JOURNAL OF BACTERIOLOGY, JUlY 1973, p. 262-267Copyright 0 1973 American Society for Microbiology

Vol. 115, No. 1Printed in U.SA.

Benzoate Metabolism in Pseudomonasputida(arvilla) mt-2: Demonstration of Two

Benzoate PathwaysTERUKO NAKAZAWA AND TAKESHI YOKOTA

Department of Bacteriology, School of Medicine, Juntendo University, Bunkyoku, Tokyo, Japan

Received for publication 28 March 1973

Benzoate-grown cells of Pseudomonas putida(arvilla) mt-2 contain bothmetapyrocatechase and pyrocatechase activities, although the former activity ismuch higher than that of the latter. A spontaneous mutant deficient inmetapyrocatechase and 2-hydroxymuconic semialdehyde hydrolyase, the firsttwo enzymes in the meta-cleavage pathway of the ring of catechol, has beenisolated from this strain. This mutant grows well on a minimal medium containingbenzoate as a sole carbon source and has the high activity of pyrocatechase.These findings indicate that the strain mt-2 possesses the genetic capacity forenzymes of both the meta- and ortho-cleavage pathways of benzoate degrada-tion, but its phenotypic expression is the meta pathway.

A fluorescent pseudomonad designated asPseudomonas putida(arvilla) mt-2 (7) was usedby Nozaki et al. (17) as a source for crystalliza-tion of metapyrocatechase (catechol 2,3-oxygenase; EC 1.13.1.2), which catalyzes theoxygenative cleavage of the ring of catechol toform 2-hydroxymuconic semialdehyde (HMS).According to an extensive taxonomic analysis ofthe fluorescent pseudomonads by Stanier et al.(20), P. putida(arvilla) mt-2 is a typical repre-sentative of the species P. putida biotype A. Itdiffers, however, from other members of thisspecies in the nature of its pathway for thebenzoate metabolism. Whereas most strains ofthis species degrade catechol by the ortho-cleavage pathway, strain mt-2 decomposes ben-zoate through the meta-cleavage pathway (Fig.1). Although strain mt-2 has been reported tocontain no pyrocatechase (catechol 1,2-oxygen-ase; EC 1.13.1.1) (7), the assay for pyrocate-chase in the presence of high levels of metapyro-catechase seemed to require some caution, sincethese two enzymes attack the same substrates,catechol and oxygen.

In this paper we present evidence indicatingthat benzoate-grown cells of P. putida(arvilla)mt-2 do contain pyrocatechase. The presence ofpyrocatechase in wild-type cells was demon-strated by the selective inactivation of meta-pyrocatechase and the isolation of a mutantwhich possesses no meta-cleavage pathway, butdoes possess the ortho-cleavage pathway.

MATERIALS AND METHODSBacterial strains. P. putida(arvilla) mt-2 (ATCC

23973) was supplied by M. Nozaki, Department ofMedical Chemistry, Kyoto University, Kyoto, Japan.Mutant P 100 was obtained spontaneously from thestrain mt-2 as a metapyrocatechase-deficient mutant(see Results). Mutant P 111, which is unable to growon benzoate as a sole carbon source, was isolated fromP 100 as described below. The identity of these strainsas P. putida was established by examining the Gramreaction, motility, microscopic morphology, ability toproduce fluorescent pigment, growth at 42 C, "eggyolk" reaction, and denitrification (20).

Media. Minimal medium M9 without carbonsources (3) was supplemented with 0.2% sodiumbenzoate and 0.05% yeast extract (Difco Laboratories,Detroit, Mich.; BY medium). L broth (11) was used insome experiments as a rich medium. Nutrient agar(Eiken Chemical Co. Ltd., Tokyo, Japan) was supple-mented with 0.2% sodium benzoate where indicated(BN agar). The M9 medium containing 1.5% agar(Eiken) was supplemented with either 1% glucose and0.4% sodium benzoate (GB agar) or 0.2% sodiumbenzoate (B agar).

Culture conditions and preparation of extracts.The cells were grown at 27 C with shaking for 18 h inBY medium. Cultures were harvested by centrifuga-tion, and the cells were washed once with 0.05 Mpotassium phosphate buffer (pH 7.5). Cells weresuspended in this buffer at a density of 7 x 109 to 10o0cells per ml and disintegrated for 1 min in an ice bathwith a 20 kc probe-type Tomy sonic oscillator, modelUR 105 P (Tomy Seiko Co. Ltd., Tokyo, Japan). Thepreparation was centrifuged at 12,000 x g for 20 minat 0 C to remove whole cells and large debris.

