Clustering of Mutations Affecting Central Pathway Enzymes

Transcription

1 JOURNAL OF BACTEROLOGY, Dec. 1983, p /83/ $02.00/0 Copyright X 1983, Americn Society for Microbiology Vol. 156, No. 3 Clustering of Muttions Affecting Centrl Pthwy Enzymes of Crbohydrte Ctbolism in Pseudomons eruginos RANDALL A. ROEHL,t THOMAS W. FEARY,t AND PAUL V. PHBBS, JR.* Deprtment of Microbiology nd mmunology, Medicl College of Virgini, Virgini Commonwelth University, Richmond, Virgini Received 5 July 1983/Accepted 12 September 1983 Muttions in crbohydrte-negtive mutnts of Pseudomons eruginos PAO1 individully deficient in glucose 6-phosphte dehydrogense (zwj), 6- phosphogluconte dehydrtse (edd), or pyruvte crboxylse (pyc) were mpped on the chromosome by plsmid R68.45-medited conjugtion nd by bcteriophge F116L-medited trnsduction. Loci for ll three genes were locted in the 45- to 55-min region of the chromosome; both zwf-l nd edd-l were linked by trnsduction to nla, wheres pyc-2 ws linked by conjugtion to rgf10. The zwf-j muttion exhibited cotrnsduction frequencies of >95% with both edd-i nd the hex-9001 mrker, muttion reported to prevent growth on hexoses. The ltter muttion ws shown to cuse specific deficiency in 2-keto-3-deoxy-6- phosphogluconte ldolse ctivity nd ws redesignted ed These results demonstrte tight clustering of the gene loci for glucose 6-phosphte dehydrogense nd for both enzymps unique to the Entner-Doudoroff-pthwy in P. eruginos. Our evidence suggests suproperonic clustering of these nd other inducible crbohydrte ctbolic genes in the 45- to 55-min region of the chromosome. The ctbolism of glucose, gluconte, nd mnnitol occurs vi the inducible Entner-Doudoroff pthwy in Pseudomons eruginos (Fig. 1). An essentil role for this pthwy in the metbolism of these substrtes is evident from the growth properties of mutnts tht cnnot convert these substrtes to the centrl metbolite 6-phosphogluconte or tht fil to form ctive enzymes of the Entner-boudoroff pthwy. For exmple, mutnts deficient in inducible glucose 6-phosphte dehydrogense (zwf muttions) fil to grow in mnnitol miniml medium, lthough they grow erobiclly t nerly wildtype rtes on glucose or gluconte (20) by utilizing the membrne-ssocited direct oxidtive pthwy (8). However, zwf muttions totlly block nerobic glucose utiliztion by denitrifying cells in which membrne}ssocited glucose dehydrogense is not expressed (8). Mutnts tht re deficient in the first enzyme of the Entner-Doudoroff pthwy, 6-phosphogluconte dehydrtse (EDD) (edd muttions), re unble to convert 6-phosphogluconte to 2-keto- 3-deoxy-6-phosphogluconte nd thus re unble to utilize glucose, gluconte, or mnnitol (2), Other pleiotropic crbohydrte-negtive t Present ddress: Genex Corp., Githersburg, MD t Present ddress: Wrner Lmbert Co., Morris Plins, NJ mutnt strins of P. eruginos tht re deficient in pyruvte crboxylse (pyc muttions) fil to grow on ll six-crbon crbohydrtes, glycerol, lctte, nd pyruvte (19). Although the physiologicl nd enzymtic chrcteristics of severl mutnt strins tht re defective in crbohydrte utiliztion hve been described, virtully nothing is known of the rrngement of crbohydrte ctbolic genes on the P. eruginos chromosome. Trnsductionl nlyses by two-fctor crosses hve shown tht the edd nd pyc muttions re not linked (19). However, linkge reltionships between these muttions nd other genetic mrkers hve not been estblished. n this study, we mpped the zwf, edd, nd pyc muttions on the P. eruginos chromosome, determined