Growth of Pseudomonas Aeruginosa on Nitrous Oxide DENNIS A

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Growth of Pseudomonas aeruginosa on Nitrous Oxide DENNIS A. BAZYLINSKI, CARLTON K. SOOHOO, AND THOMAS C. HOLLOCHER* Department ofBiochemistry, Brandeis University, Waltham, Massachusetts 02254 Received 16 October 1985/Accepted 24 February 1986

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N20 in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N20. Growth on N20 was cleanly blocked by acetylene. Cell yields, CO2 production, and N20 uptake all increased with initial PN2O at PN2O ' 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N20 as the sole oxidant. N20 was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N20 efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N20 had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N20 neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N20 when small and growth-limiting concentrations of nitrate or nitrite (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N20 was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N20. It would seem that nitrate, nitrite, or a metabolic product can stimulate strain P2 (but not PAO1 or P1) to grow abundantly on exogenous N20. The phenotype of strain P2 suggests that the ability of strains PAO1 and P1 to grow on endogenous N20 may also be under control of nitrate or a metabolic product of nitrate. The metabolic defect that prevents abundant growth of strains PAO1 and P1 on exogenous N20 was not traceable to dysfunction of the respiratory proton pump, high proton permeability of the membrane, failure to form or maintain a proton motive force, or a nutritional requirement. The metabolic defect is not understood at present.

Although many N2-producing denitrifying bacteria, such reducing activity (25). N20 appears not to be highly toxic to as Pseudomonas denitrificans, P. stutzeri, P. perfec- P. aeruginosa, because it can grow on nitrate under 1 atm (= tomarinus, and Paracoccus denitrificans, grow vigorously 109.29 kPa) of N20 (4, 6). on exogenous N20 (4, 6, 15) P. aeruginosa appears to be an In this report we re-examine with improved methods the exception (4-6, 25; B. A. Bryan, Ph.D. thesis, University of apparent inability of P. aeruginosa to grow on exogenous California, Davis, 1980). Growth of P. aeruginosa on N20 N20 and find that the organism can in fact grow to the extent has not been reported previously. Another possible excep- of two to three doublings. The nature of the defect that tion is Aquaspirillum magnetotacticum, a magnetic bacte- prevents abundant growth on N20 was investigated but rium, which reduces nitrate to N2 in growing cultures but remains obscure. We also observe that growth of one strain does not appear to grow on exogenous N2O (1; D. A. of P. aeruginosa on N20 can be enhanced considerably by Bazylinski and R. P. Blakemore, unpublished results). Not nitrate or nitrite. only can N2-producing denitrifiers generally grow on N2O, but their molar growth yields on nitrate, nitrite, and N20 are MATERIALS AND METHODS proportional to the oxidation number of nitrogen in these N-oxides (4, 15, 26; Bryan, Ph.D. thesis). Thus growth Bacteria and cultures. Three strains of P. aeruginosa were yields per electron are virtually identical among these N- used in this study: strain PAO1 was supplied by B. W. oxides. Moreover, the 'H+/2e- ratios measured in oxidant Holloway, Monash University, Clayton, Victoria, Australia; pulse studies of N2-producing denitrifiers are similar among strain P1 was isolated from the infected ear of a domestic dog these N-oxides (3, 7, 16, 18) and with NO as well (8); and the and characterized at the Department of Microbiology, N-oxides, including NO, support in these cells the active University of New Hampshire, Durham, from which it was transport of L-proline (8, 27), the uptake of which is driven obtained; strain P2 was isolated at Brandeis University from by the proton motive force (19). The failure of P. aeruginosa the infected ear of an adult human female. Strain P2 is an to grow on N20 is particularly puzzling, inasmuch as N20 asporogenous gram-negative rod with single polar flagella, has been shown to be an obligatory, freely diffusible inter- positive for oxidase, catalase, gelatinase (slow), and mediate in the reduction of nitrate or nitrite by nongrowing assimilation of acetamide and able to grow at 42°C but not at cells (25), and this endogenous N20 would appear to be used 4°C (2). Growth was obligately aerobic for all sugars, for growth with the same efficiency applicable to other carbohydrates, and media tested. Although it produced denitrifiers and other N-oxides (Bryan, Ph.D. thesis). In soluble fluorescent yellow-green pigment, it failed to produce addition, denitrifying P. aeruginosa has considerable N20 pyocyanin or other chloroform-soluble pigments on several media, including that of King et al. (14). These and other characteristics indicate that P2 is a strain of P. aeruginosa * Corresponding author. unable to produce pyocyanin. It is clearly distinguished from 1239 1240 BAZYLINSKI ET AL. APPL. ENVIRON. MICROBIOL. the various biotypes of P. fluorescens and P. putida (2, 12, ments, using N20 as oxidant. The most decisive control was 24). Isolation of