JOURNAL OF BACTERIOLOGY, Dec. 1983, p. 1123-11290021-9193/83/121123-07$02.00/0Copyright X 1983, American Society for Microbiology

Vol. 156, No. 3

Clustering of Mutations Affecting Central Pathway Enzymesof Carbohydrate Catabolism in Pseudomonas aeruginosa

RANDALL A. ROEHL,t THOMAS W. FEARY,t AND PAUL V. PHIBBS, JR.*Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth

University, Richmond, Virginia 23298

Received 5 July 1983/Accepted 12 September 1983

Mutations in carbohydrate-negative mutants of Pseudomonas aeruginosaPAO1 individually deficient in glucose 6-phosphate dehydrogenase (zwJ), 6-phosphogluconate dehydratase (edd), or pyruvate carboxylase (pyc) were mappedon the chromosome by plasmid R68.45-mediated conjugation and by bacterio-phage F116L-mediated transduction. Loci for all three genes were located in the45- to 55-min region of the chromosome; both zwf-l and edd-l were linked bytransduction to nalA, whereas pyc-2 was linked by conjugation to argF10. Thezwf-J mutation exhibited cotransduction frequencies of >95% with both edd-i andthe hex-9001 marker, a mutation reported to prevent growth on hexoses. Thelatter mutation was shown to cause a specific deficiency in 2-keto-3-deoxy-6-phosphogluconate aldolase activity and was redesignated eda-9001. These resultsdemonstrate tight clustering of the gene loci for glucose 6-phosphate dehydroge-nase and for both enzymps unique to the Entner-Doudoroff-pathway in P.aeruginosa. Our evidence suggests supraoperonic clustering of these and otherinducible carbohydrate catabolic genes in the 45- to 55-min region of thechromosome.

The catabolism of glucose, gluconate, andmannitol occurs via the inducible Entner-Dou-doroff pathway in Pseudomonas aeruginosa(Fig. 1). An essential role for this pathway in themetabolism of these substrates is evident fromthe growth properties of mutants that cannotconvert these substrates to the central metabo-lite 6-phosphogluconate or that fail to form ac-tive enzymes of the Entner-boudoroff pathway.For example, mutants deficient in inducible glu-cose 6-phosphate dehydrogenase (zwf muta-tions) fail to grow in mannitol minimal medium,although they grow aerobically at nearly wild-type rates on glucose or gluconate (20) by utiliz-ing the membrane-associated direct oxidativepathway (8). However, zwf mutations totallyblock anaerobic glucose utilization by denitrify-ing cells in which membrane}associated glucosedehydrogenase is not expressed (8). Mutantsthat are deficient in the first enzyme of theEntner-Doudoroff pathway, 6-phosphogluco-nate dehydratase (EDD) (edd mutations), areunable to convert 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate and thus are un-able to utilize glucose, gluconate, or mannitol(2), Other pleiotropic carbohydrate-negative

t Present address: Genex Corp., Gaithersburg, MD 20877.t Present address: Warner Lambert Co., Morris Plains, NJ

07950.

mutant strains of P. aeruginosa that are defi-cient in pyruvate carboxylase (pyc mutations)fail to grow on all six-carbon carbohydrates,glycerol, lactate, and pyruvate (19).Although the physiological and enzymatic

characteristics of several mutant strains that aredefective in carbohydrate utilization have beendescribed, virtually nothing is known of thearrangement of carbohydrate catabolic genes onthe P. aeruginosa chromosome. Transductionalanalyses by two-factor crosses have shown thatthe edd and pyc mutations are not linked (19).However, linkage relationships between thesemutations and other genetic markers have notbeen established. In this study, we mapped thezwf, edd, and pyc mutations on the P. aerugin-osa chromosome, determined the specific defectcaused by the previously uncharacterized hex-9001 mutation (7, 25), and demonstrated tightlinkage of this allele with zwf and edd (R. A.Roehl, P. V. Phibbs, Jr., and T. W. Feary,Abstr. Annu. Meet. Am. Soc. Microbiol. 1982,K2, p. 136).