the specific defect cused by the previously unchrcterized hex muttion (7, 25), nd demonstrted tight linkge of this llele with zwf nd edd (R. A. Roehl, P. V. Phibbs, Jr., nd T. W. Fery, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, K2, p. 136). MATERALS AND METHODS Bcteril strns nd growth medi. All bcteril strins (Tble 1) were derived from P. eruginos PA01 (7). Procedures for nitrosogunidine mutgenesis nd isoltion of mutnt strins contining zwf, edd, nd pyc muttions hve been described (19, 20). Strin PA01838 contining the ed-9001 (hex-9001) mrker 1123

2 1124 ROEHL, FEARY, AND PHBBS J. BACTEROL. CYTOPLA SM C MEMBRANE MANNTOL -,- MANNTOL GLUCOSE GLUCONATE 2-KETOGLUCONATE <> \7\(ll GLUCOSE ATP GLUCOSE-6- P GLUCONATE l ATP AD (P) 2-KETOGLUCO NATE ~~~~~~ATP dh,nad FRUCTOSE fat FRUCTOSE-6-P 6-P-GLUC Pt$ FRUCTOSE - GLYCEROL-3-P - " F- N-pYN. - GLYCEROL - CONATE ( NAD(P) 2-KETO-6- PHOSPHOGLUCONATE 6PEP fpk dd k FRUCTOSE --P Tp> FRUCTOSE- 1,6-P2 2-KETO-33-DEOXY- 6-P-GLUiJCONATE PYRUVATE NPs do fd~~~~~~e DHYDROXYACETONE- P GLYCERALDEHYDE -; -X-----> GLYCEROL-3-P 1,3-P2-GLYCERATE /X pk ATPX At G-O 0~GLYCEROL 3- P- GLYCERATE NAB EMP PYRUVATE ppe PHOSPHOENOLPYRUVATE 2-P-G LYCERAT E FG. 1. Crbohydrte ctbolic pthwys in P. eruginos. Abbrevitions: pgi, phosphoglucoisomerse; zwf, glucose 6-phosphte dehydrogense; edd, 6-phosphogluconte dehydrtse; ed, 2-keto-3-deoxy-6-phosphogluconte ldolse; tpi, triosephosphte isomerse; fd, fructose-1,6-diphosphte ldolse; fdp, fructose-1,6- diphosphte phosphtse; fpk, fructose 1-phosphte kinse; pts, phosphotrnsferse system; mtr, mnnitol trnsport system; mdh, mnnitol dehydrogense; frk, fructokinse; pyc, pyruvte crboxylse. PEP, phosphoenolpyruvte; TCA, tricrboxylic cid; EMP, Embden-Meyerhoff pthwy. ws kindly provided by H. Mtsumoto, Shinshu University, Mtsumoto, Jpn. Spontneous nlidixic cid-resistnt mutnts were selected on succinte miniml gr medium tht contined nlidixic cid (400 ptg/ml). Strin PRP894 (his-50) ws isolted by treting strin PFB98 with ethyl methnesulfonte s described previously (24) nd spreding the mutgenized cells on succinte miniml gr medium contining limiting mounts of histidine (2.5 Lg/ml). Smll colonies were purified nd tested for histidine uxotrophy. Cells were cultured routinely in either bsl slts medium (4) or in complex (T) medium tht contined the following (grms per liter): tryptone, 5; yest extrct, 2.5; glucose, 1; nd NCl, 8.5. Stock solutions of crbon sources were filter sterilized nd dded to bsl slts medium to finl concentrtion of 20 mm. Lower finl concentrtions were employed for benzote (10 mm) nd tyrosine (5 mm). Amino cid supplements were used t 25,ug/ml in solid medium nd 50,ug/ml in liquid medium. Solid medium contined 1.5% gr. Growth conditons, extrct preprton, nd enzyme ssys. Crude cell extrcts used in enzyme ssys were prepred from cells tht were cultured in 300 ml of bsl slts medium contining 10 mm glucose plus 20 mm lctte nd hrvested in the lte exponentil phse by centrifugtion. Cells were disrupted by pssge through French pressure cell (15,000 lb/in2 t 50C), nd the soluble superntnt frctions of crude extrcts were collected fter centrifugtion (105,000 x g) for 2 h t 40C. The combined ctivity of EDD (EC ) nd the Entner-Doudoroff pthwy enzyme 2-keto-3- deoxy-6-phosphogluconte ldolse (EDA) (EC ) nd the ctivity of EDA lone were determined in soluble extrct frctions by using previously described spectrophotometric methods (2, 9, 10). The protein concentrtion