apyocyanogenic strains of P. aeruginosa is the use of acetylene which cleanly blocks reduction of N20 not uncommon, particularly from clinical sources (12, 24). by the nitrous oxide reductase of denitrifiers but has no Two other species of N2-producing denitrifying bacteria, effect on reduction of 02, nitrate, or nitrite (29; Bryan, Ph.D. Paracoccus denitrificans ATCC 19367 and Pseudomonas thesis). In addition, P. aeruginosa produced fibrous macro- denitrificans ATCC 13867, were used as reference organisms scopic aggregates of cells when grown on 02- Cells were to exemplify performance of bacteria which can grow largely monodispersed when grown on N-oxides under abundantly on N20 at 1 atm. Strains PAO1 and P1 were used anaerobic conditions. Over the course of 1 to 3 days, cells of to show that PAO1 was not a unique phenotype. At present, strains PAO1 and P1 growing on 02 were also observed to strain P2 exhibits a unique phenotype. The bacteria were produce pigment (24). Cultures under N20 did not exhibit maintained aerobically at pH 7 and 30°C on undefined medium any of these criteria to suggest leakage of 02 during growth which contained the following (grams per liter): yeast extract, experiments. 6; Bacto-Peptone (Difco Laboratories, Detroit, Mich.), 3; Survival of cells under N20 over prolonged periods. Cul- KH2PO4, 1; K2HPO4, 1.5. tures under N20 were often incubated for up to 7 days at To obtain inocula for the anaerobic growth experiments 30°C. The viability of P. aeruginosa under such conditions described below, cells were grown semianaerobically from was checked qualitatively when incubation was ended by single colonies under nitrate-limiting conditions (10 mM plating on nutrient agar plates and by the rate at which cell KNO3) at 30°C in a succinate-salts minimal medium (4). The growth resumed following addition of nitrate or admission of suggested trace mineral solution for this medium was re- air to cultures at the end of the experiment. Cells were placed by a modified mineral solution of Wolin et al. (1, 28) judged to be viable if growth resumed without delay at the that supplied 11 trace elements commonly required for rate expected for fresh cells. By these criteria, cultures ofP. bacterial growth. The term "semianaerobically," used aeruginosa were judged to be largely if not entirely viable for above, refers to cultures which contained dissolved 02 at the periods of at least 1 week in the succinate-salts medium time of inoculation but became essentially anaerobic within under N20. a few hours due to a combination of aerobic respiration and Tests for a nutritional requirement to promote growth on N2 production. Such cultures were neither shaken nor N20. Experiments were carried out with 100 ml of the stirred during growth. Cells were judged to be competent in succinate-salts medium in 250-ml Erlenmeyer flasks. Sterile denitrification and suitable for use as inocula when N2 medium was sparged with N20 for a time sufficient to evolution was vigorous and just after the nitrate and nitrite remove dissolved 02 and then inoculated by injecting 1 ml of had become exhausted. These first-stage cultures were used a first-stage culture. A flow of N20 of 30 ml min-' was to inoculate (1.8%, vol/vol) second-stage cultures, which are maintained during the subsequent period of incubation at identical to the former, except that they were made highly 30°C (usually 48 h). Positive growth controls included me- anaerobic and sealed prior to inoculation as described be- dium supplemented with nitrate or under air and Pseudomo- low. Cells from second-stage cultures were judged to be nas or Paracoccus denitrificans which grows well on N20 at competent and suitable for use as inocula if N2 evolution 1 atm. The negative growth control was the culture under toward the end of growth had been vigorous and when flowing argon. The nutrient supplements tested were (per nitrate, nitrite, and N20 were exhausted. liter): hemin at 1 mg; Asolectin at 50 jig after saponification; Anaerobic growth experiments. Anaerobic growth experi- Tween 80 at 3 ml; the vitamin elixer of Wolin et al. (28) at 10 ments were carried out with 55 ml of anaerobic succinate- ml; vitamin K (in ethanol) at 10 mg; 20 amino acids at 20 mg salts medium in stoppered 155-ml serum vials. The stoppers each. Asolectin is purified soya bean phospholipids, and used were gas-impermeable black rubber stoppers (no. Tween 80 is a nonionic detergent commonly used as a source 2048-11800; Bellco Glass, Inc., Vineland, N.J.) (see refer- of unsaturated fatty acids for bacterial growth. In addition to ence 17) which were secured with crimped aluminum rings to these defined supplements, undefined medium, such as yeast render the seals autoclavable. Medium was sparged with extract or Bacto-Peptone (Difco), was also added in some 02-free argon for 1 h at 500 ml min-' and then allowed to experiments at 3 to 4 g liter-'. The vitamin elixir supplied equilibrate overnight at 4°C with an atmosphere of 90% biotin, folic acid, pyridoxine, riboflavin, thiamine, nicotinic N2-10% H2 (02 at about 5 ppm [5 ,ul liter-'] in an anaerobic acid, pantothenic acid, vitamin B12, p-aminobenzoic acid, chamber (Coy Laboratory Products Inc., Ann Arbor, and thioctic acid. The amino acid mixture supplied all 20 