Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91:1140-1154. 1966.-The control of synthesis of the five enzymes responsible for the conversion of d(-)-mandelate to benzoate by Pseudomonas putida was investigated. The first three compounds occurring in the pathway, d(-)-mandelate, l(+)-mandelate, and benzoylformate, are equipotent inducers of all five enzymes. A nonmetabolizable inducer, phenoxyacetate, also induces synthesis of these enzymes; but, unlike the metabolizable inducer-substrates, it does not elicit synthesis of enzymes that mediate steps in the pathway beyond benzoate. Under conditions of semigratuity, dl-mandelate elicits immediate synthesis at a steady rate of the first two enzymes of the pathway, but two enzymes which act below the level of benzoate are synthesized only after a considerable lag. Succinate and asparagine do not significantly repress the synthesis of the enzymes responsible for mandelate oxidation.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. II. Isolation and properties of blocked mutants

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. II. Isolation and properties of blocked mutants. J. Bacteriol. 91:1155-1160. 1966.-Mutants of Pseudomonas putida blocked in early reactions of the pathway for oxidation of d-mandelate were isolated and partially characterized. The specific genetic lesions in these mutants made normal inducer-metabolites of the pathway nonmetabolizable. Under the conditions of gratuitous enzyme synthesis so obtained, it could be shown that the d and l isomers of mandelate are equipotent inducers, and that the synthesis of the first five enzymes of the mandelate pathway is coordinate. Further experiments with the blocked mutants showed that benzoylformate, the third intermediate of the pathway, acts as an inducer without prior conversion to mandelate, and that there is no inducible, concentrating permease for mandelate.     

Induction and multi-sensitive end-product repression in the enzymic pathway degrading mandelate in Pseudomonas fluorescens

1. The first five enzymes involved in the degradation of mandelate in Pseudomonas fluorescens have been examined. 2. Induction is not significantly affected by glucose. 3. The first three enzymes form a group inducible by mandelate and repressible by benzoate, catechol and succinate. 4. The possibility that benzoate and catechol act as repressors only after they have been degraded to succinate is unlikely since mutants blocked at suitable points in the pathway have the same repression pattern as the wild type. 5. It is concluded that synthesis of the enzymes is subject to a multi-sensitive repression mechanism that can be independently activated by benzoate or catechol or succinate. 6. In each case the repression can be largely overcome by increasing the concentration of the inducer. 7. The enzymes of the first group are thus controlled by a dual system in which induction by the first substrate is opposed by repression exerted by the end product of the first group and by the products of succeeding groups.     

Metabolism of Benzoate and the Methylbenzoates by Pseudomonas putida (arvilla) mt-2: Evidence for the Existence of a TOL Plasmid

Mutant strains of Pseudomonas putida (arvilla) mt-2 which have lost the ability to grow at the expense of m- or p-toluate (methylbenzoate) but retain the ability to grow with benzoate arise spontaneously during growth on benzoate; this genetic loss occurs to a lesser extent during growth on nonaromatic carbon sources in the presence of mitomycin C. The mutants have totally lost the activity of the enzymes of the divergent meta pathway with the possible exception of 2-oxopent-4-enoate hydratase and 4-hydroxy-2-oxovalerate aldolase; unlike the wild type they utilize benzoate by the ortho pathway. Evidence is presented that these mutants have lost a plasmid coding for the enzymes of the meta pathway, which may be transmitted back to them or into other P. putida strains. Preliminary results from these mutants and from a mutant defective in the regulation of the plasmid-carried pathway suggest that the wild type contains two benzoate oxidase systems, one on the plasmid which is nonspecific in both its catalysis and its induction and one on the chromosome which is more specific to benzoate as substrate and is specifically induced by benzoate.     