in cell extrcts ws determined by the direct spectrophotometric method of Klb nd Bernlohr (11). Genetic procedures. Procedures for R68.45-medited chromosome mobiliztion nd for conjugtive selftrnsfer of the plsmid hve been described (24). Generlized trnsducing bcteriophge F116L (13) ws employed in trnsduction experiments on solid medium s described previously (19). Chemicls. Pyridine nucleotides, commercil coupling enzymes, enzyme substrtes, nd other regents used in enzyme ssys were of the highest purity vilble from either P-L Biochemicls, nc., Milwukee, Wis., or Sigm Chemicl Co., St. Louis, Mo. Other chemicls were of regent-grde purity, purchsed from J. T. Bker Chemicl Co., Phillipsburg, N.J., Fisher Scientific Co., Pittsburgh, P., or Sigm Chemicl Co. Tryptone, yest extrct, nd gr were purchsed from Difco Lbortories, Detroit, Mich. RESULTS Growth properties nd enzyme deficiencies in mutnt strins. The growth chrcteristics nd specific defects of strins tht contin zwf, edd, nd pyc muttions hve been described (2, 19, 20). The hexose-negtive mutnt PA01838 contined muttion designted hex-9001, nd its growth phenotype ws found to be essentilly identicl to tht of strins which contin edd muttions (inbility to utilize glucose, gluconte, )

3 VOL. 156, 1983 TABLE 1. CLUSTERED CATABOLC GENES N P. AERUGNOSA 1125 Description of bcteril strins of P. eruginos Strin Donor Genotype' Derivtion or source' Reference PA01 FP- Prototroph 7 PAO8 FP- (R68.45) met-28 ilv-202 str-l(r68.45) (CbF Tcr 5 Kmr PA0236 FP- his-4 ilv-226 lys-12 met-28 nla2 proa82 5 trp-6 PA01632 FP- mi-1sl hutc107 hutu108 Clrkec PA01838 FP- met-9020 ed-9001 Mtsumotod PA02369 FP- ctal cnu-9001 met-9020 nr-9011 Mtsumoto puue8 tyu-9025 PFB9 FP- edd-l 2 PFB14 FP- pyc-2 19 PFB34 FP- pyc-4 19 PFB52 FP- edd-2 2 PFB98 FP- zwf-1 20 PFB131 FP- pgi-3 20 PFB811 FP- rgf1o leu-10 nl-s4 24 PFB818 FP- rgf1o leu-10 nl-54 rif PFB851 FP- ctal cnu-9001 met-9020 nl-71 nr- PA02369, Nlr, spon. mut puue8 tyu-9025 PFB890 FP- mi-1s hutc107 hutu108 nl-73 PA01632, Nlr, spon. mut. PFB894 FP- his-50 zwf-l PFB98, His-, EMS All gene designtions re bsed on the P. eruginos chromosoml mp of Royle et l. (25), except for mi- 151 (mie), zwf-j nd pgi-3 (20), nl-54 nd rif-50 (24), nd new gene designtions edd, pyc, nd ed b Abbrevitions: Nlr, nlidixic cid resistnce; spon. mut., spontneous muttion; His-, histidine requirement; EMS, ethyl methnesulfonte mutgenesis. c P. H. Clrke, University College, London, Englnd. d H. Mtsumoto, Shinshu University, Mtsumoto, Jpn. or mnnitol nd very slow growth on fructose or glycerol). A soluble cell extrct of strin PA01838 ws found to lck the combined ctivity of EDD-EDA (Tble 2). However, the extrct from PA01838 complemented extrcts from edd mutnts PFB9 nd PFB52 in ssys for EDD- EDA ctivity, indicting the presence of EDD ctivity in the PA01838 extrct. Direct ssy of EDA ctivity, using 2-keto-3-deoxy-6-phosphogluconte s substrte, showed specific deficiency of EDA ctivity in the extrct of strin PA01838 (Tble 2). Thus, we hve replced the originl hex-9001 designtion with ed-9001 to reflect the specific mutnt phenotype. Conjugtionl mpping. Chromosome-mobilizing plsmid R68.45 ws used in mpping genetic loci for the zwf-l nd pyc-2 muttions by identifying linkge with other known genetic mrkers. A zwf-j donor strin crrying R68.45 ws constructed by mting PA08(R68.45) with PFB98 (zwf-j) nd selecting crbenicillin resistnce on succinte miniml medium. A PFB98(R68.45) isolte from this mting