of Mich.). Medium was added to the vials and the vials were the commonly occurring amino acids, including L-cysteine sealed in the anaerobic chamber. N20 or C2H2 or both were and L-methionine as the sulfur-containing amino acids. added aseptically by injection after autoclaving. Finally, the Estimation of growth and final cell yields. Growth and cell vials were inoculated by injection of 1.0 ml of a second-stage yields were estimated by absorbance and, where feasible, by culture per vial. Incubation was at 30°C in a water bath dry cell weights. A660 of cultures was measured in 1-cm shaker set at 100 rpm. In some experiments, nitrate or nitrite cuvettes with a Perkin-Elmer Lambda 2 spectrophotometer. (10 mM) was injected in place of or in addition to N20. Dry cell weights were determined by weight differences after Because nitrite at concentrations above 2 mM inhibited having filtered culture samples through 0.2-,um polycarbon- growth of P. aeruginosa, anaerobic KNO2 solution was ate filters (Nucleopore Corp., Pleasanton, Calif.) and dried added in increments of 2 mM during growth. Injection was the filters to constant weight at 100°C. A linear relationship made only when the concentration of nitrite from the previ- was established between cell dry weight and absorbance ous increment had reached or approached zero. Anaerobic with slope corresponding to 0.59 (PAO1 and P1) and 0.65 growth experiments were performed in triplicate, and one (P2) mg of cell dry weight ml-1 (absorbance unit)-1 for P. culture in each triplicate set was streaked onto a defined aeruginosa. These slopes are very similar to the one re- medium or nutrient agar plate to check for contamination at ported previously for strain PAO1 (4). Cell yields were the conclusion of each experiment. Contamination was not proportional to initial concentrations of nitrate of at least up observed to have occurred in this study. to 10 mM for all denitrifiers used in this study. Avoidance of oxygen leakage artifacts in growth experi- Proton translocation in oxidant pulse experiments. VOL. 51, 1986 GROWTH OF P. AERUGINOSA ON N20 1241

Denitrifying bacteria were grown at 30°C on 10 mM KNO3 in (Matheson Scientific, Inc., Elk Grove Village, Ill.) by pas- undefined medium, harvested by centrifugation, washed sage through an alkaline pyrogallol train (9). Acetylene twice with cold 150 mM KCI, and suspended in the same at (Matheson) was prepurified grade. about 3 x 1010 cells ml-' (about 6 mg of protein ml-'). The apparatus used, technique, and method of data analysis for oxidant pulse experiments were as previously described (16, RESULTS AND DISCUSSION 18, 21). Because valinomycin-K+ proved to be ineffective as Anaerobic growth of P. aeruginosa on N20. Perhaps the a permeant ion for P. aeruginosa strains PAO1 and P1, salient result of these studies is that P. aeruginosa can grow presumably due to an inability to penetrate the outer mem- reproducibly on N20, contrary to current opinion (4-6, 25; brane (7, 18), the permeant ion used throughout was Bryan, Ph.D. thesis). Thus, the difference between P. aeru- methyltriphenylphosphonium cation at 10 mM (7, 22). An ginosa and other denitrifying bacteria which can reduce ethanol solution of carbonyl cyanide m-chlorophenylhy- nitrate or N20 to N2 is quantitative rather than qualitative. drazone (CCCP) was used as an uncoupler (21, 27) and Figures 1 and 2 and Table 1 illustrate some features of KSCN was used to block N20 reduction (18). growth on N20. (i) Growth, N20 uptake, and CO2 produc- Active transport of proline. Cells were grown in defined tion (Fig. 1) are clearly inhibited by acetylene which is medium and harvested as in the preceding section, washed known to inhibit nitrous oxide reductase specifically. This twice with cold 50 mM potassium phosphate buffer (pH 7.0), establishes that growth depends on N20 reduction and is not and suspended in 50 mM potassium phosphate-10 mM due to 02 contamination. (ii) Growth depends upon addition sodium succinate (pH 7.0) at about 2 x 1010 cells ml-'. of N20 and does not occur in its absence (Fig. 1 and 2). This L-[14C]proline (1.2 nmol) and oxidant (20 pLmol of nitrate, rules out the possibility of carryover of an N-oxide in the nitrite, or N20) were injected under anaerobic conditions in inoculum or an unexpected fermentative pathway for suc- that order into 1 ml of cell suspension at 30°C. The mixture cinate, which is the sole carbon source in the defined was stirred vigorously by means of a small magnetic stirring medium. (iii) Growth is not logarithmic (Fig. 1) as it is when bar. At intervals, 25-,ul aliquots were removed by syringe 02 or nitrate serves as oxidant. Rather, the rate of growth and the cells were separated, washed, dried, and assayed for decreases with time. Typically something of the order of 14C by the rapid filtration method of Lieberman and Hong one-third of the total growth occurs within 1 day, although (19) as adapted for denitrifying bacteria (27). Uptake of subsequent growth may continue for 5 to 8 days. (iv) Cell proline through anaerobic respiration proceeded without a yield, N20 uptake, and CO2 production depend upon the detectable lag and typically reached a steady-state level amount of N20 available for growth (Fig. 2). With amounts within 6 min after the