MATERIALS AND METHODSBacterial strns and growth media. All bacterial

strains (Table 1) were derived from P. aeruginosaPA01 (7). Procedures for nitrosoguanidine mutagene-sis and isolation of mutant strains containing zwf, edd,and pyc mutations have been described (19, 20). StrainPA01838 containing the eda-9001 (hex-9001) marker

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1124 ROEHL, FEARY, AND PHIBBS

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3-DEOXY-JCONATE

PYRUVATE )

pPEPHOSPHOENOLPYRUVATE

2-P-G LYCERAT E

FIG. 1. Carbohydrate catabolic pathways in P. aeruginosa. Abbreviations: pgi, phosphoglucoisomerase;zwf, glucose 6-phosphate dehydrogenase; edd, 6-phosphogluconate dehydratase; eda, 2-keto-3-deoxy-6-phos-phogluconate aldolase; tpi, triosephosphate isomerase; fda, fructose-1,6-diphosphate aldolase; fdp, fructose-1,6-diphosphate phosphatase; fpk, fructose 1-phosphate kinase; pts, phosphotransferase system; mtr, mannitoltransport system; mdh, mannitol dehydrogenase; frk, fructokinase; pyc, pyruvate carboxylase. PEP, phospho-enolpyruvate; TCA, tricarboxylic acid; EMP, Embden-Meyerhoff pathway.

was kindly provided by H. Matsumoto, Shinshu Uni-versity, Matsumoto, Japan. Spontaneous nalidixicacid-resistant mutants were selected on succinate min-imal agar medium that contained nalidixic acid (400ptg/ml). Strain PRP894 (his-50) was isolated by treatingstrain PFB98 with ethyl methanesulfonate as de-scribed previously (24) and spreading the mutagenizedcells on succinate minimal agar medium containinglimiting amounts of histidine (2.5 Lg/ml). Small colo-nies were purified and tested for histidine auxotrophy.

Cells were cultured routinely in either basal saltsmedium (4) or in complex (T) medium that containedthe following (grams per liter): tryptone, 5; yeastextract, 2.5; glucose, 1; and NaCl, 8.5. Stock solutionsof carbon sources were filter sterilized and added tobasal salts medium to a final concentration of 20 mM.Lower final concentrations were employed for benzo-ate (10 mM) and tyrosine (5 mM). Amino acid supple-ments were used at 25 ,ug/ml in solid medium and 50,ug/ml in liquid medium. Solid medium contained 1.5%agar.Growth conditons, extract preparton, and enzyme

assays. Crude cell extracts used in enzyme assays wereprepared from cells that were cultured in 300 ml ofbasal salts medium containing 10 mM glucose plus 20mM lactate and harvested in the late exponential phaseby centrifugation. Cells were disrupted by passagethrough a French pressure cell (15,000 lb/in2 at 50C),and the soluble supernatant fractions of crude extractswere collected after centrifugation (105,000 x g) for 2h at 40C. The combined activity of EDD (EC 4.2.1.12)and the Entner-Doudoroff pathway enzyme 2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) (EC4.1.2.14) and the activity of EDA alone were deter-

mined in soluble extract fractions by using previouslydescribed spectrophotometric methods (2, 9, 10). Theprotein concentration in cell extracts was determinedby the direct spectrophotometric method of Kalb andBernlohr (11).

Genetic procedures. Procedures for R68.45-mediatedchromosome mobilization and for conjugative self-transfer of the plasmid have been described (24).Generalized transducing bacteriophage F116L (13)was employed in transduction experiments on solidmedium as described previously (19).

Chemicals. Pyridine nucleotides, commercial cou-pling enzymes, enzyme substrates, and other reagentsused in enzyme assays were of the highest purityavailable from either P-L Biochemicals, Inc., Milwau-kee, Wis., or Sigma Chemical Co., St. Louis, Mo.Other chemicals were of reagent-grade purity, pur-chased from J. T. Baker Chemical Co., Phillipsburg,N.J., Fisher Scientific Co., Pittsburgh, Pa., or SigmaChemical Co. Tryptone, yeast extract, and agar werepurchased from Difco Laboratories, Detroit, Mich.

