Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa.

A Mercenier, J P Simon, C Vander Wauven, D Haas and V Stalon J. Bacteriol. 1980, 144(1):159. Downloaded from http://jb.asm.org/ on February 24, 2012 by guest

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JOURNAL OF BACTERIOLOGY, Oct. 1980, p. 159-163 0021-9193/80/10-0159/05$02.00/0

Vol. 144, No. 1

Regulation of Enzyme Synthesis in the Arginine Deiminase Pathway of Pseudomonas aeruginosa

ANNICK MERCENIER,' JEAN-PAUL SIMON,2 CORINNE VANDER WAUVEN,1 DIETER HAAS,3 AND VICTOR STALON'*

Laboratoire de Microbiologie, Faculte des Sciences, Universite Libre de Bruxelles, Brussels, Belgium'; Institut de Recherches du Centre d'Enseignement et de Recherches des Industries Alimentaires et Chimiques, B-1070 Brussels, Belgium2; and Department ofMicrobiology, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland'

The three enzymes of the arginine deiminase pathway in Pseudomonas aerugistrain PAO were induced strongly (50- to 100-fold) by a shift from aerobic growth conditions to very low oxygen tension. Arginine in the culture medium was not essential for induction, but increased the maximum enzyme levels twofold. The induction of the three enzymes arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3) appeared to be coordinate. Catabolic ornithine carbamoyltransferase was studied in most detail. Nitrate and nitrite, which can replace oxygen as terminal electron acceptors in P. aeruginosa, partialy prevented enzyme induction by low oxygen tension in the wild-type strain, but not in nar (nitrate reductase-negative) mutants. Glucose was found to exert catabolite repression of the deiminase pathway. Generally, conditions of stress, such as depletion of the carbon and energy source or the phosphate source, resulted in induced synthesis of catabolic ornithine carbamoyltransferase. The induction of the deiminase pathway is thought to mobilize intra- and extracellular reserves of arginine, which is used as a source of adenosine 5'-triphosphate in the absence of respiration.
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Two pathways participate in arginine catabolism in Pseudomonas aeruginosa. In the arginine deiminase pathway, arginine is degraded via citrulline to ornithine and carbamoylphosphate, which serves to generate ATP from ADP (13, 14).This pathway was found to provide energy for motility in a fluorescent Pseudomonas sp. strain under anaerobic conditions (11). A second catabolic pathway has been characterized recently; it involves the decarboxylation of arginine to agmatine, which is converted to Ncarbamoylputrescine and putrescine (8, 9). A mutant lacking the catabolic ornithine carbamoyltransferase, the second enzyme of the arginine deiminase pathway, is able to grow aerobically on arginine as the only carbon and nitrogen source at the same rate as the wild-type strain (4). Thus, we have suggested (4) that the arginine deiminase pathway is not primarily concerned with the utilization of arginine. In this paper, we describe the regulation of this pathway, as determined by studying enzyme formation under a variety of growth conditions in the presence of oxygen and other terminal electron acceptors. It appears that depletion of terminal electron acceptors leads to an induction of the deiminase pathway, enabling the cells to generate energy from arginine.

MATERIALS AND METHODS

Bacterial strains and media. All mutants were derived from P. aeruginosa strain PA01 (5). These strains were routinely grown at 370C on minimal nitrogen salt-free medium 154 (13) supplemented with trace elements as follows: 28 mg of H3B03 per liter, 7.5 mg of Na2MoO4.2H20 per liter, 2.4 mg of ZnSO4 per liter, and 2.5 mg of CuSO4*5H20 per liter. Carbon and nitrogen sources were added at concentrations of 25 mM each after sterilization. N03- and N02 concentrations were 100 and 10 mM, respectively, as suggested by Williams et al. (19). For limitation of growth by phosphate, the phosphate in the medium was replaced by 50 mM Tris-hydrochloride buffer (pH 7.0). Growth of bacteria. Cells were grown in 1.3 liters of medium in a 2-liter microfermentor (Biolafitte, Maisons-Lafitte, France). Oxygen tension was measured with a sterilizable oxygen electrode, which was calibrated in sterile medium before inoculation. The system was allowed to equilibrate with air under agitation (200 rpm), and this oxygen tension was designated as 100%. Aerobic conditions were achieved by stirring the culture at a constant rate of 200 rpm, and air was sparged through the culture vessel to maintain air saturation (i.e., 100% oxygen tension). Exponentially growing cultures were used as inocula; the initial cell density in the fermentor was approximately 3 x 107 cells per ml. Growth limitation experiments. In experiments with a limiting essential nutrient, there was a linear