reaction was initiated (8, 27). below 400,umol of N20 vial-' (initial PN,O = 0.1 atm) the Analyses. Nitrite was determined colorimetrically (23). N20 was quantitatively reduced to N2 in the case of strains Nitrate was determined by the nitrite assay after its reduc- PAO1 and P1 and cell yield increased as the amount of N20 tion to nitrite by zinc metal. N20 and CO2 were sampled by increased (with strain P2, N20 uptake was quantitative means of gas-tight syringes (series A-2; Precision Sampling below 270Fmol of N20). In this low range of PN20, CO2 Corp.) and measured by gas chromatography with a production also increased with increasing cell yield. Above Shimadzu GC-9A gas chromatograph equipped with a ther- 400 ,umol of N20, the cell yield and CO2 production de- mal conductivity detector and a Porapak Q column (2.4 m by creased somewhat. From qualitative or semiquantitative 3.2 mm). Carrier gas was helium at 30 ml min-1, and the analyses it is clear that N20 uptake also decreased in the operating temperatures were as follows: detector, 140°C; range of 600 to 4,000 pLmol of N20. The inaccuracy in the column, 30°C; injector, 50°C. Areas under peaks were esti- determinations comes from the fact that only a small fraction mated with a Shimadzu C-RiB Chromatopac integrator and of the total N20 is reduced to N2 above 400 p.mol of N20 standard curves were prepared with pure gases. The amount vial-'; thus the determination depended on the difference of CO2 or N20 in a system was the sum of that in the between two large numbers. Because N2O was the sole headspace plus that in solution. The latter was calculated oxidant, the production of little CO2 clearly implied reduc- from the respective aqueous solubility constants and Hen- tion of little N2O. Acetylene at 0.1 atm inhibited growth and ry's law (20). Bicarbonate in solution was estimated by using CO2 evolution at 4,000,umol of N20 as cleanly as it did at 400 an apparent pK of 6.37 for CO2 (10). Total CO2 was the sum ,umol (data not shown). (v) The growth yield per electron of CO2 and bicarbonate. equivalent of N20 (2 equivalents mol-1) at 136,umol of N20 The amounts and 15N content of N20 and N2 in the (Table 1) was nearly equal to that per electron equivalent of headspace of cultures were determined by means of a nitrate (5 equivalents mol-1), but the cell yield on N2O Hewlett-Packard 5992A gas chromatograph/mass spectrom- relative to that on nitrate decreased with greater amounts of eter equipped with a Porapak Q column (2.4 m by 3.2 mm) N20. Thus, growth on 400,umol of N20 (800 micro-electron operating at 40°C (9). Samples of 0.15 ml were expanded into equivalents) gave a growth yield only 60% that expected for a gas chromatography sample loop as previously described 800 micro-electron equivalents of nitrate. (vi) Growth on (13). Absolute amounts of gases were estimated by use of N2O never exceeded the equivalent of two to about three external standards. The amount of gas in solution was replications (three to ninefold increase in cell density). A estimated by use of Henry's law as described above. direct comparison is made between P. aeruginosa (Fig. 1) Reagents. Amino acids, valinomycin, carbonic anhydrase, and Paracoccus denitrificans (Fig. 3). The growth limit CCCP, hematin, and methyltriphenylphosphonium bromide of Paracoccus denitrificans on N20 is an A660 of about were from Sigma Chemical Co. (St. Louis, Mo.). L- 1.4. [14C]proline (250 Ci mol-1) was from ICN Radiochemicals In some cases, P. aeruginosa that had grown on 4,000 (Irvine, Calif.), [15N]NaNO3 (99 atom %) was from KOR ,umol (1.0 atm) of N20 per vial was used to inoculate Isotopes (Cambridge, Mass.), Asolectin was from Associ- identical cultures. Cell yields from second serial cultures ated Concentrates (Long Island, N.Y.), undefined media were smaller than those from the inoculum. From these and were from Difco, and Tween 80 was from Fisher Scientific other experiments, we have no evidence currently to suggest Co., Pittsburgh, Pa. Trace 02 was removed from N20 that serial transfer of P. aeruginosa on N2O or very long 1242 BAZYLINSKI ET AL. APPL. ENVIRON. MICROBIOL. A B

0.10 0.10 400

E 0.08 E-6 0.08 300 _0 0 lav u 0 E 0.06 E 0.06 b0 0 a. S 0 N U 4 200 0 V -) S 0_o ._>- 0.04 0 ';- 0.04 C. n Iz0 0 100 0 0.02 0.02 z

0 0 0 0 2 3 4 5 6 7 8 0 1 2 3 4 5 6 Days Days c 0.10

0.08 E 300 -

- 0

V t 0.06 0 0 0 0. 0 4 200 o C-) c of P. strains PAO1 (A), P1 (B), and 0 FIG. 1. Growth aeruginosa oj 0.04 P2 (C) on N20. Symbols: Solid lines, 0.1 atm of N20; dashed lines, 0.1 atm of N20 plus 0.1 atm of acetylene; broken lines, no gaseous additions. Broken lines for CO2 production were omitted for clarity in (A) and (B), inasmuch as they essentially superimposed on the 100 0 dashed lines. N20 uptake and CO2 evolution in the acetylene and 0.02 zN minus-N20 controls were not recorded in the experiment of (C). Other experiments similar to that of (C) show that N20 uptake and CO2 evolution in those controls were negligible. CO2 refers to CO2 plus bicarbonate. Cells were incubated anaerobically at 30°C in 155-ml serum vials as described in the text. The above pressures of O 0 0 1 2 3 4 5 6 7 N20 and acetylene refer to the initial pressures before equilibration Days with the aqueous phase; thus, 0.1 atm = 400 ,umol of a gas.