ws crossed with recipient strins PA0236 nd PRP811 contining vrious mpped uxotrophic mrkers (Tble 1; Fig. 2), nd the inheritnce of wild-type lleles ws selected in trnsconjugnts. Coinheritnce of zwf-l in progeny cells ws scored by testing growth on mnnitol miniml medium. No conjugtionl linkge of zwf-l ws observed with ilv-226, his4, lys-12, met-28, trp-6, or pro-82 (dt not shown). However, when Arg+ ws selected in mtings with recipient strin PRP811, zwf nd rgf loci were coinherited t frequency of 91% (Tble 3). Since zwf-1 ppered to be locted in the lte region of the chromosome, Leu+ trnsconjugnts of recipient strin PRP811 lso were selected. However, Leu+ trnsconjugnts were obtined only t very low frequency in PFB98(R68.45) x PRP811 crosses. This result most likely ws cused by the frequent coinheritnce of Nls donor DNA with the Leu+ llele (Fig. 2). This would be lethl event for trnsconjugnts, since nlidixic cid ws employed to counterselect donor cells. As n lterntive counterselection strtegy, n uxotrophic Hisderivtive of zwf-l mutnt strin PFB98 ws isolted (strin PRP894) nd R68.45 ws trnsferred s before by conjugtion from PA08(68.45) to PRP894. Strin PRP894(R68.45) ws then crossed with recipient strin PRP851 contining mpped genetic mrkers in the lte region of the chromosome. The results (Tble 3) show tht zwf-l exhibited 40% linkge with met- 9020, confirming loction in the 45- to 55-min region of the chromosome. The pyc-2 muttion in strin PFB14 ws mpped in similr mnner by crossing PFB14(R68.45) with pproprite recipient

4 1126 ROEHL, FEARY, AND PHBBS TABLE 2. Complementtion nlysis of Entner- Doudoroff pthwy enzyme ctivities in cell extrcts of mutnt strins Sp ct (nmol min' nig Strins (genotype) of protein-') EDD-EDA EDA PA01 (wild type) PFB9 (edd-1) <lb 159 PFB52 (edd-2) <1 -c PA01838 (ed-9001) <1 3 PFB9 (edd-1) + 9 PFB52 (edd-2) PA01838 (ed-9001) PA01 (wild type) PA01838 (ed-9001) PFB9 (edd-1) PA01838 (ed-9001) PFB52 (edd-2) Enzyme ctivity ws determined in soluble frctions of crude extrcts prepred from cells grown in bsl slts medium contining 20 mm lctate plus io mm glucose. For complementtion ssys, equl volumes of extrcts (50 >J ech) contining pproximtely equl concentrtions of protein were dded to rection mixtures. b <1, Activity below the limit of detection. c, Activity not determined. strins. Trnsconjugnts were scored for coinheritnce of pyc-2 by testing for growth on gluconte miniml medium.,the pyc-2 muttion exhibited n 8% frequency of coinheritnce with the rgf locus t 45 min on the chromosome mp, but no linkge ws observed with either cta or tyu-9025 in conjugtions performed with recipient strin PRP851 (Tble 3; Fig. 2). Trnsductionl mpping. Two-fctor trnsductionl crosses between mutnts tht contined zwf, edd, pyc, nd pgi muttions were performed to ssess linkge between these mrkers. Ech of these muttions prevented growth of P. eruginos on mnnitol miniml medium; pgi muttions cuse deficiency in the constitutively expressed phosphoglucoisomerse (20). Bcteriophge F116L ws propgted on mutnt strins contining ech muttion. The phge lystes were djusted to contin 1010 PFU/ml nd then used to trnsduce the vrious strins. Trnsductnts of ech recipient strin were selected by growth on mnnitol miniml medium (Tble 4). The extremely low frequency of occurrence of mnnitol-positive trnsductnts in crosses between strins contining edd-j nd edd-2 demonstrted tht these muttions re tightly linked nd re probbly in the sme gene. Mnnitol-positive