RESULTS

Growth properties and enzyme deficiencies inmutant strains. The growth characteristics andspecific defects of strains that contain zwf, edd,and pyc mutations have been described (2, 19,20). The hexose-negative mutant PA01838 con-tained a mutation designated hex-9001, and itsgrowth phenotype was found to be essentiallyidentical to that of strains which contain eddmutations (inability to utilize glucose, gluconate,

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CLUSTERED CATABOLIC GENES IN P. AERUGINOSA 1125

TABLE 1. Description of bacterial strains of P. aeruginosa

Strain Donor Genotype' Derivation or source' Reference

PA01 FP- Prototroph 7PAO8 FP- (R68.45) met-28 ilv-202 str-l(R68.45) (CbF Tcr 5

KmrPA0236 FP- his-4 ilv-226 lys-12 met-28 nalA2 proA82 5

trp-6PA01632 FP- ami-1Sl hutC107 hutU108 ClarkecPA01838 FP- met-9020 eda-9001 MatsumotodPA02369 FP- catAl cnu-9001 met-9020 nar-9011 Matsumoto

puuE8 tyu-9025PFB9 FP- edd-l 2PFB14 FP- pyc-2 19PFB34 FP- pyc-4 19PFB52 FP- edd-2 2PFB98 FP- zwf-1 20PFB131 FP- pgi-3 20PFB811 FP- argF1O leu-10 nal-S4 24PFB818 FP- argF1O leu-10 nal-54 rif-50 24PFB851 FP- catAl cnu-9001 met-9020 nal-71 nar- PA02369, Nalr, spon. mut.

9011 puuE8 tyu-9025PFB890 FP- ami-1SI hutC107 hutU108 nal-73 PA01632, Nalr, spon. mut.PFB894 FP- his-50 zwf-l PFB98, His-, EMS

a All gene designations are based on the P. aeruginosa chromosomal map of Royle et al. (25), except for ami-151 (amiE), zwf-J and pgi-3 (20), nal-54 and rif-50 (24), and new gene designations edd, pyc, and eda-9001.

b Abbreviations: Nalr, nalidixic acid resistance; spon. mut., spontaneous mutation; His-, histidine require-ment; EMS, ethyl methanesulfonate mutagenesis.

c P. H. Clarke, University College, London, England.d H. Matsumoto, Shinshu University, Matsumoto, Japan.

or mannitol and very slow growth on fructose orglycerol). A soluble cell extract of strainPA01838 was found to lack the combined activi-ty of EDD-EDA (Table 2). However, the extractfrom PA01838 complemented extracts from eddmutants PFB9 and PFB52 in assays for EDD-EDA activity, indicating the presence of EDDactivity in the PA01838 extract. Direct assay ofEDA activity, using 2-keto-3-deoxy-6-phospho-gluconate as substrate, showed a specific defi-ciency of EDA activity in the extract of strainPA01838 (Table 2). Thus, we have replaced theoriginal hex-9001 designation with eda-9001 toreflect the specific mutant phenotype.

Conjugational mapping. Chromosome-mobi-lizing plasmid R68.45 was used in mapping ge-netic loci for the zwf-l and pyc-2 mutations byidentifying linkage with other known geneticmarkers. A zwf-J donor strain carrying R68.45was constructed by mating PA08(R68.45) withPFB98 (zwf-J) and selecting carbenicillin resist-ance on succinate minimal medium. APFB98(R68.45) isolate from this mating wascrossed with recipient strains PA0236 andPRP811 containing various mapped auxotrophicmarkers (Table 1; Fig. 2), and the inheritance ofwild-type alleles was selected in transconju-gants. Coinheritance of zwf-l in progeny cellswas scored by testing growth on mannitol mini-mal medium. No conjugational linkage of zwf-l