incubations (weeks) with N20 will induce an ability to grow experimnental variation in the range 0.1 to 1.0 atm of N20 is abundantly on high partial pressures of N20. believed to be related to differences of a few hours in the age The results illustrated in Fig. 2 are typical of individual of inocula. experiments in which triplicate cultures give reasonably A number of possibilities were entertained to rationalize small standard deviations, but are not entirely representative the curious growth characteristics of P. aeruginosa on N20. of the picture presented from a large number of such These included a nutritional deficiency (i.e., the inability to experiments. We believe that the maximum in cell yield, synthesize an essential component but only when N20 N20 uptake, and CO2 production occurs at about 0.1 atm served as the oxidant in respiration), failure of the respira- (0.07 atm with strain P2) and that these parameters decrease tory proton pump or collapse of the proton motive force more or less smoothly to the values seen at 1 atm. Thus, the when reducing N20, a direct inhibitory action of N20 on maximum is not particularly prominent. The reason for growth, and the possibility that N20 is not an obligatory free VOL. 51, 1986 GROWTH OF P. AERUGINOSA ON N20 1243 0.10 r 400 _ A Eo TABLE 1. Observed and expected final cell yields of P. :t aeruginosa strains PAO1 and P1 and Paracoccus denitrificans 0 when grown with N20 as sole oxidant at 30°C 0.08 F Cell yields (A66o) 300 D Incuba- N20 added tion Observeda Expected 0E (p.mol) time (either 02 m Strain PA01 Strain P1 strain)b 0 0C) 06 0.06 F 0 N P. aeruginosa 200 0 7 days 0.009 ± 0.002 0.006 ± 0.001 0.009c z 136 1-1.5 0.033 ± 0.001 0.033 ± 0.003 0.035 I days 0.04 1 I I ± 0.050 ± 0.001 0.070 o 272 2-3 0.052 0.000 C.) A II ar-i2 days [I N 1-1 1-1 XN 408 5-7 0.066 ± 0.001 0.063 ± 0.001 0.104 1-1 100 n 1-1 E days 0.02 I 1-1 o. 1-1 ri O E.) "I 0 a. Paracoccus I ,l + 1-1 .11 denitrificansd I., 408 18-20 h 0.24 0.23 0 la 0 1,224 18-20 h 0.32 0.35 o &O IC 0 0 0 N IC 2,041 18-20 h 0.56 0.59 o o2 o 0 0 o 0 0 0 a Values for PA01 and P1 represent means ± standard deviations with Initial Partial Pressure N20,atm triplicate cultures; values for Paracoccus denitrificans represent a single determination. See text for description of anaerobic growth experiments. 0.10 400 Cultures were incubated until the N20 was exhausted. b B EE6 Based on the observation that cultures grew to an Aw0 of 0.35 in 20 h with :. 10 mM nitrate as the sole oxidant and the assumption that nitrate will contribute 5 electron equivalents mol-' to growth-related respiration and N20 *- will contribute 2 electron equivalents mol-1. See text. It was observed that N2 0.08 !q was the sole final product of reduction of these two N-oxides by P. 300 n aeruginosa. E c Absorbance immediately following inoculation. c d As footnote b, except that A60 = 0.79 in 20 h with 10 mM nitrate. The S c..o absorbance of Paracoccus denitrificans in cell suspensions was found to be o 0.06 o about twice that for P. aeruginosa per unit of dry weight and probably reflects 0 0 z cell size differences between the organisms. .4 200 V

.-I 0 .11 >0 004 1.1 Possibility that N20 directly inhibits growth. The anaerobic .-I I I 0 "I .U growth experiments summarized in Table 2 show that N20 at .11 CY) 11 I., E 1 atm does not inhibit growth of PAO1 and P1 on nitrate and 1.1 .-I o "I may increase the cell yield slightly, although the increases .1, 0.02 11 O L were not highly significant. Typically the increases in cell 11 "I N "I yield were in the range of 5 to 10%. These results expand "I C. .-I upon confirm those of Carlson and Ingraham (6). "I o6 and largely I., .-I Growth of strain P2 on limiting nitrate or nitrite and 0 EL Jei exogenous N20 is, however, at least threefold greater than o v Oa 0 U) o o o o ft 0 - c e _ on nitrate or nitrite alone. More remarkable was the on0 oD 0 oo 0 that ci observation that strain P2 could grow on N20 for at least 24 Initial Partial Pressure N20,atm h when the N20 was added to the culture immediately after FIG. 2. Growth yields of P. aeruginosa strains PAO1 (A) and P1 the quantitative exhaustion of nitrate, nitrite, and N20 (if an to after (B) when grown on various amounts of N20. Blocks and bars any). Cultures typically grew to A660 of 0.75 0.80 represent means and standard deviations, respectively, of triplicate 24 h under anaerobic N20 which had been introduced to the cultures. Cells were incubated anaerobically at 30°C in 155-ml serum culture at an Aw0 of 0.20 immediately after growth on 5 mM vials, as described in the text. Incubation continued either until N20 nitrate had ceased. Similarly, strain P2 grew to an A660 of was exhausted (N20 pressures in the range of 0.034 to 0.1 atm) or for 0.225 in 48 h on N20 after the N20 had been added at an Aw0 1 week (N20 pressures > 0.1 atm). Negative growth controls were of 0.051 just after exhaustion of 2 mM nitrite. incubated as long as the experimental vials. Cultures containing Unsuccessful experiments were carried out on filter- as in .0.15 atm of N20 failed to consume all of the N20, discussed sterilized spent medium in an effort to detect a factor that the text. The pressures of N20 and acetylene refer to the initial would stimulate growth of fresh inocula on N20. pressures before equilibration with the aqueous phase; thus, 0.1 atm Possibility that N20 is not an obligatory free intermediate = 400 ,umol of a gas. The experiments depicted with the two strains were performed separately at different times. Initial A60 was about between nitrate and NI. In Table 2 it is shown that 0.1 atm of 0.008. acetylene lowers the growth yield of P. aeruginosa by about one-fifth when grown on nitrate and by one-third or slightly more when grown on nitrite. A sitnilar observation was made intermediate between nitrate and N2, so that growth (or its with P. aeruginosa and P. stutzeri grown on nitrate (4). In lack) on exogenous N20 may not be of general physiological our experiments, nitrate (or nitrite) was converted quantita- relevance. Investigations of these several possibilities are tively to N20, as determined by gas chromatography, in the summarized below. presence of acetylene. Because the reduction of nitrate, 1244 BAZYLINSKI ET AL. APPL. ENVIRON. MICROBIOL.