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TWO BENZOATE PATHWAYS IN P. PUTIDA mt-2

Enzyme assays. Enzyme assays were performed at27 C with a Hitachi Perkin-Elmer spectrophotometer,model 139 (Hitachi Ltd., Tokyo, Japan). All measure-ments were made in cuvettes with a 1-cm light pathin a total volume of 3.0 ml.

Metapyrocatechase was assayed by measuring therate of formation of HMS (16). Cell-free extracts wereprepared in phosphate buffer containing 10% acetoneto stabilize the enzyme (17). The reaction mixturecontained 0.05 M potassium phosphate buffer (pH7.5), 0.33 mM catechol, and appropriately dilutedcell-free extracts. A 1-Amol amount of HMS gives anoptical density of 14.7 at 375 nm under these condi-tions (16).HMS hydrolyase was determined by measuring the

rate of decrease in the absorbance at 375 nm as thestrongly absorbing substrate was converted to nonab-sorbing products. The substrate for this reaction wasproduced in a cuvette by the incubation of 34 gg ofcrystalline metapyrocatechase (kindly supplied by M.Nozaki) with 33 MM cateehol in 3 ml of 0.05 Mpotassium phosphate buffer (pH 7.5). After the reac-tion was completed, a sample of cell-free extract wasadded to the mixture, and the time course of thereaction was followed.

Pyrocatechase was assayed by measuring the rateof formation of cis,cis-muconate (14). Cell-free ex-tracts were prepared in phosphate buffer withoutacetone. The reaction mixture contained 0.05 Mpotassium phosphate buffer (pH 7.5), 0.33 mM cate-chol, and cell-free extracts. The activity of pyrocate-chase in the extracts of metapyrocatechase-deficientmutants was determined by the addition of theextracts directly to the reaction mixture. To measurethe enzyme activity in the extracts containing highlevels of metapyrocatechase, cell-free extracts weretreated with H202 before assay (see Results). Theconversion of 1 Mmol of catechol to cis, cis-muconatecauses an increase in the absorbance at 260 nm of 5.33optical density units (14).One unit of these enzyme activities was defined as

that which catalyzes either the formation of 1 Mmol ofthe product or the degradation of 1 Mmol of thesubstrate per min. Protein concentrations were deter-mined by the biuret method with bovine serumalbumin (Sigma Chemical Co., St. Louis, Mo.) as astandard (10). The specific activity was defined as 1unit of enzyme activity per milligram of protein.

Fluctuation analysis. The fluctuation analysiswas performed by the method of Luria and Delbriick(12). An overnight culture of the strain mt-2 in Lbroth was diluted in saline to 1.5 x 102 cells per ml.Then 0.1-ml portions were transferred into 11 identi-cal tubes containing 5 ml of L broth. After incubationat 27 C for 15 h with shaking, cultures were dilutedwith saline. A 0.1-ml portion of 10-2 dilutions wasspread on GB agar for the determination of mutants,while a 0.1-ml portion of 10-i dilutions was spriead onnutrient agar for the determination of viable cells. Totest the reliability of the plating method, 10 identicaltubes of 10-2 dilutions were made from one inoculum,and a 0.1-ml portion from each tube was spread on GBagar. The mutation rate was calculated by the for-mula; r = aNtln(NtCa), where r, a, N,, and C

[

coo

benzoate

meta cleavage OHcatechol.

lortho cleavage

metapyrocatechase pyrocatechase

¢>H t cooo- ~~~~coo-C=Os, mCOnat2-hydroxymuconic cis ,ci s-muconate

semialdehyde(HMS)___ ~~~~~~~coo

HO-( o formateCH COO muconolactone3

4-hydroxy-2--ketoval erate

acetaldehyde pyruvate ,o-ketoadipateenol lactone

o =rcoocoo

p-ketoadi pate

succinate acetyl

FIG. 1. The meta- and ortho-cleavage pathways foroxidation of benzoate in P. putida.

represent an average of the number of mutants insamples, the mutation rate, the number of bacteria ina sample, and the number of samples, respectively(12).

Elimination experiments. The effect of "curing"drugs on the mutation frequency was examined asfollows. An overnight culture of strain mt-2 in L brothwas diluted to 2 x 103 cells per ml in L broth. A 0.5-mlportion was transferred into 5 ml of L broth (pH 7.6)without or with either 50 Mug of acridine orange perml, 10% (wt/vol) sodium dodecyl sulfate, or 1 mMethidium bromide. After incubation at 27 C for 24 hwith shaking, cells were plated on both GB agar andnutrient agar after appropriate dilutions in saline.

Mutagenesis and selection of benzoate-nondeg-radative mutants. N-Methyl-N'-nitro-N-nitroso-guanidine (Daiichi Pure Chemicals Co. Ltd., Tokyo,Japan) was used at a final concentration of 150 Mg/ml in 0.1 M sodium citrate buffer (pH 5.5). D-Cyclo-serine screening was carried out as described byCurtiss et al. (4). After the treatment of the strain P100 with the mutagen at 27 C for 90 min, the cells werewashed once with and resuspended in 10 ml of M9medium supplemented with 1% sodium citrate. Af-ter incubation overnight with shaking, collectedand washed cells were suspended in 200 ml of M9medium supplemented with 0.2% sodium benzoate.The culture was aerated for 2 h to starve the mutantcells that were unable to grow on benzoate and to al-low the parent to begin growing. Freshly preparedD-cycloserine in sterile 0.1 M phosphate buffer (pH

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8.0) was added to the culture at a final concentrationof 2 mM. After incubation for 3 h at 27 C with shak-ing, unlysed cells were collected by centrifugation,washed once with 50 ml of the M9 medium, and re-

suspended in 10 ml of the same medium. After ap-

propriate dilution in saline, cells were plated on anM9 agar plate supplemented with 1% sodium cit-rate, and the plates were incubated to permitgrowth. The plates were then velvet-replicated ontoB agar. Mutants which were unable to grow on benzo-ate were selected and purified. One of these mu-

tants lacking pyrocatechase, designated as P 111,was used in this study.

RESULTS

Demonstration of pyrocatechase in P. pu-tida(arvilla) mt-2. Since pyrocatechase andmetapyrocatechase share the common sub-strates, catechol and oxygen, the former activ-ity, even if present in the extracts of P. putida(arvilla) mt-2, might not be detected becauseof the competition of high levels of the latter.In addition, the strongly light-absorbing prod-uct of the latter reaction might interfere withthe spectrophotometric assay of the former. Ithas been established that the active form ofiron in metapyrocatechase is in a ferrous stateand the enzyme activity is quantitatively inac-tivated by H202 due to the conversion of theactive form to the inactive ferric form (18). Incontrast, the active form of pyrocatechase isferric and thus resistant to the oxidant (15).Metapyrocatechase, therefore, could be selec-tively inactivated before carrying out an assayof pyrocatechase.When the cell-free extract of the strain mt-2

grown in BY medium was treated with 40 mMH202, more than 99% of metapyrocatechase was

inactivated (Table 1). On the other hand, the

TABLE 1. Metapyrocatechase and pyrocatechaseactivities in extracts ofP. putida(arvilla) mt-2 before

and after H202 treatment

Sp actaExtracts Metapyrocate-

chase Pyrocatechase

Untreated 0.622 0.138bH202-treatedc 0.002 0.107

a For definition of enzyme activity see Materialsand Methods.

'Initial velocity was determined, since the reactionreached the plateau in approximately 15 s.

c H202 at a final concentration of 40 mM was addedto the extracts (6.8 mg/ml) and incubated at 27 C for30 min. Then bovine liver catalase (BDH ChemicalsLtd., Poole, England) was added (40 enzyme units/ml) followed by incubation for another 10 min toinactivate excess H202.

rate of increase in the absorbance at 260 nm wasreduced by only 20%. The reaction product ofcatechol with the H202-treated extract showedan absorption maximum at 260 nm and thuswas identified as cis, cis-muconate (8), whilethat with the untreated extract showed an

absorption maximum at 375 nm due to HMS(Fig. 2). The possibility of the oxidative conver-sion of metapyrocatechase to pyrocatechasewas excluded, since a completely inactivatedpreparation of crystalline metapyrocatechaseby H202 had no activity of pyrocatechase.Above findings may indicate that the benzoate-grown cells of P. putida(arvilla) mt-2 containpyrocatechase in addition to metapyrocate-chase, which is in contrast to the results ofFeist and Hegeman (7).

Isolation of a mutant defective in the meta-cleavage pathway. The strain mt-2 forms yel-low colonies due to the accumulation of HMSwhen grown on BN agar. We found that a fewbrown colonies appeared among the numerousyellow colonies on this plate. The brown colo-nies were picked up and purified twice bystreaking on nutrient agar plates, one of whichwas designated as P 100. Mutant P 100 and thewild type of the strain mt-2 were grown in BYmedium, and cell-free extracts were prepared.In Table 2 are shown the specific activities ofmetapyrocatechase, HMS hydrolyase, and py-

rocatechase in these extracts. Mutant P 100

1.0

0.6L/)

Lu

-j

< 0.4P-10-C_

0.2

0

250 300 350 400 450WAVE LENGTH (nm)

FIG. 2. Absorption spectra of the reaction productsof catechol with untreated and H202-treated extractsof P. putida(arvilla) mt-2. Catechol (33 ,uM) in 3 ml of0.05 M potassium phosphate buffer (pH 7.5) wasincubated at 27 C for 5 min with either 0.36 mg of theuntreated ( ) or 0.72 mg of the H202-treatedextracts (--- -) prepared as described in Table 1.

,,^\%I %I I

I I

I II I

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TABLE 2. Specific activities of enzymes in extracts ofthe wild type and mutant P 100 of P. putida(arvilla)

mt-2

Enzymes Wild type Mutant P 100

Metapyrocatechase 0.944 < 0.0001bHMS hydrolase 0.0048 < 0.0001Pyrocatechase 0.059 0.x254

a For definition of enzyme activity see Materialsand Methods.bThe minimum detectable levels of activity are

indicated by the less-than-or-equal-to signs (<).

contained no detectable level of either metapy-rocatechase or HMS hydrolase, the first twoenzymes concerned in the meta cleavage of thering of catechol. On the other hand, the activityof pyrocatechase in P 100 was much higher thanthat of the wild-type strain. More than fiveclones which formed brown colonies on BN agarwere selected, and the enzyme activities weredetermined in the extracts, all of which gave thesame results as P 100. Although P 100 had noenzyme activity concerned in the meta-cleavagepathway, it grew much better than the wild-type strain in the M9 medium containing ben-zoate as a sole carbon source (Table 3). Thus, itwas reasonable to conclude that benzoate isdegraded through the ortho-cleavage pathwayin mutants defective in the enzymes of themeta-cleavage pathway.Analysis of the meta-cleavage-deficient

mutation. To analyze the mutation of thedeficiency of the meta-cleavage pathway moreprecisely, a selection medium for mutants wassought. In Table 3 are shown the effects ofbenzoate on the growth of the wild type and P100 of the strain mt-2. It is evident that thegrowth of the wild type in M9 medium contain-ing 0.2% sodium benzoate as a sole carbonsource was much slower than that of P 100.Furthermore, the growth of the wild type onglucose was inhibited by the addition of benzo-ate more severely than that of strain P 100. Theaddition of benzoate to M9 medium containingcitrate completely inhibited the growth of bothstrains. GB agar which contains 1% glucose and0.4% sodium benzoate was chosen for the selec-tion of the meta-cleavage-deficient mutants. InFig. 3 are shown large and small colonies on aGB plate which correspond to the mutant andthe wild-type cells of the strain mt-2, respec-tively.To establish that the observed mutation is

really a spontaneous one, a fluctuation analysiswas performed. The number of mutants appear-ing on plates of GB agar fluctuated widely from

tube to tube (x2 = 164, where X2 = Z(Xi -_) 2/5ZIn the control experiment in which sampleswere plated from a single culture, the narrowstatistical distribution of about the mean (x2 =3.6) was observed. The mutation rate wascalculated to be 4.2 x 10-4 mutations perbacterium per generation. The mutation ratewas not accelerated by the addition of acridineorange, sodium dodecyl sulfate, or ethidiumbromide.Mutant P 100 was stable during 20 successive

transfers on solid media. To determine whetherthe spontaneous mutation in P 100 is a single-site mutation or a deletion, further mutantswere isolated from P 100 which were unable togrow on benzoate. Strain P 111, one of thesemutants lacking pyrocatechase, was tested forits ability to revert spontaneously. Spontaneousrevertants appeared on B agar with the rever-sion frequency of 3 x 10-9, but none of these

TABLE 3. Effect of benzoate on growth of thewild type and mutant P 100 of P. putida(arvilla) mt-2

Doubling time (min)Growth substrate(s)a

Wild type Mutant P 100

Benzoate in M9 225 94Glucose in M9 82 76Glucose + benzoate in M9 105 80Citrate in M9 72 66Citrate + benzoate in M9 00 00

a Substrate concentrations are 0.2% for sodiumbenzoate and 1% for the other carbon sources.

FIG. 3. Appearance of large colonies among thenumerous small colonies on a GB agar plate. Largecolonies were isolated and identified as meta-cleav-age-deficient mutants by the formation of browncolonies on BN agar.

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formed yellow colonies on BN agar. Therefore,these revertants regained the ability to grow on

benzoate using the ortho pathway like P 100,the parent strain from which P 111 was derived.Thus, the spontaneous mutation from mt-2 to P100 was tentatively assumed to be a deletionmutation.

DISCUSSION

It has occasionally been reported that some

strains of the fluorescent pseudomonad capableof decomposing naphthalene, salicylate, ben-zenesulfonate, and/or phenol by the meta-cleav-age pathway decompose benzoate through theortho-cleavage pathway (1, 5, 6). These obser-vations were interpreted to mean that thesestrains possess the genetic capability to decom-pose catechol through both the meta- andortho-cleavage pathways and that the pheno-typic expression is determined by the chemicalnature of the aromatic precursor with which theorganisms are grown (6).The strain mt-2 of P. putida(arvilla) is an

exception since this fluorescent pseudomonaddecomposes catechol through the meta-cleavagepathway even though grown on benzoate. Evi-dence is presented in this paper indicating thatstrain mt-2 has both the ortho and meta path-ways genetically, but the phenotypic expressionof the former is repressed. These results are

consistent with the recent report by Murray etal. (13) which provides further evidence for theoperation of the ortho pathway in the mt-2strain.

Regulation of the synthesis of the enzymes ofthe ortho pathway has been studied in strains P.putida (19) and P. aeruginosa (9), which de-grade catechol exclusively through the orthopathway. In both cases, it has been shown thatpyrocatechase is product induced by cis, cis-muconate and that catechol itself is not an

inducer. Accordingly, when such strains are

grown at the expense of benzoate, the inductionof pyrocatechase results from the intracellularaccumulation of cis, cis-muconate, which isformed metabolically from catechol through thebasal activity of the oxygenase. The enzymes ofthe meta pathway, however, are induced by theprimary substrate, cresols or phenol in P. pu-

tida (6). It has also been reported that catecholand HMS cannot serve as functional inducers ofthe meta pathway in P. putida (7).

It is possible, therefore, to explain the repres-sion of the ortho pathway in strain mt-2 on thebasis of the differences in the mode of regulationof these two pathways. When cells are grown on

benzoate, the meta pathway is induced bybenzoate, and catechol is degraded mainly

through this pathway. Accordingly, cis, cis-muconate does not accumulate enough to in-duce pyrocatechase. In contrast, the loss of themeta pathway in P 100 causes the accumulationof catechol and cis, cis-muconate as well, whichleads to the induction of pyrocatechase (Table2).

It is of interest that, although the specificactivity of metapyiocatechase in the wild-typestrain is higher than that of pyrocatechase inmutant P 100, the mutant grows much betterthan the wild type on a medium containingbenzoate as a sole carbon source (Table 3). Onepossible explanation for this finding is that thebenzoate oxygenase system which catalyzes theconversion of benzoate to catechol is repressedsomehow in the wild-type strain.A similar complete shift from use of the meta

pathway to the ortho pathway to that caused bythe mutation of mt-2 to P 100 was observed byFeist and Hegeman while studying phenol deg-radation through the meta pathway by P.putida U (6). A mutant unable to grow onphenol yielded a revertant which is able to growon phenol but uses the ortho pathway. In theircase, the revertant is derived from the wild-typeP. putida U by two mutational events, while P100 is derived from P. putida(arvilla) mt-2 by asingle spontaneous mutation.The fact that none of the spontaneous revert-

ants derived from P 111, a pyrocatechase-defi-cient mutant, possesses the meta pathway sug-gests that the mutation in P 100 is a deletionrather than a point mutation. No detection ofHMS hydrolyase could not be attributable to asecondary event accompanied by a loss of met-apyrocatechase, since the hydrolyase has beenshown to be induced not by the substrate but bybenzoate itself (7). Thus, the simultaneous lossof these two enzymes in P 100 might alsosuggest a deletion of the genes specifying theenzymes of the meta pathway, which may existas a gene cluster.

Recently, after this study had been com-pleted, Chakrabarty (2) reported that the genesof the meta-cleavage pathway concerning in thedegradation of salicylate in P. putida Rl existon an extrachromosomal element and that theloss of the salicylate gene cluster is enhancedonly by mitomycin C but not by other curingdrugs such as acridine orange, acriflavine, or so-dium dodecyl sulfate. It seems possible, there-fore, that the gene cluster of the meta-cleavagepathway of benzoate degradation in P. putida(arvilla) mt-2 may also exist as a plasmid. Thehigh spontaneous mutation rate observed by thefluctuation analysis for the spontaneous metapathway mutants is further suggestive of plas-mid loss.

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2. Chakrabarty, A. M. 1972. Genetic basis of the biodegra-dation of salicylate in Pseudomonas. J. Bacteriol.112:815-823.

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5. Davis, J. I., and W. C. Evans. 1964. Oxidative metabo-lism of naphthalene by soil pseudomonads. The ringfission mechanism. Biochem. J. 91:251-261.

6. Feist, C. F., and G. D. Hegeman. 1969. Phenol andbenzoate metabolism by Pseudomonas putida; regula-tion of tangenitial pathways. J. Bacteriol. 100:869-877.

7. Feist, C. F., and G. D. Hegeman. 1969. Regulation of themeta cleavage pathway for benzoate oxidation byPseudomonas putida. J. Bacteriol. 100:1121-1123.

8. Hayaishi, O., M. Katagiri, and S. Rothberg. 1957. Stud-ies on oxygenases. Pyrocatechase. J. Biol. Chem.229:905-919.

9. Kemp, M. B., and G. D. Hegeman. 1968. Genetic controlof the ,-ketoadipate pathway in Pseudomonasaeruginosa. J. Bacteriol. 96:1488-1499.

10. Layne, E. 1957. Spectrophotometric and turbidimetricmethods for measuring proteins, p. 447. In S. P.Colowick and N. 0. Kaplan (ed.), Methods in en-

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13. Murray, K., C. J. Duggleby, J. M. Sala-Trepat, and P. A.Williams. 1972. The metabolism of benzoate andmethylbenzoates via the meta-cleavage pathway by.Pseudomonas arvilla mt-2. Eur. J. Biochem.28:301-310.

14. Nakazawa, T., and A. Nakazawa. 1970. Pyrocatechase(Pseudomonas), p. 518. In H. Tabor and C. W. Tabor(ed.), Methods in enzymology, vol. 17A. AcademicPress Inc., New York.

15. Nakazawa, T., M. Nozaki, 0. Hayaishi, and T. Yamano.1969. Studies on pyrocatechase. II. Electron spin reso-nance and other properties of iron in the active center.J. Biol. Chem. 244:119-125.

16. Nozaki, M. 1970. Metapyrocatechase (Pseudomonas), p.

522. In H. Tabor and C. W. Tabor (ed.), Methods inenzymology, vol. 17A. Academic Press Inc., New York.

17. Nozaki, M., H. Kagamiyama, and 0. Hayaishi. 1963.Metapyrocatechase. I. Purification, crystallization andsome properties. Biochem. Z. 338:582-590.

18. Nozaki, M., K. Ono, T. Nakazawa, S. Kotani, and 0.

Hayaishi. 1968. Metapyrocatechase. II. The role of ironand sulfhydryl groups. J. Biol. Chem. 243:2682-2690.

19. Omston, L. N. 1966. The conversion of catechol andprotocatechuate to ,-ketoadipate by Pseudomonas pu-tida. IV. Regulation. J. Biol. Chem. 241:3800-3801.

20. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966.The aerobic pseudomonads: a taxonomic study. J. Gen.Microbiol. 43:159-271.

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