was observed with ilv-226, his4, lys-12, met-28,trp-6, or pro-82 (data not shown). However,when Arg+ was selected in matings with recipi-ent strain PRP811, zwf and argF loci werecoinherited at a frequency of 91% (Table 3).Since zwf-1 appeared to be located in the lateregion of the chromosome, Leu+ transconju-gants of recipient strain PRP811 also were se-lected. However, Leu+ transconjugants wereobtained only at a very low frequency inPFB98(R68.45) x PRP811 crosses. This resultmost likely was caused by the frequent coinheri-tance of Nals donor DNA with the Leu+ allele(Fig. 2). This would be a lethal event for trans-conjugants, since nalidixic acid was employed tocounterselect donor cells. As an alternativecounterselection strategy, an auxotrophic His-derivative of zwf-l mutant strain PFB98 wasisolated (strain PRP894) and R68.45 was trans-ferred as before by conjugation fromPA08(68.45) to PRP894. Strain PRP894(R68.45)was then crossed with recipient strain PRP851containing mapped genetic markers in the lateregion of the chromosome. The results (Table 3)show that zwf-l exhibited 40% linkage with met-9020, confirming a location in the 45- to 55-minregion of the chromosome.The pyc-2 mutation in strain PFB14 was

mapped in a similar manner by crossingPFB14(R68.45) with appropriate recipient

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1126 ROEHL, FEARY, AND PHIBBS

TABLE 2. Complementation analysis of Entner-Doudoroff pathway enzyme activities in cell extracts

of mutant strainsaSp act (nmol min' nig

Strains (genotype) of protein-')EDD-EDA EDA

PA01 (wild type) 115 184PFB9 (edd-1) <lb 159PFB52 (edd-2) <1 -cPA01838 (eda-9001) <1 3PFB9 (edd-1) + 9PFB52 (edd-2)

PA01838 (eda-9001) + 280PA01 (wild type)

PA01838 (eda-9001) + 157PFB9 (edd-1)

PA01838 (eda-9001) + 355PFB52 (edd-2)a Enzyme activity was determined in soluble frac-

tions of crude extracts prepared from cells grown inbasal salts medium containing 20 mM lactAte plus iomM glucose. For complementation assays, equal vol-umes of extracts (50 >J each) containing approximatelyequal concentrations of protein were added to reactionmixtures.

b <1, Activity below the limit of detection.c, Activity not determined.

strains. Transconjugants were scored for coin-heritance of pyc-2 by testing for growth ongluconate minimal medium.,The pyc-2 mutationexhibited an 8% frequency of coinheritance withthe argF locus at 45 min on the chromosomemap, but no linkage was observed with eithercatA or tyu-9025 in conjugations performed withrecipient strain PRP851 (Table 3; Fig. 2).

Transductional mapping. Two-factor trans-ductional crosses between mutants that con-tained zwf, edd, pyc, and pgi mutations wereperformed to assess linkage between thesemarkers. Each of these mutations preventedgrowth of P. aeruginosa on mannitol minimalmedium; pgi mutations cause a deficiency in theconstitutively expressed phosphoglucoisomer-ase (20). Bacteriophage F116L was propagatedon mutant strains containing each mutation. Thephage lysates were adjusted to contain 1010PFU/ml and then used to transduce the variousstrains. Transductants of each recipient strainwere selected by growth on mannitol minimalmedium (Table 4). The extremely low frequencyof occurrence of mannitol-positive transductantsin crosses between strains containing edd-J andedd-2 demonstrated that these mutations aretightly linked and are probably in the same gene.Mannitol-positive transductants also were ob-served at low frequencies in crosses betweenstrains that contained zwf-l and either edd allele,indicating that zwf-l is in a different, but closelylinked, gene. In contrast, much higher frequen-