0.25 500 pool. (The transfer of "5N in each interval was equal to -A'4N20 observed ['5N20/'4N20 mol ratio].) (ii) The total amount of 15N to pass into the N20 pool {A15N20 observed - [A'4N20 observed (15N20/'4N20 mol ratio)] summed over all intervals to 18 to 19 h} agreed with the initial amount of 0.20 400 15N in nitrate at the outset. (iii) The maximum rate of E appearance of '5N2 coincided roughly with the maximum 15N20 content in the N20 pool, and the latter corresponded to the time of exhaustion of nitrate and nitrite. (iv) Aftet 0 exhaustion of nitrate and nitrite at 18 to 19 h, the rates of E 0.15 300 n disappearance of 15N20 and appearance of '5N2 become o ~~~~~~~0 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~00~~~~~~~~~~~~~~~~~.essentially equal. Because there was not detectable scram- bling of 14N20 and 15N compounds to form 14'15N20 (or 0 1415N2), the experiment also showed that N20 does not 0 equilibrate with any mononitrogen precursor of N20, 'in O 0.10 200 agreement with studies on nongrowing denitrifiers (25; 0 ~~~~~~~~~~~~~~~EGarber and Hollocher, Fed. Proc. 39:1773, 1980). Essen- 0 tially identical results were obtained with strain PA01. The 0 isotope studies and the decrease in cell yields caused by 0 acetylene argue strongly that N20 is produced quantitatively 0.05 100 from nitrate by P. aeruginosa and is used normally and efficiently for growth. Because endogenous 15N20 from ['5N]nitrate freely mixes with exogenous '4N20, the organism cannot in fact distinguish between endogenous and exogenous N20. 0 0 Possibility that respiration with N20 is not linked to proton 0 4 8 12 16 translocation. Oxidant pulse experiments, using 10 mM Hours methyltriphenylphosphonium cation as the permeant ion and FIG. 3. Growth, N20 uptake, and CO2 production by P. aeruginosa strains PAO1 and P1 grown with nitrate, Paracoccus denitrificans on 0.1 atm of N20 as the sole oxidant. with the usual Conditions were as in the legend to Fig. 1. Note that the unit of time resulted in transient proton translocation is hours, not days. Growth, N20 uptake, and CO2 were nil in the characteristics (7, 8, 16, 18), i.e., a rise time of about 1 s and presence of 0.1 atm of acetylene. passive recovery with half-times of about 2 min at room temperature. There was little or no difference between P. aeruginosa and the reference species (Pseudomonas nitrite, and N20 to N2 involves 5, 3, and 1 electron per N denitrificans and Paracoccus denitrificans also grown on atom, respectively, inhibition of N20 reduction during nitrate) with respect to passive proton permeability and the growth on nitrate or nitrite may be expected to decrease cell 'HI/2e- ratios. For example, the 'H+/2e- ratio was 6 to 7 yield by one-fifth or one-third, respectively. It has been for 02 and about 3 for N20 among the reference species and shown for several N2-producing denitrifiers (15, 26) that the cell yield per electron is very similar among nitrate, nitrite, and N20, and this is confirmed for nitrate and nitrite with P. aeruginosa (Table 2). The above results suggest that N20 is 0.4 a normal intermediate in the reduction of nitrate or nitrite by P. aeruginosa and that this endogenous N20 can be used efficiently for growth. . A more critical assessment of whether N20 is an obliga- E 150 0.3 tory free intermediate in the reduction of nitrate by P. -i aeruginosa was made by means of an isotope experiment. The experiment, which involves the trapping of 15N20 from z I [15N]nitrate in a pool of '4N20, had been used previously to &o oo100 0.2 1 0 show that N20 is indeed an obligatory free intermediate in 4. the reduction of nitrate or nitrite by nongrowing P. aerugi- z nosa (25) and other N2-producing, denitrifiers (E. Garber and T. C. Hollocher, Fed. Proc. 39:1773, 1980). Figure 4 sum- o 50 0.1 marizes an analogous experiment during growth of strain P1 z on [15N]nitrate plus 14N20. When P. aeruginosa grows anaerobically on 10 mM nitrate in defined medium, N20 is normally not detected in the headspace or, if detected, is 0 present in very small amounts (data not shown). In Fig. 3, it 0 4 8 12 16 20 24 can be seen that 15N appears in the N20 pool before it Hours appears in the N2 pool and that 15N20 appears in large FIG. 4. Denitrification of 15N03- in the presence of '4N20 during amounts. Moreover, the system showed those criteria ex- growth ofP. aeruginosa strain P1. The initial amounts of "5N03- (99 atom % of '5N) and N20 were 500 and 220 ,umol, respectively. The pected for a precursor-terminal product relationship be- anaerobic culture procedure and isotope methods were as described tween N20 and N2. (i) During each time interval, the in the text. Nitrate and nitrite were exhausted at 18 to 19 h. A6W was calculated transfer of 15N from the N20 pool into the N2 pool not a good measure of growth beyond 14 h because of formation of closely agreed with the observed increase of i5N in the N2 N2 bubbles. VOL. 51, 1986 GROWTH OF P. AERUGINOSA ON N20 1245