trnsductnts lso were observed t low frequencies in crosses between strins tht contined zwf-l nd either edd llele, indicting tht zwf-l is in different, but closely linked, gene. n contrst, much higher frequen- J. BACTEROL. cies of trnsduction to the mnnitol-positive phenotype indicted tht pyc4 nd pgi-3 re unlinked muttions nd tht neither llele is closely linked with zwf-1, edd-1, or edd-2. The specific frequency of cotrnsduction of zwf-l nd edd-j ws determined by trnsducing strin PFB9 (edd-l), using phge F116L propgted on strin PFB98 (zwf-1). Trnsductnts were selected on gluconte miniml medium, n4 the coinheritnce of zwf-1 ws scored by testing for th; bility to grow on mnnitol miniml medium. edd-l nd zwf-j loci were coinherited t frequency of 99% (Tble 5), which is consistent with the tight linkge tht ws observed in quntittive 2-fctor crosses (Tble 4). The ed-9001 muttioth hs beehi mpped by plsmid FP5-medited conjugtion in the lte region of the P. eruginos chromosome ner met-9020 nd leu-10 (7, 21). The tight trnsductionl linkge of zwf-j nd edd-j nd the conjugtionl mppipg of zwf-j nd ed-9001 indicted tht ed-9q0q might be cotrnsducible with zwf-l nd edd-l. Linkge of zwf-j nd ed-9001 ws tested by trnsducing strin PA01838 (ed- 9001) to gluconte positive, using F116L propgted on strin PFB98 (zwf-1), nd scoring trnsductnts for coinheritnce of zwf-l on mnnitol miniml medium. The results showed 96% cotrnsduction of zwf-j nd ed-9001 (Tble 5). Therefore, zwf-1, edd-l, nd ed-9001 re clustered in the 50- to 55-min region of the chromosome ner leu-10 nd met The cluster ws mpped more precisely by determining trnsductionl linkge of these muttions to other previously mpped mrkers in this region of the chromosome. The results from tyu-9029 ilv / his-4 -ys cota met ~ ~~~ trp-6 met ^ leu-10 rgf Hmi-151 proa edd- ptsj ed-9001 pyc-2 zwf- nla FG. 2. Chromosoml mp of P. eruginos PA01 bsed on the mp of Hollowy nd Crockett (6). Linked genetic mrkers re enclosed in brckets.

5 VOL. 156, 1983 CLUSTERED CATABOLC GENES N P. AERUGNOSA 1127 TABLE 3. Conjugtionl mpping of zwf-l nd pyc-2" Coinheritnce frequency Donor Recipient Selection zwf-l pyc-2 PFB98(R68.45) PRP811 (rgfo) Arg (87/96)b PRP894(R68.45) PRP851 (met-9020) Met (93/230) PFB14(R68.45) PRP851 (ctal) Ben+c <0.01 (0/72) PFB14(R68.45) PRP851 (tyu-9025) Tyu+d <0.01 (0/70) PFB14(R68.45) PRP818 (rgfio) Arg (12/150) Conjugtions with chromosome-mobilizing plsmid R68.45 were done s described previously (24). Donor cells were counterselected with nlidixic cid (400,ug/ml) in the selective solid medi. b Numbers in prentheses indicte the number of trnsconjugnts contining the unselected mrker per number of trnsconjugnts scored. c Ben', Benzote utiliztion. d Tyu+, Tyrosine utiliztion. F116L-medited trnsductions (Tble 5) showed tht zwf-l ws not linked to either mi-151 (PFB98 x PRP890 cross) or leu-10 (PRP811 x PFB98 cross); ed-9001 lso ws not linked to met-9020 (PAQ1 x PA01838 cross). However, the zwf-1, ed-9001, nd edd-i muttions did exhibit 44, 39, nd 31% cotrnsduction frequencies, respectively, with nla54 in strin PRP811. Nerly identicl linkge vlues were obtined when coinheritnce ws determined with the nla2 muttion in strin PA0236. Thus, the zwfed-edd cluster is linked to the nla locus nd is locted between mi-151 nd leu-10. The proposed gene order is shown in Fig. 3. DSCUSSON Four genes corresponding to inducible enzymes of the centrl pthwys of crbohydrte ctbolism were locted in the 45- to 55-min