J. BACTERIOL.

cies of transduction to the mannitol-positivephenotype indicated that pyc4 and pgi-3 areunlinked mutations and that neither allele isclosely linked with zwf-1, edd-1, or edd-2.The specific frequency of cotransduction of

zwf-l and edd-J was determined by transducingstrain PFB9 (edd-l), using phage F116L propa-gated on strain PFB98 (zwf-1). Transductantswere selected on gluconate minimal medium,an4 the coinheritance of zwf-1 was scored bytesting for th; ability to grow on mannitol mini-mal medium. edd-l and zwf-J loci were coinher-ited at a frequency of 99% (Table 5), which isconsistent with the tight linkage that was ob-served in quantitative 2-factor crosses (Table 4).The eda-9001 mutatioth has beehi mapped by

plasmid FP5-mediated conjugation in the lateregion of the P. aeruginosa chromosome nearmet-9020 and leu-10 (7, 21). The tight transduc-tional linkage of zwf-J and edd-J and the conju-gational mappipg of zwf-J and eda-9001 indicat-ed that eda-9Q0QI might be cotransducible withzwf-l and edd-l. Linkage of zwf-J and eda-9001was tested by transducing strain PA01838 (eda-9001) to gluconate positive, using F116L propa-gated on strain PFB98 (zwf-1), and scoring trans-ductants for coinheritance of zwf-l on mannitolminimal medium. The results showed 96% co-transduction of zwf-J and eda-9001 (Table 5).Therefore, zwf-1, edd-l, and eda-9001 are clus-tered in the 50- to 55-min region of the chromo-some near leu-10 and met-9020.The cluster was mapped more precisely by

determining transductional linkage of these mu-tations to other previously mapped markers inthis region of the chromosome. The results from

ilv-226

tyu-9029

cotA -

met-902-0 Ileu-10 Hmi-151 argF

edd-I ptsj

eda-9001 pyc-2zwf-InalA

- his-4

-Iys-12

met-28

trp-6

proA

FIG. 2. Chromosomal map of P. aeruginosa PA01based on the map of Holloway and Crockett (6).Linked genetic markers are enclosed in brackets.

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CLUSTERED CATABOLIC GENES IN P. AERUGINOSA 1127

TABLE 3. Conjugational mapping of zwf-l and pyc-2"Coinheritance frequency

Donor Recipient Selectionzwf-l pyc-2

PFB98(R68.45) PRP811 (argFIO) Arg+ 0.91 (87/96)bPRP894(R68.45) PRP851 (met-9020) Met+ 0.40 (93/230)PFB14(R68.45) PRP851 (catAl) Ben+c <0.01 (0/72)PFB14(R68.45) PRP851 (tyu-9025) Tyu+d <0.01 (0/70)PFB14(R68.45) PRP818 (argFiO) Arg+ 0.08 (12/150)

a Conjugations with chromosome-mobilizing plasmid R68.45 were done as described previously (24). Donorcells were counterselected with nalidixic acid (400 ,ug/ml) in the selective solid media.

b Numbers in parentheses indicate the number of transconjugants containing the unselected marker pernumber of transconjugants scored.

c Ben', Benzoate utilization.d Tyu+, Tyrosine utilization.

F116L-mediated transductions (Table 5) showedthat zwf-l was not linked to either ami-151(PFB98 x PRP890 cross) or leu-10 (PRP811 xPFB98 cross); eda-9001 also was not linked tomet-9020 (PAQ1 x PA01838 cross). However,the zwf-1, eda-9001, and edd-i mutations didexhibit 44, 39, and 31% cotransduction frequen-cies, respectively, with nalA54 in strain PRP811.Nearly identical linkage values were obtainedwhen coinheritance was determined with thenalA2 mutation in strain PA0236. Thus, the zwf-eda-edd cluster is linked to the nalA locus and islocated between ami-151 and leu-10. The pro-posed gene order is shown in Fig. 3.

DISCUSSIONFour genes corresponding to inducible en-

zymes of the central pathways of carbohydratecatabolism were located in the 45- to 55-minregion of the P. aeruginosa chromosome map.Three of these genes were tightly clustered, asshown by cotransduction of zwf with both edaand edd at frequencies of >95% (Table 5). Therelative gene order deduced from the presentconjugational and transductional crosses placesthe zwf-eda-edd gene cluster between ami-151and leu-10 (Fig. 3). All three genes of the cluster

were linked by transduction with nalA, whichmaps between ami-151 and leu-10, but they didnot exhibit transductional linkage with either ofthe latter markers. The previously uncharacter-ized hex-9001 mutation (redesignated eda-9001in this study) was placed between nalA and ami-151 in earlier maps of the P. aeruginosa chromo-some (6, 7, 21), based on very low frequencies ofcotransduction of eda-9001 and leu-10. Howev-er, additional carbohydrate catabolic markers,specifying glycerol utilization, recently havebeen identified in this region of the chromo-some, and they exhibit transductional linkage toami-151, nalA, and the zwf-eda-edd cluster.Those results indicated the relative gene ordershown in Fig. 3 (S. M. Cuskey and P. V. Phibbs,Jr., Abstr. Annu. Meet. Am. Soc. Microbiol.1983, K39, p. 183). The relative gene orderwithin the zwf-eda-edd cluster was based on thefrequencies of cotransduction of each locus withthe nalA marker (Table 5; Fig. 3). Due to thenearly identical effects of the edd and eda muta-tions on carbohydrate utilization, it was notpossible for us to employ three-point transduc-tional analysis to determine more precisely thegene order within this cluster.The activities of glucose 6-phosphate dehy-

TABLE 4. Quantitative two-factor transductional crossesaNo. of mannitol-positive transductants of donor strain':

Recipient strain PFB9 PFB52 PFB98 PFB34 PFB131 PA01(edd-1) (edd-2) (zwf-I) (pyc4) (pgi-3) (wild type)

PFB9 0 1 11 72 209 127PFBS2 2 1 15 95 325 168PFB98 10 8 0 181 412 248PFB34 73 107 126 0 125 93PFB131 208 227 179 162 0 189None 0 0 0 0 0 0

a Plate transductions were done by spreading 10' PFU of phage F116L on mannitol minimal medium seededwith 108 recipient cells. Donor and recipient strains have been grouped according to their proposed linkagearrangement.

I Numbers represent the average number of transductant colonies per transduction plate, after adjusting forlow frequencies of spontaneous reversion that were determined on control plates that received no phage.

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1128 ROEHL, FEARY, AND PHIBBS

TABLE 5. Frequencies of cotransduction of zwf-1, edd-1, and eda-9001 with markers in the late region of theP. aeruginosa chromosomea.

Donor Recipient strn Selec- Coinheritance with unselected marker'stan Rcpetstrain tionb zwf-1 eda-9(KI) naLA leu-10

PFB98 PFB9 (edd-1) Gnt+ 0.99 (326/329)PFB98 PA01838 (eda-9001) Gnt+ 0.96 (64/67)PFB98 PRP890 (ami-151) Act+ <0.01 (0/223)PA01 PA01838 (met-9020) Met+ <0.01 (0/171)PRP811 PFB98 (zwf-1) Mtl+ 0.44 (175/395) <0.01 (0/395)PRP811 PFB9 (edd-1) Gnt+ 0.31 (58/185) <0.01 (0/185)PRP811 PA01838 (eda-9001) Gnt+ 0.39 (70/179)PA0236 PFB98 (zwf-1) Mtl+ 0.48 (92/190)PA0236 PFB9 (edd-1) Gnt+ 0.42 (78/186)

a Plate transductions were done by spreading 109 PFU of phage F116L on selective minimal media seededwith 108 recipient cells.

b Gnt+, Gluconate utilization; Act+, acetamide utilization; Met+, no requirement for methionine on lactateminimal medium; Mtl+, mannitol utilization.

I Entries represent cotransduction frequency. Numbers in parentheses show the number of transductantscontaining the unselected marker per number of transductants scored.

drogenase, EDD, and EDA are coinduced dur-ing growth of P. aeruginosa on mannitol, glu-cose, gluconate, glycerol, glycerol 3-phosphate,and glycerate (2, 10, 14, 17, 20, 26). 6-Phospho-gluconate has been suggested as the probableinducer metabolite for all three enzymes (8, 20),but whether the corresponding genes constitutea single, coordinately expressed regulatory unithas not been established. The present demon-stration that zwf, eda, and edd are located withina single transductional linkage group supportsthe notion that these functionally related genesare structural elements of a regulatory unit.However, we have no genetic evidence at thistime that a regulatory locus governs the coordi-nate expression of all three enzymes or that thethree genes are contiguous.Most of the genes for glucose catabolism have

been mapped on the Escherichia coli chromo-some, and the possible evolutionary and func-tional significance of their arrangement into four