TABLE 3. Respiration-dependent active transport of proline by z Z(D O zDt D( denitrifying P. aeruginosaa H3 14C counts (10-3) min-' +z + + - + ni Oxidant (uncorrected for background)b _-^ ni Strain PAO1 Strain P1 B Pn 3 n rs 0- p,. - . o 3 oB3 0 Nitrate 9.6 9.0 0 0 Z oo o Nitrite 10.2 13.6 C> C) p) C N20 6.5 12.0 00w 0 Nitrate + 0.1 atm of acetylene 8.8 10.4 Nitrite + 0.1 atm of acetylene 9.7 13.7 -. C0 0> Z 1+ + 13 1+1+ N20 + 0.1 atm of acetylene 0.3 0.5 N20 + 10 ,uM CCCP 0.6 0.5 . 0. None 0.5 0.8 + O Pz No cells 0.05 0.05 Cs o 0 .0. a _ See text for a description of the method. a Q b Is.)sCD0 gQ = CQ Background counts averaged 50 min-'. The data apply to samples filtered c0. S0 6.0 min after initiation of the reaction. < 0 -. oo o oo0 _. 0 . . z o. 0 0- o 0o GO P. aeruginosa. Proton translocation was largely or entirely -5L z zw 0 abolished by 10 ,uM CCCP in all cases. That driven by N20 5C- 0- 0U was abolished by 150 mM KSCN (3, 18). So3 0Q_ cannot or 0. c _* Cs Possibility that the proton motive force develop be n O maintained. The observation above that the proton is about o z w + ~- -4 '5 as permeant to P. aeruginosa membrane as it is toward that -: 0 o 'I o) w of other denitrifiers makes the possibility that the proton C., motive force cannot develop or be maintained unlikely. OQ' o> o 0o Nevertheless, a more direct test is whether N20 can support Cs in P. aeruginosa the active transport of a substance, such as (S 1+ + 1+ 1+ so L-proline, the sequestration of which is known to be driven a O o) 00= 0 by the proton motive force (19). Proline uptake studies with 0 0 sO strains PAO1 an P1 are summarized in Table 3. The reduc- Cs 00 0 o tion of nitrate, nitrite, and N20 all supported proline uptake 1+ R - w ^ () in P. aeruginosa. Uptake supported by N20 was abolished 3 'm Cs -= F0 and that .J by acetylene CCCP; supported by nitrate and nitrite O I 1+ 1+ 1+ so was abolished by CCCP but not by acetylene. The results W0 3 < 0 with P. aeruginosa are similar to those reported for other * I 3_. I.0 I Cs denitrifying bacteria (8, 27). CS 5, Possibility of a nutritional requirement during growth on Cs _. N20. P. aeruginosa grew poorly under 1 atm of (flowing) O+ o ~-. N20 in both the defined medium and undefined media at 0 CDS- Cs 26°C. Typically the initial was about 0.005 and, after 48 (-) a 0 Aw 0z h, 0.03 or lower. None of the nutritional supplements used, WJ noI 0 alone or in combinations, stimulated growth on N20 and _3< o S several (vitamin K, Asolectin hydrolysate, and hemin) may I+ l+I+ _. have had an inhibitory effect. In control experiments, Pseu- o o 0 100 grew I+ domonas and Paracoccus denitrificans abundantly in 0z defined or undefined medium with N20. Little or no bubble 1+ 1+o was P. 0Z formation observed under these circumstances with I+ .+0I 0 aeruginosa. If P. aeruginosa requires a nutrient for abundant growth under N20, that nutrient would appear not be o o 0e 00w ON0 or q, among the vitamins, amino acids, lipids, etc., tested 0 Cs among the ingredients to be found in undefined media. 0 n Conclusions. The results of this study extend and in part OQ 0 o oN z Zz 1+ a 'm confirm those of Bryan and co-workers (4; Bryan, Ph.D. R R B_ 0 0 0 o a-1 thesis), St. John and Hollocher (25), and Carlson and O < 01 _ 0F 00 8 Ingraham (6) regarding the growth and energetics of P. 0 Cs aeruginosa on N-oxides. We establish that P. aeruginosa 00o oo can in fact on an observation hitherto and + + grow N20, missed, s that its growth is most efficient at low PN,O. Growth at higher W oo PN,O is poor, with cell yields being only a small fraction of those realized by Pseudomonas or Paracoccus denitrificans r) a(o5 under comparable conditions. The notable exception to this z z result is the observation that strain P2 can grow well on N20 0. I +- oC. O in the presence of (or for some time after the exhaustion of) modest concentrations of nitrate or nitrite. In addition, 1246 BAZYLINSKI ET AL. APPL. ENVIRON. MICROBIOL. isotope studies show that N20 is an obligatory, free inter- Biochem. Biophys. Res. Commun. 107:1504-1507. mediate in the reduction of nitrate during growth of P. 9. Garber, E. A. E., and T. C. Hollocher. 1982. '5N, 180 tracer aeruginosa, and cell yields in the presence and absence of studies on the activation of nitrite by denitrifying bacteria. J. acetylene confirm that endogenous N20 derived from nitrate Biol. Chem. 257:8091-8097. or 10. Gordon, A. J., and R. A. Ford. 1972. The chemists companion, nitrite is used for growth by P. aeruginosa as efficiently as p. 58. John Wiley & Sons, Inc., New York. it is by other N2-producing bacteria. Although the above 11. Hawk, P. B., B. L. Oser, and W. H. Summerson. 1954. Practical results with strains PAQ1 and P1 might be explained in part physiological chemistry, 13th ed., p. 1321. The Blakiston Co., by a general inhibition of growth by N20, no such inhibition Inc., New York. was observed in direct experiments, in confirmation of 12. Hugh, R., and G. L. Gilardi. 