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ess was as

J. BACTERIOL. determined by using

an

relationship between the bacterial cell concentration and the particular nutrient in the culture. For carbonlimited growth 8 mM pyrvate was used; 0.2 mM phosphate was used for phosphate-limited growth. The conditions were chosen to give 3 x i0W to 4 x 108 cells per ml in starved cultures. Preparation of cell extracts. Samples of the culture were pumped out of the fermentor with a peristaltic pump and chilled on ice, and 200 Ag of chloramphenicol per ml was added. After the bacteria were harvested by centrifugation at 4C, they were washed once with 0.9% (wt/vol) NaCl. Cell extracts were prepared as described previously (4). Assay of catabolic ornithine carbamoyltransferase activity in cell extracts. Catabolic ornithine carbamoyltransferase was assayed by colorimetric determination of the citrulline formed from ornithine and carbamoylphosphate, as previously described (4). Assay of arginine deiminase. Arginine deiminase enzyme was aLso assayed by colorimetric determination of citrulline. The standard incubation mixture contained the following (in 2 ml): 200 ,mol of citrateNaOH buffer (pH 5.5), 10 umol of MnCl2, 10 umol of arginine, and extract. The reaction was started with arginine and stopped after 15 nin at 37C by adding 2.0 ml of 1 N hydrochloric acid. Carbamate kinase determination. Carbamate kinase was assayed in the direction of ATP synthesis. The assay mixture (1 ml) contained 50 mM citrateNaOH (pH 6.15), 40 mM MgCl2, 7.5 mM ADP, 5 mM carbamoylphosphate, and cell extract. The reaction was started by adding ADP and stopped after 15 min of incubation by heating at 100C for 3 min. The interfering adenylate kinase activity was determined under these same conditions in the absence of carbamoylphosphate. ATP formation was measured in a coupled assay mixture containing glucose and hexokinase, which resulted in the formation of glucose 6phosphate; this compound was detected by the conversion of NADP to NADPH by glucose 6-phosphate dehydrogenase. The net increase in absorbance at 340 nm was measured with a Beckman DU spectrophotometer. The reaction mixture (1.0 ml) for the coupled assay contained 75 mM Tris-hydrochloride (pH 7.5), 0.37 mM NAD, 8 mM MgCl2, 300 mM glucose, 6 U of yeast hexokinase (Boehringer), and 30 U of yeast glucose-6-phosphate dehydrogenase (grade I; Boehringer). The reaction was initiated by adding the sample containing ATP, and incubation was at 30C for 60 min. The assay was proportional to ATP concentration from 0.01 to 0.15,umol. The amount of carbamate kinase activity was obtained after subtraction of the interfering adenylate kinase activity, which appeared constitutive under the growth conditions examined (specific activity, approximately 10 ,umol/h per mg of protein). Protein concentrations. The protein concentrations of extracts were estimated by the method of Lowry et al. (7). Specific activities. Specific activities are expressed as the amount of enzyme that catalyzed the formation of 1 pmol of product per h per mg of protein. Arginine consumption. In some experiments, the amount of arginine consumed during the growth proc-

amino acid analyzer,

previously described (8). RESULTS

Effect of oxygen tension on enzyme formation in the arginine deiminase pathway. The levels of the arginine deiminase pathway enzymes in P. aeruginosa cultures grown aerobically were difficult to reproduce. The specific activities of arginine deiminase and catabolic ornithine carbamoyltransferase appeared to depend on the form of the flask, the volume of the culture medium, and the culture density at harvesting time. The rate of catabolic ornithine carbamoyltransferase synthesis was low during exponential growth and at the end of the exponential phase in a variety of growth media with or without arginine (Table 1). In a minimal medium containing arginine and glutamate, decreasing the oxygen tension in the culture medium led to a strong induction of the arginine deiminase pathway (Fig. 1). When the air supply was cut off, the oxygen tension in the medium fell below the limit of detection (i.e., <1% saturation) within a few minutes, although the culture remained in contact with air at atmospheric
TABLE 1. Regulation of specific catabolic ornithine carbamoyltransferase activities in strain PAQI Sp act
Growth medium'

