50 Amoles of phosphate, and 25,moles of the test compound. At these concentrations, formaldehyde, ethyl alcohol, and methanol entirely abolished nitrite oxidation. Permeability to organic compounds. In a relatively short period of time, N. agilis incubated in reaction mixtures containing acetate-1-c'4 incorporated the label into the cells. Commonly, the maximal absorption of acetate was attained in less than 30 min, and no further uptake of C14 was found upon prolonged incubation. Indeed, a slight loss of radioactivity was noted from cells incubated for more than 60 min (Fig. 1). Glycine, although not assimilated as readily as acetate, was also incorporated in appreciable amounts by N. agilis. Hypoxanthine and glycerol were also readily taken up by the autotroph, the radioactivity in the bacterial pellet increasing progressively with time. By contrast, the uptake of C'4 by N. agilis cells supplied with labeled pyruvate, xanthine, alanine, formate, and methanol was relatively small (Fig. 2). The failure of the organism to retain the radioactivity from formate is rather surprising in view of the slight, but apparently significant, oxygen uptake noted in the presence of formate, a finding reported earlier by Silver (1960). In spite of the structural relationship of xanthine and hypoxanthine, the former w C i GLYCINE oacetate -1-C14/ TIME (MIN) H.POXANTHINE GLYCEROL FIG. 1. Uptake of labeled compounds by Nitrobacter agilis cell suspensions. The reaction mixture contained 50,moles of phosphate (ph 7.2), 0.20,uc of substrate, and 0.50 to 1.1 mg of cells per ml. -J -J w C. (0 0. C.) TIME (MIN) FIG. 2. Uptake of labeled compounds by Nitrobacter agilis cell suspensions. The reaction mixture contained 50 Amoles of phosphate (ph 7.2), 0.20,uc of substrate, and 0.50 to 1.1 mg of cells per ml.

4 154 IDA AND ALEXANDER J. BACTERIOL. was taken up by the cells far more slowly than the latter. A comparison was made of the uptake of acetate-i-c'4 with that of acetate-2-c'4 to determine whether there was a preferential absorption of carbon from the carboxyl group. The activity of both methyl- and carboxyl-labeled acetate was taken up readily by the cells, but the results presented in Table 2 demonstrate that there was little difference in the absorption pattern, particularly in view of the 68% higher specific activity of the methyl-labeled substrate. The absorption of acetate was markedly affected by the temperature of the reaction mixture. Thus, the rate of labeling of cells incubated at 4 C with acetate-i -C14 was only about 15% of that of a suspension incubated at 25 C. This suggests that the uptake at the higher temperature resulted largely from an enzymatically effected incorporation rather than nonenzymatic adsorption. Acetate incorporation. Because of the rapid uptake of acetate by the autotroph, the fate of the carbon derived from this compound was investigated further. The radioactivity after 20 min of incubation was largely associated with the lipid constituents of the organism (Table 3). A lesser amount of the acetate-carbon was recovered in the amino acid-containing fraction, whereas only a relatively minor proportion of acetate was incorporated into the organic acid-containing and neutral fractions. Despite the small quantity of acetate-carbon taken up during the 20-min test period, these observations demonstrate that at least one exogenously supplied organic compound, acetate. is metabolized. Chromatography of the amino acid-containing fraction revealed a number of ninhydrin-reactive spots, and a scan of the TABLE 2 Comparison of absorption of acetate-i-c14 with that of acetate-2-c14 by Nitrobacter agilis* Time Acetate-i-C'4 Acetate-2-C'4 min count/min count/min 5 3,230 4, ,275 4, ,512 6, ,449 7, ,562 7, ,212 7, ,037 6,720 * The reaction mixture contained (per milliliter): 1.0 mg of cells, 50,umoles of phosphate buffer (ph 7.2), and 0.20,uc of carrier-free acetate. The values represent the radioactivity equivalent to 1.0 mg of cells. TABLE 3. Incorporation of acetate-2-c'4 into major cellular fractions of Nitrobacter agilis* Fraction Activity Percentage distribution count/min Lipid... 30, Amino acids... 8, Organic acids... 2, Neutral... 1, Total... 42, * The data refer to the total radioactivity in cell fractions obtained from a reaction mixture containing 314 mg of cells, 1.0 mmole of phosphate buffer (ph 7.4), and 20,c of acetate-2-c'4 in a total volume of 20 ml. paper chromatograms revealed significant radioactivity associated with the glutamate and aspartate spots. Glutamate in particular showed considerable incorporation of the label. Nitrite did not enhance the rate of incorporation of the organic compounds. For example, C'4 uptake from labeled pyruvate or methanol (1.0 jac/ml) and from xanthine or hypoxanthine (0.2,uc/ml) by N. agilis cell suspensions (2.14 and 1.28 mg of cells per ml, respectively) was not stimulated by the addition of nitrite, although the nitrite was rapidly oxidized under the experimental conditions. DISCUSSION Although the most striking characteristic of obligate chemoautotrophs is their reliance upon inorganic molecules as sources of carbon and energy, the inability of these organisms to make use of the energy or carbon provided in exogenous organic compounds has never been adequately explained. Nitrobacter is particularly suited for such investigations inasmuch as it is restricted to one energy source, nitrite, and a single carbon source, carbon dioxide. The data presented here demonstrate that a number of inorganic ions and organic compounds would neither serve as energy sources for growth nor as substrates for oxidation by intact cells or extracts of N. agilis. The results also show the failure of the organism to make use of organic compounds as sole carbon sources. However, Delwiche and Finstein (1965) demonstrated that not only is N. agilis permeable to certain organic acids, but that the extent of growth is increased by them, provided the bacterium is supplied with nitrite and carbon dioxide. As a first approximation, it can be propose(d that any one of three physiological characteristics