region of the P. eruginos chromosome mp. Three of these genes were tightly clustered, s shown by cotrnsduction of zwf with both ed nd edd t frequencies of >95% (Tble 5). The reltive gene order deduced from the present conjugtionl nd trnsductionl crosses plces the zwf-ed-edd gene cluster between mi-151 nd leu-10 (Fig. 3). All three genes of the cluster TABLE 4. were linked by trnsduction with nla, which mps between mi-151 nd leu-10, but they did not exhibit trnsductionl linkge with either of the ltter mrkers. The previously unchrcterized hex-9001 muttion (redesignted ed-9001 in this study) ws plced between nla nd mi- 151 in erlier mps of the P. eruginos chromosome (6, 7, 21), bsed on very low frequencies of cotrnsduction of ed-9001 nd leu-10. However, dditionl crbohydrte ctbolic mrkers, specifying glycerol utiliztion, recently hve been identified in this region of the chromosome, nd they exhibit trnsductionl linkge to mi-151, nla, nd the zwf-ed-edd cluster. Those results indicted the reltive gene order shown in Fig. 3 (S. M. Cuskey nd P. V. Phibbs, Jr., Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, K39, p. 183). The reltive gene order within the zwf-ed-edd cluster ws bsed on the frequencies of cotrnsduction of ech locus with the nla mrker (Tble 5; Fig. 3). Due to the nerly identicl effects of the edd nd ed muttions on crbohydrte utiliztion, it ws not possible for us to employ three-point trnsductionl nlysis to determine more precisely the gene order within this cluster. The ctivities of glucose 6-phosphte dehy- Quntittive two-fctor trnsductionl crosses No. of mnnitol-positive trnsductnts of donor strin': Recipient strin PFB9 PFB52 PFB98 PFB34 PFB131 PA01 (edd-1) (edd-2) (zwf-) (pyc4) (pgi-3) (wild type) PFB PFBS PFB PFB PFB None Plte trnsductions were done by spreding 10' PFU of phge F116L on mnnitol miniml medium seeded with 108 recipient cells. Donor nd recipient strins hve been grouped ccording to their proposed linkge rrngement. Numbers represent the verge number of trnsductnt colonies per trnsduction plte, fter djusting for low frequencies of spontneous reversion tht were determined on control pltes tht received no phge.

6 1128 ROEHL, FEARY, AND PHBBS TABLE 5. Frequencies of cotrnsduction of zwf-1, edd-1, nd ed-9001 with mrkers in the lte region of the P. eruginos chromosome. Donor Recipient strn Selec- Coinheritnce with unselected mrker' stn Rcpetstrin tionb zwf-1 ed-9(k) nla leu-10 PFB98 PFB9 (edd-1) Gnt (326/329) PFB98 PA01838 (ed-9001) Gnt (64/67) PFB98 PRP890 (mi-151) Act+ <0.01 (0/223) PA01 PA01838 (met-9020) Met+ <0.01 (0/171) PRP811 PFB98 (zwf-1) Mtl (175/395) <0.01 (0/395) PRP811 PFB9 (edd-1) Gnt (58/185) <0.01 (0/185) PRP811 PA01838 (ed-9001) Gnt (70/179) PA0236 PFB98 (zwf-1) Mtl (92/190) PA0236 PFB9 (edd-1) Gnt (78/186) Plte trnsductions were done by spreding 109 PFU of phge F116L on selective miniml medi seeded with 108 recipient cells. b Gnt+, Gluconte utiliztion; Act+, cetmide utiliztion; Met+, no requirement for methionine on lctte miniml medium; Mtl+, mnnitol utiliztion. Entries represent cotrnsduction frequency. Numbers in prentheses show the number of trnsductnts contining the unselected mrker per number of trnsductnts scored. drogense, EDD, nd EDA re coinduced during growth of P. eruginos on mnnitol, glucose, gluconte, glycerol, glycerol 3-phosphte, nd glycerte (2, 10, 14, 17, 20, 26). 