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tension'

Oxygen

formed per h per mg of protein)

(pAmol of citrulline

22c Glutamate ................. High 14 Arginine ................... High 15 Glutamate + arginine ....... High 740 Glutamate ................. Low 448 Citrate + NH4 . ............. Low 270 Glucose + NH4+ ............. Low 1,212 Glutamate + arginine ....... Low 850 Pyruvate + arginine ......... Low 770 Succinate + arginine ........ Low 560 -Citrate + arginine .......... Low 486 Fumarate + arginine ....... Low 320 Glucose + arginine .......... Low aMedium 154 was supplemented with a carbon source (25 mM) and/or a nitrogen source (25 mM). b High oxygen tension means air saturation; cultures were harvested during the exponential growth phase. Low oxygen tension means <1% saturation. Cultures were grown aerobically to a concentration of approximately 2.5 x 108 cells per ml and then incubated without aeration for 2.5 h, as described in the legend to Fig. 1. 'Basal enzyme level. Some of the enzyme activity may have been due to anabolic ornithine carbamoyltransferase, which is formed at a high rate in medium containing glutamate (17).

VOL. 144, 1980

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sumption of these two compounds continued for about 10 h without change in the bacterial mass. Although P. aeruginosa grows well on arginine as the only nutrient (17), cell lysis often occurred ~~~~~~~~~1000under high oxygen tension, and hence experiments in which arginine was used as the sole ~50~ carbon and nitrogen source were abandoned. / 5500 i .1 Catabolite repression of catabolic ornithine carbamoyltransferase occurred when P. aeruginosa was cultivated in media containing arginine and 4. 00 other carbon sources (Table 1). The lowest level Time (hours) of catabolic ornithine carbamoyltransferase was FIG. 1. Induction ofcatabolic ornithine carbam- obtained in the presence of glucose; fumarate oyltransferase during growth of P. aeruginosa strain and citrate gave specific activities which were PAOI on medium containing 25 mM glutamate and about 40 to 50% of the maximum level obtained 25 mM arginine under low oxygen tension. The with medium containing glutamate plus argidashed line shows the oxygen tension. Symbols: 0, nine. growth given as the extinction at 660 nm; *, catabolic Effect of nitrate and nitrite. P. aeruginosa ornithine carbamoyltransferase specific activity. Aeration was stopped at the time indicated by the arrow. is able to grow anaerobically with nitrate or nitrite as terminal electron acceptor (15, 16). This fact was exploited to study the regulation pressure. As soon as the oxygen tension became undetectable, all three enzymes of the pathway of the deiminase pathway during different (arginine deiminase [EC 3.5.3.6], catabolic omi- modes of respiration. In the presence of nitrate, thine carbamoyltransferase [EC 2.1.3.3], and ashift from full aerobiosis to low oxygen tension carbamate kinase [EC 2.7.3.2]) were formed rapidly, and coordinate synthesis of these enzymes 10~~~~0 was observed (Fig. 2). In the experiments described below, the levels of catabolic ornithine carbamoyltransferase and arginine deiminase -!4 4 were assayed under a variety of conditions, but 100 ro~~~~~~~~~~~~~~~ carbamate kinase was not studied in such detail. When chloramphenicol (final concentration, ~o 200 ,ug/ml) was added to a culture 30 min after the interruption of the oxygen supply, the levels of arginine deiminase and catabolic ornithine Carbomatekinaselsp. act.) Arginine deiminse(sp.act.) carbamoyltransferase did not increase (data not FIG. 2. Coordinate regulation of the enzymes of shown). Thus, de novo protein synthesis is re- the arginine deiminase pathway in an experiment quired for the induction of the deiminase path- identical to that described in the legend to Fig. 1. way. In a culture induced for the arginine deiminase pathway by low oxygen tension, restora100 tion of aeration resulted in a resumption of fast growth, and catabolic ornithine carbamoyltransferase formation was repressed (data not shown). Effect of arginine. Induction of catabolic 5OC ornithine carbamoyltransferase occurred under 0 50 Y low oxygen tension even in the absence of argiI nine (Table 1). However, the addition of 10 mM arginine to an induced culture of strain PAO1 o (growing on medium containing citrate and 8 10 20 2 6 4 NH4+) increased the level of this enzyme by a lime (hours ) factor of two (Fig. 3). Maximum enzyme formaFIG. 3. Induction of catabolic ornithine carbamtion was observed during growth on medium oyltransferase during growth of P. aeruginosa on containing glutamate and arginine about 2 h medium containing 25 mM citrate and 20 mM NH4' after the air supply was cut off (Table 1). Slow under low oxygen tension. At the times indicated by growth continued for 6 h under low oxygen the open arrows, 10 mM arginine was added as a tension. When growth had stopped, 40% of the supplement to the growth medium. Symbols: 0, initial glutamate and 40% of the initial arginine growth given as the extinction at 660 nm; *, catabolic ornithine carbamoyltransferase specific activity. were still present in the medium, and the conIx
.

b100

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*~~~~~~~~100

200

6~ ~ ~ ~ ~ ~ ~ ~r

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J. BACTERIOL.

100 caused only a slight reduction in the doubling time; the catabolic ornithine carbamoyltransfer1.0 ase level increased for 1 h after the oxygen c tension became zero (Fig. 4). However, the max- f0 g 600; imum level of activity with nitrate was much 50,j 4 lower than the maximum level obtained without nitrate, and after 1 h the specific activity decreased slowly to a final level corresponding to Y o. 200 y about 20% of the maximally induced activity (Fig. 1 and 4). 4 2 6 10 8 With nitrite as the electron acceptor, some Time hours) inhibition of catabolic ornithine carbamoylFIG. 4. Induction of transferase induction was also observed (Table oyltransferase during catabolic ornithine carbamP. on 2). We may conclude that both nitrate and ni- medium containing 25 growth of aeruginosa mM glutamate, 25 mM argitrite prevent induction of the arginine deiminase nine, and 100 mM nitrate under low oxygen tension. pathway, but do less efficiently than oxygen. Aeration was stopped at the time indicated by the van Hartingsveldt et al. (15, 16) isolated muarrow. Symbols: 0, growth given as extinction at 660 tants of strain PAO impaired in the disimilatory nm; *, catabolic ornithine carbamoyltranserase spenitrate reductase (nar) and anaerobic growth cific activity. (ana). In four nar mutants tested, nitrate did not prevent the strong induction of catabolic TABLE 2. Regulation of specific catabolic ornithine carbamoyltransferase activities in cultures grown omithine carbamoyltransferase under low oxyon medium containing glutamate and arginine gen tension (Table 2). Similarly, in an anaA under low oxygen tension mutant, which has nitrate and nitrite reductase Sp act (pmol activities but does not grow anaerobically (16), of citrulline Straina Relevant geno- Electron acnitrate and nitrite failed to interfere with cataper h per mg ceptor type bolic ornithine carbamoyltransferase induction. of protein)b These findings indicate that the terminal elec333 NO3PAO1 Wild type tron acceptors themselves are not directly in620 Wild type N02PA01 volved in the regulation of the deiminase path943 narA N03S1130 way. narB 1,086 N03S1128 1,325 N03Effect of nutrient depletion. If induction of S1133 narC 875 narE N03S1241 catabolic ornithine carbamoyltransferase occurs 1,250 anaA NO3S1125 in P. aeruginosa during removal of oxygen as a 1,220 anaA N02S1125 result of energy limitation, then other conditions which produce an energy deficiency might also aThe strains of the S series were derived from strain be expected to lead to induction of the arginine PAO by van Hartingsveldt et al. (15, 16) and were deiminase pathway. P. aeruginosa was grown kindly supplied by A. Stouthamer. nar mutants lack aerobically with limiting amounts of either py- the dissimilatory nitrate reductase. The anaA (nirB) ruvate (8 mM) as the. only carbon source or mutant is affected in anaerobic growth, but has both nitrite reductase activities inorganic phosphate (0.2 mM); growth came to nitrate andvalues were obtained after(15, 16). under b These growth a stop after about five generations. The level of low oxygen tension for 6.5 h, as described in the legend ornithine carbamoyltransferase, which was low to Fig. 1. during exponential growth, increased during the first 30 min of the stationary phase and then TABLE 3. Regulation of specific catabolic ornithine remained at this level for at least 4 h (Table 3). carbamoyltransferase activity by nutrient depletion during conditions of aerobiosis DISCUSSION Sp act (pmol What is the physiological role of the arginine of Growth phase' percitmlline Growth medium deiminase pathway in P. aeruginosa? Previous h per mg of protein) work (4, 8) has shown that under aerobic conditions a block in catabolic ornithine carbamo- Glutamate (25 mM) + 22 Exponential yltransferase does not prevent the utilization of 496 Stationary arginine (25 mM) + arginine as the only nutrient; in fact, arginine phosphate (0.2 mM) 20 Exponential Pyruvate (8 mM) + can be degraded to putrescine via the arginine 240 NH4+ (25 mM) Stationary decarboxylase pathway. Since ATP is one of the end products of the deiminase pathway, this Exponential phase, Activity measured during the pathway can provide energy in microorganisms five generations after inoculation; stationary phase, such as Bacillus licheniformis (3), Streptococ- maximum activity reached 30 min after cessation of growth (cell density, 3 x 108 cells per ml). cus faecalis, and Mycoplasma arthritidis (1).
4D (D 9
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VOL. 144, 1980

P. AERUGINOSA ARGININE DEIMINASE PATHWAY

LITERATURE CITED

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The present study supports the idea that the deiminase pathway of P. aeruginosa is used to obtain energy under extreme conditions of nutrient depletion. In full aerobiosis, the levels of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase were 1 to 2% of the levels obtained under conditions of induction. Depletion of oxygen as the terminal electron acceptor resulted in the strongest induction. Terminal electron acceptors themselves (oxygen, nitrate, nitrite) do not regulate the deiminase pathway directly. Thus, in the wild type, but not in nar mutants, nitrate partially inhibited the induction by oxygen limitation, indicating that a functional nitrate reductase is required for this inhibition. Depletion of the carbon and energy or phosphate source (conditions of stress) also leads to partial induction. Glucose, fumarate, citrate, and pyruvate repressed the deiminase pathway during the shift from aerobiosis to anaerobiosis (Table 1). Glucose was most effective in this respect, although in P. aeruginosa tricarboxylic acid cycle intermediates are generally more effective in catabolite repression of inducible enzymes (10, 12, 17). The arginine decarboxylase pathway (9) was not induced by oxygen depletion (unpublished data). Kight-Olliff and Fitzgerald (6) found that exogenous ATP inhibited the induction of alkylsulfatase in P. aeruginosa. In much the same way, ATP (6 mM) and UTP (6 mM) attenuated the induction of catabolic ornithine carbamoyltransferase under low oxygen tension, whereas AMP (6 mM) stimulated enzyme synthesis (unpublished data). Possibly, exogenous nucleotides modify the energy charge (2), which may be a critical signal in the induction of the arginine deiminase pathway. It has been shown that during aerobic growth of P. aeruginosa on a good carbon source (e.g., succinate) the energy charge is high (18). Depletion of an essential nutrient should result in a rapid decrease in the ATP level (18) and induction of the arginine deiminase pathway. In addition, ATP has been shown to be an allosteric inhibitor of catabolic ornithine carbamoyltransferase in Pseudomonas putida (14). This control may have an important function under conditions of noninduction.
ACKNOWLEDGMENTS
We are grateful to T. Leisinger and A. Pierard for advice and helpful discussions. This work was supported by grant 2.4549.79 from the Fonds de la Recherche Fondamentale Collective. C.V. is aspirante and V.S. is chercheur qualifie at the Fonds National de la Recherche Scientifique. D.H. was supported by the Swiss National Foundation for Scientific Research under project 3.204-0.77.

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