Aromatic acids are chemoattractants for Pseudomonas putida

A quantitative capillary assay was used to show that aromatic acids, compounds that are chemorepellents for Escherichia coli and Salmonella sp., are chemoattractants for Pseudomonas putida PRS2000. The most effective attractants were benzoate; p-hydroxybenzoate; the methylbenzoates; m-, p-, and o-toluate; salicylate; DL-mandelate; beta-phenylpyruvate; and benzoylformate. The chemotactic responses to these compounds were inducible. Taxis to benzoate and m-toluate was induced by beta-ketoadipate, a metabolic intermediate formed when benzoate is dissimilated via enzymes specified by chromosomal genes. Benzoylformate taxis was induced by benzoylformate and L(+)-mandelate. Taxis to mandelate, benzoylformate, and beta-phenylpyruvate was exhibited by cells grown on mandelate, but not by cells grown on benzoate. Cells grown on benzoate were chemotactic to benzoate, the toluates, p-hydroxybenzoate, and salicylate. These results show that P. putida synthesizes at least two distinct chemoreceptors for aromatic acids. Although DL-mandelate was an effective attractant in capillary assays, additional experiments indicated that the cells were actually responding to benzoylformate, a metabolite formed from mandelate. With the exception of mandelate taxis, chemotaxis to aromatic acids was not dependent on the expression of pathways for aromatic degradation. Therefore, the tactic responses exhibited by cells cannot be attributed to an effect of the oxidation of aromatic acids on the energy metabolism of cells.     

Isolation and characterization of a benzoylformate decarboxylase and a NAD+/NADP+-dependent benzaldehyde dehydrogenase involved in D-phenylglycine metabolism in Pseudomonas stutzeri ST-201

Following induction with D-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD+/NADP+-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.     

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.     

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.     

Regulatory mutations of the Pseudomonas plasmid alk regulon.

Pseudomonas putida strains with plasmids carrying pleiotropic alk mutations gave rise to alkane-positive "revertants," which differ from wild type. Some had restricted ability to utilize alkane and primary alcohol growth substrates, and others could grow on undecane and dodecanol, which are not utilized by alk+ strains. These revertants showed altered responses to normal inducers of alkA+, alkB+, and alkC+ activities. Some revertants were constitutive for these activities. Constitutive mutants could also be isolated directly from wild type, but they appeared spontaneously at a frequency of less than 2 X 10(-8). Regulatory mutations of all three types, pleiotropic negative, altered inducer specificity, and constitutive, were tightly linked in transduction crosses with a polar alkB mutation. These results demonstrate that the IncP-2 plasmid alk gene cluster constitutes a regulon. They also permit the identification of at least one cistron whose gene product participates in inducer recognition and suggest that the alkABC regulon is not under simple repressor control.     

Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida.

Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source. The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system. The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate. The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains. Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes. Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively. Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type. Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems.     

Hyperinduction of enzymes of the phosphorylative pathway of glucose dissimilation inPseudomonas cepacia

Glucose-dehydrogenase-deficient (Gcd^–) strains ofPseudomonas cepacia 249 compensated for loss of operation of the direct oxidative pathway by expanding the phosphorylative pathway. When grown on glucose, they had between two- and fourfold higher than normal levels of glucokinase and NAD-linked glucose-6-phosphate dehydrogenase activity and a comparable increase in capacity to transport glucose. Similar expansion of the phosphorylative pathway was noted when the wild type was grown on cellobiose or trehalose. Gcd^– strains grew normally on cellobiose and trehalose, but not if also deficient in glucokinase; this indicates that the disaccharides were converted to glucose and metabolized via the phosphorylative pathway. The expansion of the phosphorylative pathway during growth of the wild type on disaccharides or of Gcd^– mutants on glucose was a consequence of hyperinduction of pathway enzymes. Other compounds that promoted such hyperinduction included aromatic conjugates of glucose such as arbutin and salicin, and mannose. Under conditions leading to expansion of the phosphorylative pathway, enzymes related to the direct oxidative pathway, such as gluconate dehydrogenase and the 6-phosphogluconate dehydrogenase active with NAD, were not formed. The results indicate that intracellular glucose and extracellular glucose are metabolized to 6-phosphogluconate via different routes.     

Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.     

Regulation of enzymes of the 3,5-xylenol-degradative pathway in Pseudomonas putida: evidence for a plasmid.

Constitutive synthesis of enzymes responsible for methyl group oxidation in 3,5-xylenol degradation and an associated p-cresol methylhydroxylase in Pseudomonas putida NCIB 9869 was shown by their retention at high specific activities in cells transferred from 3,5-xylenol medium to glutamate medium. The specific activities of other enzymes of the 3,5-xylenol pathway declined upon removal of aromatic substrate, consistent with their inducible control. Specific activities of the methyl-oxidizing enzymes showed an eventual decline concomitant with a decrease in the fraction of bacteria capable of growth with 3,5-xylenol; a simultaneous loss of the ability to grow with m-hydroxybenzoate was also observed. The property of 3,5-xylenol utilization could be transferred to another strain of P. putida. It is proposed that enzymes of the 3,5-xylenol pathway and those for conversion of p-cresol to p-hydroxybenzoate are plasmid encoded, that the early methyl-oxidizing enzymes are expressed constitutively, and that the later enzymes are inducible.     

Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida

The gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) was cloned in Pseudomonas aeruginosa (ATCC 15692). The selection for the cloned gene was based upon the inability of P. aeruginosa to grow on (R)-mandelate as sole carbon source by virtue of the absence of mandelate racemase in its mandelate pathway. Fragments of P. putida DNA obtained by digestion of chromosomal DNA with Sau3A were ligated into the BamHI site of the Gram-negative vector pKT230 and transformed into the P. aeruginosa host. A transformant able to utilize (R)-mandelate as sole carbon source was characterized, and the plasmid was found to contain approximately five kilobase pairs of P. putida DNA. Subcloning of this DNA revealed the position of the gene for the racemase within the cloned DNA from P. putida. The dideoxy-DNA sequencing procedure was used to determine the sequence of the gene and its translated sequence. The amino acid sequence and molecular weight for mandelate racemase deduced from the gene sequence (38 570) are in excellent agreement with amino acid composition and molecular weight data for the polypeptide recently determined with enzyme isolated from P. putida; these recent determinations of the polypeptide molecular weight differ significantly from the originally reported value of 69,500 [Fee, Judith A., Hegeman, G.D., & Kenyon, G.L. (1974) Biochemistry 13,2528], which was used to demonstrate that alpha-phenylglycidate, an active site directed irreversible inhibitor, binds to the enzyme with a stoichiometry of 1:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of pathways degrading aromatic substrates in Pseudomonas putida. Enzymic response to binary mixtures of substrates

1. Induction constants (K(ind)) and repression constants (K(rep)), which are a measure of the affinity of the inducers or repressors for the induction systems, were measured for mandelate, benzoate and p-hydroxybenzoate in Pseudomonas putida. 2. From these results, the enzymic response of the organism to media containing pairs of these substrates was predicted. Nitrogen-limited chemostats, operated at high growth rates, were used to investigate these predictions in cells grown first on one aromatic substrate with the second added later. 3. In general, the values of K(ind) and K(rep) predicted quite accurately the response to substrate mixtures. Thus, in the presence of mandelate and either benzoate or p-hydroxybenzoate, the enzymes of mandelate metabolism were repressed almost completely, and the bacteria were fully induced for the alternative substrate (benzoate or p-hydroxybenzoate), which was preferentially utilized for growth. When benzoate and p-hydroxybenzoate were the two substrates in the mixture, the enzymes for metabolism of the latter were strongly repressed and growth took place mainly on benzoate. 4. The enzymic response to mixed substrates did not result in the metabolism of the better growth substrate, but in the substrate requiring the synthesis of fewer enzymes. Thus benzoate is used in preference to mandelate although the latter supports a faster growth rate. It is nevertheless considered that, with our present knowledge of the natural habitat of the organism, it is impossible to decide whether protein economy or growth rate was the factor determining the evolution of this control system.     

Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida.

Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.     

Regulation of the β-Ketoadipate Pathway in Alcaligenes eutrophus

The regulation of the synthesis of the inducible enzymes that mediate the reactions of the beta-ketoadipate pathway in Alcaligenes eutrophus has been examined by determining the inductive responses of the wild type and of mutants derived from it to metabolites of the pathway. The system of control differs in many respects from those which operate in the genera Pseudomonas and Acinetobacter.     

Metabolism of resorcinylic compounds by bacteria: alternative pathways for resorcinol catabolism in Pseudomonas putida.

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol. One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway. After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase. Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate. Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction. P. putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates. The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described. Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P. putida ORC grown with resorcinol. Its further metabolism was reduced nicotinamide adenine dinucleotide dependent.     

Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme

The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic Control of the β-Ketoadipate Pathway in Pseudomonas aeruginosa

The regulation and genetic control of the beta-ketoadipate pathway in Pseudomonas aeruginosa were investigated. The pattern of enzyme induction is apparently the same as in P. putida. Mutants were obtained for all but 1 of the 11 structural genes; the proximity of these genes on the chromosome was examined by transduction of the mutants with phage F116. If a group of enzymes was induced by the same compounds, the corresponding genes were closely clustered. Surprisingly, some locispecifying enzymes not sharing a common inducer were also clustered. It is suggested that this latter finding may indicate a degree of chromosomal specialization.