50

ami-151 nalA zwf-I

gene clusters has been discussed (22, 23). In-spection of the chromosome map of E. colireveals tight clustering of zwf, eda, and edd thatis remarkably similar to the clustered arrange-ment of these three genes in P. aeruginosa.deTorrontegui et al. (3) have reported the su-praoperonic clustering of five genes specifyingglucose utilization in Pseudomonas putida, in-cluding cotransducible eda and edd markers.The location of the five clustered genes, relativeto other chromosomal markers, could not beestablished in that study, and the zwf gene hasnot been mapped in P. putida.Pyruvate carboxylase is an inducible anapler-

otic enzyme that is essential for the utilization ofall carbohydrates by P. aeruginosa. Indepen-dently isolated pyc alleles fall into a singletransductional linkage group, and the gene ap-pears to be expressed as an independent regula-tory unit (19). The pyc mutations did not exhibittransductional linkage with the zwf-eda-edd

do-9001edd-l/

L 0.31 i i i1

, I I0.39 :;;1 0.990.44 F<0.96 1

<0.01 I,IiI <0.01 I

I !-, <0.01 I

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55'

leu-10 met-9020

FIG. 3. Transductional linkage map of the 50- to 55-min region of the P. aeruginosa chromosome. Numbersrepresent cotransduction frequencies, and arrowheads indicate the unselected markers.

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CLUSTERED CATABOLIC GENES IN P. AERUGINOSA 1129

cluster (Table 4); however, they were mapped inthe 45-min region of the P. aeruginosa chromo-some by demonstrating conjugational linkagewith argF10 (Table 3). We also reported recentlythe transductional linkage of tightly clusteredstructural and regulatory genes for the phospho-enolpyruvate-fructose 1-phosphotransferasesystem (pts markers) with both argF10 and lys-9015 in this region of the chromosome (24).Therefore, at least seven different genes specificfor carbohydrate catabolic functions, whichcomprise at least three separate regulatory units(pts loci, pyc, and zwf-eda-edd), have beenmapped within the 45- to 55-min region of the P.aeruginosa chromosome.A number of chromosomal catabolic genes

that have been mapped in Pseudomonas speciesare known to exhibit extensive clustering (1, 3,12, 15, 16, 18, 27). The present results provideanother such example' and provide additionalevidence for the proposed phenomenon of su-praoperonic clustering of dissimilatory path-ways in Pseudomonas species (27, 33).

ACKNOWLEDGMENTSWe are grateful to H. Matsumoto and R. H. Olsen for

providing valuable bacterial cultures.This work was supported by research grant PCM 80-03887

from the National Science Foundation. R.A.R. received sup-port from Public Health Service Grant A107086 from the.National Institute of Allergy and Infectious Diseases. T.W.F.was the recipient ofan A. D. Williams Visiting Scholar Awardat the Medical College of Virginia, Virginia CommonwealthUniversity, Richmond.

LITERATURE CITED1. Betz, J. L., J. E. Brown, P. H. Clarke, and M. Day. 1974.

Genetic analysis of amidase mutants of Pseudomonasaeruginosa. Genet. Res. 23:335-359.

2. Blevins, W. T., T. W. Feary, and P. V. Phibbs, Jr. 1975.6-Phosphogluconate dehydratase deficiency in pleiotropiccarbohydrate-negative mutant strains of Pseudomonasaeruginosa. J. Bacteriol. 121:942-949.

3. deTorrontegul, R. D., M. L. Wheells, and J. L. Canovas.1976. Supra-operonic clustering of genes specifying glu-cose dissimilation in Pseudomonas putida. Mol. Gen.Genet. 144:307-311.

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VOL. 156, 1983

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