6-Phosphogluconte hs been suggested s the probble inducer metbolite for ll three enzymes (8, 20), but whether the corresponding genes constitute single, coordintely expressed regultory unit hs not been estblished. The present demonstrtion tht zwf, ed, nd edd re locted within single trnsductionl linkge group supports the notion tht these functionlly relted genes re structurl elements of regultory unit. However, we hve no genetic evidence t this time tht regultory locus governs the coordinte expression of ll three enzymes or tht the three genes re contiguous. Most of the genes for glucose ctbolism hve been mpped on the Escherichi coli chromosome, nd the possible evolutionry nd functionl significnce of their rrngement into four 50 mi-151 nla zwf- gene clusters hs been discussed (22, 23). nspection of the chromosome mp of E. coli revels tight clustering of zwf, ed, nd edd tht is remrkbly similr to the clustered rrngement of these three genes in P. eruginos. detorrontegui et l. (3) hve reported the suproperonic clustering of five genes specifying glucose utiliztion in Pseudomons putid, including cotrnsducible ed nd edd mrkers. The loction of the five clustered genes, reltive to other chromosoml mrkers, could not be estblished in tht study, nd the zwf gene hs not been mpped in P. putid. Pyruvte crboxylse is n inducible nplerotic enzyme tht is essentil for the utiliztion of ll crbohydrtes by P. eruginos. ndependently isolted pyc lleles fll into single trnsductionl linkge group, nd the gene ppers to be expressed s n independent regultory unit (19). The pyc muttions did not exhibit trnsductionl linkge with the zwf-ed-edd do-9001 edd-l / L 0.31 i i i 1, 0.39 :;; F< <0.01,i <0.01!-, <0.01 <0.01 leu-10 55' met-9020 J. BACTEROL. FG. 3. Trnsductionl linkge mp of the 50- to 55-min region of the P. eruginos chromosome. Numbers represent cotrnsduction frequencies, nd rrowheds indicte the unselected mrkers.

7 VOL. 156, 1983 cluster (Tble 4); however, they were mpped in the 45-min region of the P. eruginos chromosome by demonstrting conjugtionl linkge with rgf10 (Tble 3). We lso reported recently the trnsductionl linkge of tightly clustered structurl nd regultory genes for the phosphoenolpyruvte-fructose 1-phosphotrnsferse system (pts mrkers) with both rgf10 nd lys in this region of the chromosome (24). Therefore, t lest seven different genes specific for crbohydrte ctbolic functions, which comprise t lest three seprte regultory units (pts loci, pyc, nd zwf-ed-edd), hve been mpped within the 45- to 55-min region of the P. eruginos chromosome. A number of chromosoml ctbolic genes tht hve been mpped in Pseudomons species re known to exhibit extensive clustering (1, 3, 12, 15, 16, 18, 27). The present results provide nother such exmple' nd provide dditionl evidence for the proposed phenomenon of suproperonic clustering of dissimiltory pthwys in Pseudomons species (27, 33). ACKNOWLEDGMENTS We re grteful to H. Mtsumoto nd R. H. Olsen for providing vluble bcteril cultures. This work ws supported by reserch grnt PCM from the Ntionl Science Foundtion. R.A.R. received support from Public Helth Service Grnt A from the. Ntionl nstitute of Allergy nd nfectious Diseses. T.W.F. ws the recipient of n A. D. Willims Visiting Scholr Awrd t the Medicl College of Virgini, Virgini Commonwelth University, Richmond. LTERATURE CTED 1. Betz, J. L., J. E. Brown, P. H. Clrke, nd M. Dy Genetic nlysis of midse mutnts of Pseudomons eruginos. Genet. Res. 23: Blevins, W. T., T. W. Fery, nd P. V. Phibbs, Jr Phosphogluconte dehydrtse deficiency in pleiotropic crbohydrte-negtive mutnt strins of Pseudomons eruginos. J. Bcteriol. 