1980. Pseudomonas, p. 288-317. In previous observations (4, 6). Set against these results is our E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant inability as yet to discover a dysfunction in P. aeruginosa (ed.), Manual of clinical microbiology, 3rd ed. American Soci- that might explain its inability to grow abundantly on N20. ety for Microbiology, Washington, D.C. Thus the metabolic defect remains obscure. Similarly, it is 13. Kim, C.-H., and T. C. Hollocher. 1984. Catalysis of nitrosyl transfer reactions by a dissimilatory nitrite reductase (cyto- unknown how nitrate and nitrite may permit strain P2 to chrome c,dj). J. Biol. Chem. 259:2092-2099. grow well with N20. The phenotype of strain P2 suggests at 14. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two simple least that the ability of strains PAQ1 and P1 to grow on media for the demonstration of pyocyanin and fluoroscein. J. endogenous N20 may be under control of nitrate, nitrite, or Lab. Clin. Med. 44:301-307. a metabolic product thereof. 15. Koike, I., and A. Hattori. 1975. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseu- ACKNOWLEDGMENTS domonas denitrificans under nitrate-, nitrite-, and nitrous oxide- Strain P1 was isolated by E. Renshaw ofthe Veterinary Diagnostic limited conditions. J. Gen. Microbiol. 88:11-19. Laboratory at the University of New Hampshire. It was identified by 16. Kristjansson, J. K., B. Walter, and T. C. Hollocher. 1978. R. Mooney of the Microbiology Department, University of New Respiration-dependent proton translocation and the transport of Hampshire, and D. A. Bazylinski. We thank R. P. Blakemore for use nitrate and nitrite in Paracoccus denitrificans and other of certain equipment and K. L. Nazaretian and J. Goretski for denitrifying bacteria. Biochemistry 17:5014-5019. technical assistance. We are also grateful to E. Palome and W. Fowle 17. Lacy, D., and D. Wessels. 1984. A method for the spectropho- of the Biology Department of Northeastern University for electron tometric assay of anaerobic enzymes. Anal. Biochem. 141: microscopy. 232-237. This work was supported by grant 82-18000 from the National 18. Leibowitz, M. R., E. A. E. Garber, J. K. Kristjansson, and T. C. Science Foundation and Public Health Service Biomedical Research Hollocher. 1982. Artifacts associated with the use of thiocyanate Support Grant S07-RR07044 from the National Institutes of Health. and valinomycin/K+ as permeant ions in oxidant pulse experiments on denitrifying bacteria. Curr. Microbiol. 7:305-310. 19. Lieberman, M. A., and J.-S. Hong. 1974. A mutant of Esche- LITERATURE CITED richia coli defective in the coupling of metabolic energy to 1. Bazylinski, D. A., and R. P. Blakemore. 1983. Denitrification and active transport. Proc. Natl. Acad. Sci. USA 71:4395-4399. assimilatory nitrate reduction in Aquaspirillum magne- 20. Linke, W. F. 1965. Solubilities of inorganic and metal-organic totacticum. Appl. Environ. Microbiol. 46:1118-1124. compounds, vol. 1 and 2, 4th ed., p. 460 and 794. American 2. Bergan, T. 1981. Human- and animal-pathogenic members of Chemical Society, Washington, D.C. the genus Pseudomonas, p. 666-700. In M. P. Starr, H. Stolp, 21. Scholes, P., and P. Mitchell. 1970. Respiration-driven proton H. G. Triiper, A. Balows, and H. G. Schlegel (ed.), The translocation in Micrococcus denitrificans. J. Bioenerg. prokaryotes, vol. 1. Springer-Verlag, New York. 1:309-323. 3. Boogerd, F. C., H. W. van Verseveld, and A. H. Stouthamer. 22. Schuldiner, S., and H. R. Kaback. 1975. Membrane potential 1981. 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C. studies on the pathway of denitrification in Pseudomonas aeru- Huffaker (ed.), Genetic engineering of symbiotic nitrogen fixa- ginosa. J. Biol. Chem. 252:212-218. tion and conservation of soil nitrogen. Plenum Publishing Corp., 26. van Verseveld, H. W., E. M. Meijer, and A. H. Stouthamer. New York. 1977. Energy conservation during nitrate respiration in Para- 6. Carlson, C. A., and J. L. Ingraham. 1983. Comparison of coccus denitrificans. Arch. Microbiol. 112:17-23. denitrification by Pseudomonas stutzeri, Pseudomonas aerugi- 27. Walter, B., E. Sidransky, J. K. Kristjansson, and T. C. nosa, and Paracoccus denitrificans. Appl. Environ. Microbiol. Hollocher. 1978. Inhibition of denitrification by uncouplers of 45:1247-1253. oxidative phosphorylation. Biochemistry 17:3039-3045. 7. Castignetti, D., and T. C. Hollocher. 1983. Proton translocation 28. Wolin, E. A., M. J. Wolin, and R. S. Wolfe. 1963. Formation of during denitrification by a nitrifying-denitrifying Alcaligenes sp. methane by bacterial extracts. J. Biol. Chem. 238:2882-2886. Antonie van Leeuwenhoek J. Microbiol. Serol. 49:61-68. 29. Yoshinari, T., R. Hynes, and R. Knowles. 1977. Acetylene 8. Garber, E. A. E., D. Castignetti, and T. C. Hollocher. 1982. inhibition of nitrous oxide reduction and measurement of Proton translocation and proline uptake associated with reduc- denitrification and nitrogen-fixation in soil. Soil Biol. Biochem. tion of nitric oxide by denitrifying Paracoccus denitrificans. 9:177-183.