5 N' OL. 90, 1965 PERMEABILITY OF N. AGILIS 155 may account for the unique behavior of this autotroph in regard to the few exogenous substrates available to it: (i) a permeability barrier to organic molecules, effectively a hindrance to entry of all but a few substances; (ii) the absence from the cell of the appropriate enzymes to metabolize or oxidize permeable organic molecules; and (iii) the inability of the organism to couple phosphorylation with, or get, sufficient energy from the oxidation of organic compounds penetrating into the cell. The results presented herein and by Delwiche and Finstein (1965) demonstrate that the first hypothesis is untenable in view of the permeability of N. agilis to certain labeled organic compounds. Thus, there is a portal of entry for organic carbon. Certain strains among the thiobacilli likewise are unable to use organic materials in place of sulfur as sole energy sources or in place of carbon dioxide as the sole carbon source, although a number of carbon compounds stimulate respiration or sulfur oxidation, and several penetrate the cell surface (Vishniac and Santer, 1957; Vishniac and Trudinger, 1962; Vogler, LePage, and Umbreit, 1942). Hence, for Thiobacillus as well, the restriction to chemoautotrophy is not a result of the lack of permeability of the cell to organic compounds. The conversion of exogenously supplied acetate into a variety of other compounds in the cell indicates that the autotroph is indeed capable of metabolizing substrates provided from without, and at least some of the enzymes for organic metabolism are present. This is not at all unexpected in view of the known biochemical pathways and cellular constituents in the bacterium (Malavolta, Delwiche, and Burge, 1960). It is of interest in this regard that no stimulation of nitrite oxidation by acetate was observed in the present investigation, whereas Delwiche and Finstein (1965) noted that the N. agilis population was greater in nitrite media containing acetate than in solutions free from organic carbon. Since neither permeability barriers nor the absence of enzymes capable of metabolizing exogenously supplied organic substrates accounts for the specificity of the bacterium for nitrite and carbon dioxide, it seems likely that the obligate chemoautotrophic habit is associated either with the absence of the appropriate oxidizing enzymes or with the inability of the organism to derive sufficient energy to sustain growth from the organic oxidations that it can perform. It may be that the obligate autotrophic habit is associated with the inability of the microorganism to couple phosphorylation to a significant extent with the oxidation of any other substrate than nitrite, an ion which is not only a rare energy source but also is characterized in the nitrite-nitrate couple by an Eh uncommonly high for growth substrates. Thus, the failure of acetate to support growth of the bacterium, even in a medium exposed to a gas phase containing sufficient carbon dioxide to allow for nitritelinked growth, may be associated with the inability of the organism to oxidize the organic acid and the products formed from it, or to gain sufficient energy by the oxidations to sustain growth. The relationship of acetate to the metabolism and photoautotrophy of algae and photosynthetic bacteria has already been considered (Elsden, 1962; Eppley, Gee, and Saltman, 1963). ACKNOWLEDGMENTS This investigation was supported by National Science Foundation grants G18480 and GB2321, and by Cooperative Regional Research Project NE-39. We are grateful to R. E. MacDonald for his counsel. We also thank G. E. Shattuck, Jr., for his valuable assistance. LITERATURE CITED ALEEM, M. I. H., AND M. ALEXANDER Cellfree nitrification by Nitrobacter. J. Bacteriol. 76: ARNON, D. I Microelements in culturesolution experiments with higher plants. Am. J. Botany 25: BASSHAM, J. A., AND M. CALVIN The path of carbon in photosynthesis. Prentice-Hall Inc., Engelwood Cliffs, N.J. DELWICHE, C. C., AND M. S. FINSTEIN Carbon and energy sources for the nitrifying autotroph Nitrobacter. J. Bacteriol. 90: ELSDEN, S. R Assimilation of organic compounds by photosynthetic bacteria. Federation Proc. 21: EPPLEY, R. W., R. GEE, AND P. SALTMAN Photometabolism of acetate by Chlamydomonas mundana. Physiol. Plantarum 16: MALAVOLTA, E., C. C. DELWICHE, AND W. D. BURGE Carbon dioxide fixation and phosphorylation by Nitrobacter agilis. Biochem. Biophys. Res. Commun. 2: MORRILL, L. G An explanation of the nitrification patterns observed when soils are perfused with ammonium sulfate. Ph.D. Thesis, Cornell University, Ithaca, N.Y. REMSEN, C. C., AND D. G. LUNDGREN The heterotrophic growth of the chemoautotroph Ferrobacillus ferrooxidans. Bacteriol. Proc., p. 33. SILVER, W. J Exogenous respiration in Nitrobacter. Nature 185: VISHNIAC, W., AND M. SANTER The thiobacilli. Bacteriol. Rev. 21: VISHNIAC, W., AND P. A. TRUDINGER Sym-

156 IDA AND ALEXANDER J. BACTERIOL. posium on autotrophy. V. Carbon dioxide fixation and substrate oxidation in the chemosynthetic sulfur and hydrogen bacteria. Bacteriol. Rev. 26:168-175. VOGLER, K.

6 156 IDA AND ALEXANDER J. BACTERIOL. posium on autotrophy. V. Carbon dioxide fixation and substrate oxidation in the chemosynthetic sulfur and hydrogen bacteria. Bacteriol. Rev. 26: VOGLER, K. G., G. A. LEPAGE, AND W. W. UMBREIT Metabolism of autotrophic bacteria. I. The respiration of Thiobacillus thiooxidans on sulfur. J. Gen. Physiol. 26: ZAVARZIN, G. A The incitant of the second phase of nitrification. IV. The dehydrogenase activity of a washed suspension of Nitrobacter winogradskyi. Mikrobiologiya 29:

colorimetrically by the methylene blue method according to Fogo and manometrically. In the presence of excess sulfur the amount of oxygen taken up

colorimetrically by the methylene blue method according to Fogo and manometrically. In the presence of excess sulfur the amount of oxygen taken up GLUTA THIONE AND SULFUR OXIDATION BY THIOBACILLUS THIOOXIDANS* BY ISAMU SUZUKI AND C. H. WERKMAN DEPARTMENT OF BACTERIOLOGY, IOWA STATE COLLEGE Communicated December 15, 1958 The ability of Thiobacillus