121: detorrontegul, R. D., M. L. Wheells, nd J. L. Cnovs Supr-operonic clustering of genes specifying glucose dissimiltion in Pseudomons putid. Mol. Gen. Genet. 144: Durhm, D. R., nd P. V. Phibbs, Jr Frctiontion nd chrcteriztion of the phosphoenolpyruvte:fructose 1-phosphotrnsferse system from Pseudomons eruginos. J. Bcteriol. 149: Hs, D., nd B. W. Hollowy R fctor vrints with enhnced sex fctor ctivity in Pseudomons eruginos. Mol. Gen. Genet. 144: Hollowy, B. W., nd R. J. Crockett Pseudomons eruginos. Genet. Mps 2: Hollowy, B. W., V. Krlshnpilll, nd A. F. Morgn Chromosoml genetics of Pseudomons. Microbiol. Rev. 43: Hunt, J. C., nd P. V. Phibbs, Jr Regultion of CLUSTERED CATABOLC GENES N P. AERUGNOSA 1129 lternte peripherl pthwys of glucose ctbolism during erobic nd nerobic growth of Pseudomons eruginos. J. Bcteriol. 154: Hylemon, P. B., N. R. Krieg, nd P. V. Phibbs, Jr Trnsport nd ctbolism of D-fructose by Spirillum itersonii. J. Bcteriol. 117: Hylemon, P. B., nd P. V. Phibbs, Jr ndependent regultion of hexose ctbolizing enzymes nd glucose trnsport ctivity in Pseudomons eruginos. Biochem. Biophys. Res. Commun. 48: Klb, V. F., Jr., nd R. W. Bernlohr A new spectrophotometric ssy for protein in cell extrcts. Anl. Biochem. 82: Kemp, M. B., nd G. D. Hegemn Genetic control of the,b-ketodipte pthwy in Pseudomon eruginos. J. Bcteriol. 96: Krlsbnpilll, V:'1971. A novel trnsducing phge. ts role in recognition of possible new host-controlled modifiction system in Pseudomons eruginos. Mol. Gen. Genet. 114: Lessie, T., nd F. C. Neidhrdt Adenosine triphosphte-linked control of Pseudomons eruginos glucose- 6-phosphte dehydrogense. J. Bcteriol. 93: Mtsumoto, H., T. Nkzw, S. Obt, nd Y. Terwki Chromosoml loctions of cta, poba, pca, dcu nd chu genes in Pseudomons eruginos. Genet. Res. 38: Mtsumoto, H., S. Oht, R. Kobyshi, nd Y. Terwki Chromosoml loction of genes prticipting in the degrdtion of purines in Pseudomons eruginos. Mol. Gen. Genet. 167: McCowen, S. M., P. V. Phibbs, Jr., nd T. W. Fery Glycerol ctbolism in wild-type nd mutnt strins of Pseudomons eruginos. Curr. Microbiol. 5:191-1%. 18. Ornston, L. N Regultion of ctbolic pthwys in Pseudomons. Bcteriol. Rev. 35: Phibbs, P. V., Jr., T. W. Fery, nd W. T. Blevins Pyruvte crboxylse deficiency in pleiotropic crbohydrte-negtive mutnt strins pf Pseudomon eruginos. J. Bcteriol. 118: Phlbbs, P. V., Jr., S. M. McCowen, T. W. Fery, nd W. T. Blevlns Mnnitol nd fructose ctbolic pthwys of Pseudomons eruginos crbohydrte-negtive mutnts nd pleiotropic effects of certin enzyme deficiencies. J. Bcteriol. 133: Rei, M., nd D. Hs Resistnce of Pseudomons eruginos PAO to nlidixic cid nd low levels of,blctm ntibiotics: mpping of chromosoml genes. Antimicrob. Agents Chemother. 22: Riley, M., nd A. Anilionis Evolution of the bcteril genome. Annu. Rev. Microbiol. 32: Riley, M., L. Solomon, nd D. Zipks Reltionship between gene function nd gene loction in Escherichi coli. J. Mol. Evol. 11: Roehl, R. A., nd P. V. Phibbs, Jr Chrcteriztion nd genetic mpping offructose phosphotrnsferse muttions in Pseudomons eruginos. J. Bcteriol. 149: Royle, P. L., H. Mtsumoto, nd B. W. Hollowy Genetic circulrity of the Pseudomon eruginos PAO chromosome. J. Bcteriol. 145: Vincente, M., nd J. L. Cnovs Regultion of glucolytic enzymes in Pseudomons eruginos. Arch. Microbiol. 93: Wheelts, M. L The genetics of dissimiltory pthwys in Pseudomons. Annu. Rev. Microbiol. 29: