APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1978, p. 955-9610099-2240/78/0035-0955$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 35, No. 5

Printed in U.S.A.

Pathway of Degradation of Nitrilotriacetate by a

Pseudomonas SpeciestMARY K. FIRESTONE AND JAMES M. TIEDJE*

Departments of Crop and Soil Sciences and ofMicrobiology and Public Health, Michigan State University,

East-Lansing, Michigan 48824

Received for publication 30 December 1977

The pathway of degradation of nitrilotriacetate (NTA) was determined byusing cell-free extracts and a 35-fold purification of NTA monooxygenase. Thefirst step in the breakdown was an oxidative cleavage of the tertiary amine by themonooxygenase to form the aldo acid, glyoxylate, and the secondary amine,iminodiacetate (IDA). NTA N-oxide acted as a substrate analog for induction ofthe monooxygenase and was slowly metabolized by the enzyme, but was not an

intermediate in the pathway. No intermediate before IDA was found, but an

unstable a-hydroxy-NTA intermediate was postulated. IDA did undergo cleavagein the presence of the purified monooxygenase to give glyoxylate and glycine, butwas not metabolized in cell-free extracts. Glyoxylate was further metabolized bycell-free extracts to yield CO2 and glycerate or glycine, products also found fromNTA metabolism. Of the three bacterial isolates in which the NTA pathway hasbeen studied, two strains, one isolated from a British soil and ours from a

Michigan soil, appear to be almost identical.

Nitrilotriacetic acid (NTA), N(CH2COOH)3,is used as the major phosphate substitute indetergents in Canada and Sweden. Its use forthis purpose has been restricted in the U.S. since1970 by the Environmental Protection Agency.Scientific task forces have recently been reeval-uating the U.S. and Canadian positions. In July1977, the Great Lakes Research Advisory Boardof the International Joint Commission of Can-ada and the U.S. concluded ".... that on thebasis of human health hazard there is no reason-able cause for restricting the use of NTA as areplacement for phosphate in detergents ..(11). Current annual use of NTA in Canada isabout 60 million pounds. Though still restrictedfrom use in detergents in the U.S., about 10million pounds are consumed annually for avariety of other uses (11).Much work has been done on the biodegrad-

ability of the compound, and a number of orga-nisms have been isolated that can use NTA assole carbon source (3-7, 13; R. E. Cripps and A.S. Noble, Biochem. J. 130:31p-32p, 1972). Theaerobic metabolism of NTA has been investi-gated by three groups, using Pseudomonas spp.isolated from sewage effluent (6) and soils (3, 13;Biochem. J. 130:31p-32p, 1972). Cripps and No-ble (3; Biochem. J. 130:31p-32p, 1972) andTiedje and co-workers (13; J. M. Tiedje, M. K.Firestone, B. B. Mason, and C. B. Warren, Abstr.

t Journal article no. 8305 of the Michigan AgriculturalExperiment Station.

955

Annu. Meet. Am. Soc. Microbiol. 1973, P183, p.171), both working with crude cell-free extracts,have proposed similar pathways involving theoxidative cleavage ofNTA to form iminodiaceticacid (IDA) and glyoxylate. This step requiredreduced nicotinamide adenine dinucleotide(NADH), Mn2", and 02 (Mg2" in the organismused by Cripps and Noble). Glyoxylate was fur-ther metabolized to glycerate which accumu-lated. Since N-oxide formation has been re-ported to occur before oxidative cleavage in sev-eral tertiary amines including trimethylamine(2; P. C. Large, C. A. Boulton, and M. J. C.Crabbe, Biochem. J. 128:137p-138p, 1972), thepossibility of an N-oxide intermediate was pos-tulated by Cripps and Noble (3).

In this paper we provide evidence for thepathway of NTA degradation proposed in theabstract cited above, as well as evidence for theoxidative cleavage of IDA, and for glycine pro-duction from glycoxylate. We also investigatedwhether NTA N-oxide is an intermediate, andwe compared the three cultures that have beenused in NTA pathway studies. The pathwayshown in Fig. 1 was derived from the evidenceherein and from the data of Cripps and Noble(3).

MATERIALS AND METHODS

Culture and medium. The Pseudomonas sp.(ATCC 27109) used by Focht and Joseph (6) waspurchased from the American Type Culture Collec-tion. The Pseudomonas sp. (T23) used by Cripps and

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956 FIRESTONE AND TIEDJE

o Co2 ° OH

C1COOC(2)-'' CHCHCOO ,YOXYLATE TARTRONIC

* SEMIALDEHYDE

XCHZCOO NADHHN-CH2COO\ CH2COOH 02

Mn'2NITRILOTRIACETATE

(NTA)

OH

CHCOO-N-CH2COO-

CH2COOH

a(- HYDROXYL NTA

GLYCERATE

p

/NADH

(HYDRO)Y PYRUVATE)

I,

N02

INE (2) J-w(SERINE) "'

FIG. 1. Pathway ofNTA degradation. Intermediates inparentheses have not been isolated. Portions shownby dotted lines have been implicated, but have not been directly demonstrated.

Noble (3) was a gift from Roger Cripps. The Pseudo-monas sp. used in this laboratory (ATCC 29600) wasisolated from soil as previously reported (13). It wasgrown on a mineral salts medium with NTA as thesole carbon source as previously described (13). Themedia and methods used for characterization of theorganisms are explained elsewhere (8).

Preparation of cell-free extracts. For cell-freeexperiments, large quantities of cells (10 liters) wereharvested by centrifugation at 4°C, washed once withcold 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.2, and centrifuged again.The cells, resuspended in Tris-hydrochloride buffer,were broken by twice passing through a cold AmincoFrench pressure cell (American Instrument Co.) at20,000 lb/in2. Cellular debris was removed by centrif-ugation at 20,000 x g for 20 min at 2°C. Particulateprotein was then removed by centrifugation at 144,000x g for 30 min at 4°C to yield a crude cell-free extract.Purification of the NTA monooxygenase has beendescribed elsewhere (M. K. Firestone, S. D. Aust, andJ. M. Tiedje, submitted for publication).Assay conditions. Product assays using cell-free

extracts were usually run in Warburg vessels sealedwith serum stoppers. The vessels contained 0.2 ml of20% KOH in the center well, 1 ml of crude cell-freepreparation (5 to 15 mg of protein), 6 ,umol of substrate,and 0.1 mmol of Tris-hydrochloride buffer (3-ml vol-ume) in the main reservoir and 6 or 12,umol ofNADHin the side arm. Addition of NADH (rather thansubstrate) was necessary to start the reaction becauseof the presence of relatively high unrelated NADH-oxidizing activities in the crude protein preparations.The reaction vessels were gently rotated on a platformshaker. When the reaction was complete, 0.5 ml of10% trichloroacetic acid was injected into the mainreservoir to drive off 14C02 and precipitate protein.After 30 min the vessel was opened, and the contentsof the center well were quantitatively transferred to ascintillation vial for counting (13). After cooling, theprecipitated protein was removed by centrifugation,and the supernatant was collected for product deter-minations.

Assays for NADH oxidation rates and product ac-cumulation using the 35-fold-purified monooxygenasecontained 0.1 ,umol of NADH, 1.0 t,mol of NTA, 2.0ttmol of MnCl2, and 0.1 mmol of Tris-hydrochloridebuffer in a 1-ml cuvette. The reaction was initiated bythe addition of NADH, the oxidation of which wasfollowed spectrophotometrically at 340 nm on a Cole-man 124 Perkin-Elmer double-beam spectrophotom-eter.Product analyses. For thin-layer chromatography

(TLC)-autoradiography, 10 to 50 ,l of the assay su-pernatant was spotted on Avicel thin-layer plates (An-altech Inc.), and the plates were developed in twodimensions (10) with (i) 1 N HCl-isopropanol-butanone (25:60:15) and (ii) t-butyl alcohol-butanone-acetone-ammonium hydroxide-methanol (40:20:20:19:1). '4C-labeled products were located by autora-diography. Standard Rf values were determined forNTA and NTA N-oxide; these positions were locatedby spraying the plates with 0.02% NiSO4 in methanol,exposing the wet plates to an atmosphere saturatedwith ammonia for 10 min and, after drying, sprayingthe plates with 0.1% dimethylglyoxime in ethanol. IDAand amino acids were located by spraying plates with0.2% ninhydrin in n-butanol.

Glyoxylate was determined by the glyoxylate-hy-drazone colorimetric procedure (14). Glycerate wasdetermined by complexing with chromotropic acid (1).Protein was assayed by the method of Lowry et al.,using bovine serum albumin standards (12). Gas chro-matographic analysis was previously described (13).

Chemicals. [carboxyl-'4C]NTA was a gift fromProctor and Gamble Co. [carboxyl-'4C]IDA was pro-duced biologically and purified by preparative TLC.['4C]glyoxylate, -glycine, and -serine were purchasedfrom Mallinckrodt Chemical Co. NTA, trisodium saltmonohydrate (Gold Label), IDA, disodium salt, wereobtained from Aldrich Chemical Co. Glyoxylic acid,sodium salt, monohydrate; NADH, disodium saltgrade III; and bovine albumin fraction V were obtainedfrom Sigma Chemical Co. NTA N-oxide was a giftfrom Monsanto Industrial Chemical Co., St. Louis,Mo. Ethylenediaminemonoacetic acid, N,N-ethylene-

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DEGRADATION OF NTA BY PSEUDOMONAS 957

diaminediacetric acid, N,N'-ethylenediaminediaceticacid, and ethylenediaminetriacetic acid were fromEastman Kodak Co., Rochester, N.Y.

RESULTSBiochemical characterizations of three

Pseudomonas spp. known to degrade NTA aregiven in Table 1. The metabolic characteristicsand colonial morphology were identical for theorganism that we used (ATCC 29600) and thatused by Cripps and Noble (3; Biochem J.,130:31p-32p, 1972) whereas the strain used byFocht and Joseph (ATCC 27109) (6) was mor-phologically and biochemically distinct. Basedon this characterization and subsequent char-acterization by the ATCC staff, it was deter-mined that our strain did not fit any recognizedspecies of Pseudomonas.The NTA-degrading enzymes were found in

the supernatant of crude cell-free preparationsafter centrifugation at 144,000 x g. Essentiallyno NTA-modifying activity was found associatedwith the particulate fraction.The ability to metabolize NTA was induced

by growth on either NTA or IDA, but was not

TABLE 1. Characterization of NTA-degradingPseudomonas spp."

Characteristic A2TCC T23 A2T1CC

Catalase + + +Oxidase + + +Arginine dihydrolase - - -DenitrificationCarbon source

Carbohydratesh + +Acetate + + +2-Ketogluconate - - +Propionate - - -

Saccharate - - -

p-Hydroxybenzoate - - -

,B-Alanine + + -

Arginine + + +Asparagine + + +Sorbitol + + +Ethanol + + +m-Inositol + + +Propylene glycol - - +Geraniol - - -

HydrolyzedCasein - - -

Gelatin - - -

Starch - - -

PigmentKing medium B - - Green solublePeptone glucose - - Green soluble

Growth at:4°C - - _410C - - -

" All were reported to be gram-negative rods with singlepolar flagella: ATCC 29600 (13), T23 (3), ATCC 27109 (6).

h Carbohydrates tested: D-arabinose, L-arabinose, cello-biose, fructose, glucose, maltose, ribose, sucrose, trehalose,and D-xylose.

' Extracellular polymer produced from most carbohydrates.

induced by growth on glyoxylate and glycine(Table 2). The Km values from crude cell-freeprepaxations were similar whether the cells weregrown on NTA or IDA. Growth on NTA N-oxide also induced the ability to metabolizeNTA.The products resulting from incubation of cell-

free extracts with ['4C]NTA or glyoxylate wereidentified by TLC-autoradiography as shown inFig. 2. The first apparent products from NTAwere IDA and glycerate, with glycine visibleafter 60 min of incubation. The two-dimensionalchromatography system used was not definitivefor the identification of glyoxylate, since itmoved with the solvent front in the first dimen-sion and did not travel in the second dimension.Significant quantities of glyoxylate were neverdetected in the crude cell-free preparations, evenin the presence of a phenyl hydrazine-trappingsystem, presumably due to the speed with whichglyoxylate was further metabolized. No NTA N-oxide (which would have chromatographedabove NTA in the first dimension) was detectedin crude cell-free preparations from NTA metab-olism. The products found from glyoxylate me-tabolism in cell-free preparations were glycineand glycerate (Fig. 2, column 4). The glycineapparently resulted from transamination ofglyoxylate. No products were detected from[I4C]IDA, -glycine, or -serine incubations withcell-free extracts when analyzed by 14CO2 trap-ping, TLC-autoradiography, and glyoxylate orglycerate colorimetric procedures.

Production of IDA and glycine from NTA bycell-free extracts was confirmed by gas chroma-tography (Table 3). The stoichiometry showedessentially complete conversion ofNTA to IDA,

TABLE 2. Substrate-dependent NADH-oxidizingactivity in cell-free preparations induced by growth

on several carbon sources

Carbon source' on Substrate NADH oxi- Khwhich cells were added for dation t

grownNADH-oxi- (Ux102) (IsM)

dizing activityNTA NTA 5.1 30NTA IDA 4.2 690NTA None 0.5

IDA NTA 5.0 27IDA IDA 4.3 590IDA None 0.3

Glyoxylate-glycine NTA 0.2Glyoxylate-glycine IDA 0.3Glyoxylate-glycine None 0.1

" All substrates at 0.1% concentration; glyoxylate-glycinewas in a 2:1 ratio.

'Protein concentrations (micrograms/1-ml assay) were:NTA grown, 83 pg; IDA grown, 79 ,ug; glyoxylate-glycinegrown, 77,ug.

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958 FIRESTONE AND TIEDJE

FIG. 2. Autoradiogram of two-dimensional thin-layer chromatogram of products from cell-free extractsincubated with ["'CINTA and 12 iLmol ofNADH (1-3) or ["Ciglyoxylate and 6 p,mol ofNADH (4). Incubationtime and substrate are as follows: (1) 0 min, NTA; (2) 10 min, NTA; (3) 60 min, NTA; (4) 60 min, glyoxylate.The identities of the compounds are: A, IDA; B, glycine; E, NTA; F, glycerate; G, glyoxylate; 0, origin.

TABLE 3. Substrate disappearance andproductsformed by ceU-free extract as determined by gas

chromatographyIncubation ,umol detected

time Addition(h) NTA IDA Glycine

0 0 0.68 0.06 0.045 0 0.73 0.05 0.045 NADH 0 0.65 0.26

with the latter not further metabolized underthese conditions. Glycine was the only otherproduct detected by this gas chromatographicprocedure, which was capable of separating a

large number of amino and acidic compounds.The identification of IDA was confirmed by gaschromatography-mass spectrometry.

Production of glycerate and "CO2 from sev-eral potential precursors is shown in Table 4.The "4CO2 presumably resulted from the actionof glyoxylate carboligase, which condenses twomolecules of glyoxylate to form the three-carbontartronic semialdehyde and CO2. Since NTA isa symmetrical molecule, the three acetategroups are equal, and therefore only one-thirdof the carboxyl label can be further metabolizedwhen IDA accumulates. If glyoxylate either asan added substrate or from NTA were metabo-lized by the carboligase, then 50% of the glyox-

TABLE 4. Glycerate and "4CO2 production fromseveral substrates by cell-free extracts

%14C label as C02 GlyceratebSubstrate' (tmol)(6,umol) Theoretical Found Theoretical

Found maximum maximum

NTA 13.4 16.6' 1.9 3.0'IDA 0.1 25.0 0 3.0Glyoxylate 41.9 50.0 2.2 3.0NTA N-oxide 0

a 12 umol of NADH was added to the NTA and NTA N-oxide assays; 6 ,umol of NADH was added to the IDA andglyoxylate assays. Between 7 and 11 mg of protein from thesoluble portion of a crude cell-free preparation was added toeach assay.

Determined by glycerate colorimetric analysis.Assuming IDA not metabolized.

ylate label would be found as CO2. This reason-ing was also used for the theoretical maximumvalue for glycerate shown in Table 4. As shownin the table, the stoichiometry of the metabolismof NTA and glyoxylate to '4CO2 and glyceratewas nearly equal. The absence of products fromIDA confirms that it was not metabolized in cell-free preparations. Glycerate was not producedfrom NTA N-oxide; if NTA N-oxide were anintermediate in the pathway preceding thecleavage of the tertiary amine, one would expectto find glycerate production.

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DEGRADATION OF NTA BY PSEUDOMONAS 959

To determine whether IDA and glyoxylatewere indeed the first products of NTA metabo-lism, we used a 35-fold purification of the mono-oxygenase responsible for the first step in NTAbreakdown. Figure 3 shows a TLC-auto-radiogram of the products obtained by incuba-tion of "C-NTA with the purified enzyme. IDA

FIG. 3. Autoradiogram of two-dimensional thin-layer chromatogram ofproducts from partially puri-fied NTA monooxygenase incubated with ['4C]NTA.The identities of the compounds are: A, IDA; B,glycine; E, NTA; G, glyoxylate; 0, origin.

and a small amount of glycine were produced.Glyoxylate, determined by a colorimetric pro-cedure, also accumulated as an end product inincubations of the purified enzyme with NTA,IDA, and NTA N-oxide (Table 5). The quanti-ties of glyoxylate produced from a given quantityof NADH and the rate of glyoxylate productionas determined by rate of NADH oxidation were

lower for IDA and NTA N-oxide than for NTA.The quantities of glyoxylate produced from 0.1,umol of NADH are not stoichiometric due to a

substrate-dependent NADH oxidase activity in-herent to this enzyme (Firestone et al., submit-ted). Glyoxylate was not further metabolized bythe purified monooxygenase preparation. Kmvalues deternined by using the purified mono-

oxygenase were similar to those given in Table2 for crude cell-free preparations.The activity of the partially purified mono-

oxygenase was investigated with a number ofpotential substrates. Substrate-dependentNADH oxidation and a small amount of glyox-ylate were obtained from N-methyl-IDA, eth-ylenediaminemonoacetic acid, and ethylenedi-aminetriacetic acid. No activity was detected ontriethylamine, triethylamine N-oxide, trietha-nolamine, N-acetyl glycine, nitroso-IDA, N-(phosphomethyl)-glycine (a herbicide), sarco-

sine, dimethylamine, N,N-ethylenediaminedi-acetic acid, N,N'-ethylenediaminediacetic acid,or ethylenediaminetetraacetic acid in 10 or 1mM concentrations.

DISCUSSIONOf the three Pseudomonas spp. characterized,

T23, used by Cripps and Noble and isolated fromBritish soil (3; Biochem. J. 130:31p-32p, 1972),and ATCC 29600, used in the work reportedhere and isolated from Michigan soil, were mor-phologically and biochemically identical. Thepathways for NTA degradation were very simi-lar, although slight differences were apparent.Cripps and Noble (3) found no transamination

TABLE 5. Glyoxylate production and Km values forseveral substrates with purified monooxygenase

preparationsNADH oxida-

Substrate Glyoxylate pro- tion rate' Km, (MM)duced' (Amol) UX12)

NTA 0.047 1.09 31.0IDA 0.015 0.99 780.0NTA N-oxide 0.017 0.66 3550.0

' Glyoxylate production and NADH oxidation rates weredetermined by using 45 Mg of protein, 1 ,umol of substrate, 2Mmol of MnCl2, 0.1 Mmol of NADH, and 0.1 Mmol of Tris-hydrochloride buffer in the 1-ml assay.

'Determined from rates of substrate-dependent NADHoxidation.

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960 FIRESTONE AND TIEDJE

of glyoxylate, although the involvement of gly-cine was suggested by the observation of ele-vated activities of serine hydroxymethyltrans-ferase, serine-oxaloacetate aminotransferase,and hydroxypyruvate reductase in NTA-growncells. Cell-free extracts of our Pseudomonas sp.readily produced glycine from glyoxylate. In thecell-free extracts this glycine apparently resultedfrom transamination of glyoxylate, since no

products resulted from additions of ['4C]glycineor serine. Our Pseudomonas sp. grew well on

IDA as substrate, whereas theirs grew poorly.The third strain, ATCC 27109, used by Focht

and Joseph (6), was biochemically and morpho-logically different. Whole-cell studies of NTAmetabolism in this isolate suggested that IDAand glycine were involved in the pathway. How-ever, without more information it is impossibleto determine whether a pathway similar to thatreported here is operational in this organism.As shown in our work, the first detectable

products from NTA degradation were IDA andglyoxylate. In work reported elsewhere (Fire-stone et al., submitted), we have shown thisenzyme to be a monooxygenase that requires 02,

NADH, and Mn2". This enzyme was inducibleand soluble.Large and co-workers, studying metabolism of

trimethylamine by Pseudomonas aminovorans(2; Biochem. J. 128:137p-138p, 1972), haveshown trimethylamine N-oxide to be an inter-mediate in the degradation of this tertiary amineto a secondary amine and an aldehyde. Thisreaction is carried out by two enzymes: the firsta monooxygenase producing the N-oxide, andthe second a nonoxidative enzyme catalyzing thecleavage. If the first steps in NTA breakdownwere comparable to those for trimethylamine,the N-oxide would be the product of the mono-oxygenase and serve as a substrate for a secondnonoxidative enzyme. However, we have notbeen able to detect any NTA N-oxide by TLCor gas chromatography using the partially puri-fied (35-fold) monooxygenase or cell-free ex-tracts. If the monooxygenase preparation is con-taminated with a second nonoxidative enzymeresponsible for cleavage, then the rates andquantities of glyoxylate production from NTAN-oxide should be comparable to those fromNTA. This was not the case. Also, in cell-freeextracts NTA N-oxide was metabolized, but gly-cerate, the major product of NTA breakdown,was not produced. Although cell-free extracts ofcells grown on NTA N-oxide were induced forNTA metabolism (and NTA monooxygenase), itis common for substrate analogs to induce en-

zyme synthesis without being a natural substratefor the enzyme. We have concluded that NTAN-oxide is not an intermediate preceding tertiary

AppI. ENVIRON. MICROBIO1,.

amine cleavage. The N-oxide is apparently asubstrate analog that can be slowly metabolizedby the monooxygenase; this is consistent withthe very different Km values for NTA (31 ,uM)and NTA N-oxide (3,550 ,uM).

If an intermediate does occur before cleavage,it would most likely be the a-hydroxyl of NTA(Fig. 1). This type of intermediate has beenreported for a tertiary amine, N-methyl carba-zole, metabolized by liver microsomes (9). Thea-hydroxyl of NTA should spontaneously de-compose to IDA and glyoxylate (C. B. Warren,Monsanto Chemical Co., personal communica-tion), thereby not requiring a second enzyme.This instability is also consistent with the ab-sence of any other intermediate in our TLCassays.The purified NTA monooxygenase was active

on the secondary amine, IDA, producing glyox-ylate. The Km was 20-fold higher than for NTA,and turnover to product was poor. IDA didaccumulate in stoichiometric amounts fromNTA in cell-free extracts and was not metabo-lized when introduced as substrate. The mostlikely explanation for this absence of IDA me-tabolism appears to be the low concentration ofMn2" in the cell-free extracts. Since the sub-strate is bound as the substrate-Mn2" complex(Firestone et al., submitted), the accumulationwas probably due to the low IDA-Mn2" concen-tration relative to the high Km. When purifiedmonooxygenase was used, Mn2+ was alwaysadded in substrate amounts, and IDA was me-tabolized.

Glycine would be the other logical productfrom IDA cleavage. We did find small amountsof labeled glycine when quantities of labeledIDA were allowed to accumulate from NTA,probably resulting from IDA cleavage (Fig. 3).Although IDA did serve as a substrate for NTAmonooxygenase, this may not be its major fatein the intact cell. Previous work (13) indicatedthat IDA was metabolized by acetate-growncells (noninduced for NTA breakdown); appar-ently a constitutive pathway for metabolism ex-ists.In cell-free extracts, the glyoxylate produced

by cleavage of NTA was further metabolized toglycerate (via glyoxylate carboligase and tar-tronic semialdehyde reductase) and to glycine(via transmination). It is possible that in wholecells another pathway for glyoxylate metabolismmay exist, such as entry into a dicarboxylic acidcycle through malate synthetase.

In intact cells, glycine may be further metab-olized to glycerate. Evidence for this portion ofthe pathway was found in the Pseudomonas sp.used by Cripps and Noble (3). They found ele-vated levels of the enzymes responsible for gly-

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DEGRADATION OF NTA BY PSEUDOMONAS 961

cine conversion to glycerate in cells grown onNTA.The identification of essentially the same

pathway for tertiary amine metabolism in twoPseudomonas sp. of widely differing origins,Michigan and Great Britain, may indicate thegeneral importance of this pathway in NTAmetabolism.

ACKNOWLEDGMENTSWe thank B. B. Mason, who completed a portion of this

work, C. B. Warren for the gas chromatographic and massspectrometer analyses and helpful discussions, N. Bhimji fortechnical assistance, and Roger Cripps for sending us hisPseudomonas sp. We also thank S. D. Aust for his help withthe enzyme purification and for use of his laboratory facilities.

This work was supported in part by an unrestricted grantaward to J.M.T. from Eli Lilly Research Laboratories.

LITERATURE CMD1. Bartlett, G. R. 1959. Colorimetric assay method for free

and phosphorylated glyceric acid. J. Biol. Chem.234:469-471.

2. Boulton, C. A., J. C. Crabbe, and P. L. Large. 1974.Microbial oxidation of amines: partial purification of atrimethylamine mono-oxygenase from Pseudomonasaminovorans and its role in growth on trimethylamine.Biochem. J. 140:253-263.

3. Cripps, R. E., and A. S. Noble. 1973. The metabolism ofnitrilotriacetate by a pseudomonad. Biochem. J.136:1059-1968.

4. Enfors, S. O., and N. Molin. 1973. Biodegradation ofnitrilotriacetate (NTA) by bacteria-I. Isolation of bac-

teria able to grow anaerobically with NTA as a solecarbon source. Water Res. 1:881-888.

5. Enfors, S. O., and N. Molin. 1973. Biodegradation ofnitrilotriacetate (NTA) by bacteria-II. Cultivation ofan NTA-degrading bacterium in anaerobic medium.Water Res. 1:889-893.

6. Focht, D. D., and H. A. Joseph. 1971. Bacterial degra-dation of nitrilotriacetic acid (NTA). Can. J. Microbiol.17:1553-1556.

7. Forsberg, C., and G. Lindquist. 1967. Experimentalstudies on bacterial degradation of nitrilotriacetate,NTA. Vatten 23:265-277.

8. Gamble, T. N., M. R. Betlach, and J. M. Tiedje. 1977.Numerically dominant denitrifying bacteria from worldsoils. Appl. Environ. Microbiol. 33:926-939.

9. Gorrod, J. W., and D. J. Temple. 1976. The formationof an N-hydroxymethyl intermediate in the N-demeth-ylation of N-methylcarbazole in vivo and in vitro. Xe-nobiotica 6:265-274.

10. Haworth, C., and J. G. Heathcote. 1969. An improvedtechnique for the analysis of amino acids and relatedcompounds on thin layers of cellulose. J. Chromatogr.41:380-385.

11. International Joint Commission-Great Lakes Re-search Advisory Board. 1977. Annual Report of theResearch Advisory Board, July 1977. IJC Great LakesRegional Office, Windsor, Ontario.

12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. L.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

13. Tiedje, J. M., B. B. Mason, C. B. Warren, and E. J.Malec. 1973. Metabolism of nitrilotriacetate by cells ofPseudomonas species. Appl. Microbiol. 25:811-818.

14. Trijbels, F., and G. D. Vogels. 1966. Degradation ofallantoin by Pseudomonas acidovorans. Biochim. Bio-phys. Acta 113:292-301.

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