Naphthalene plasmids in pseudomonads.

A rapid method beginning with the direct lysis of bacteria in alkaline sodium dodecyl sulfate was used to detect naphthalene plasmids in pseudomonads. The strains NCIB 9816, PG, ATCC 17483, and ATCC 17484, which can grow on naphthalene as the sole source of carbon and energy, were examined. All except ATCC 17483 contained more than one plasmid. ATCC 17483 did not contain any plasmids. The largest pair of plasmids found in each of NCIB 9816 and PG(NAH2 and NAH3, respectively) determined naphthalene metabolism and could be transferred by conjugation. This also transferred the unusually regulated meta pathway enzymes for catechol metabolism. NAH2 determines the constitutive production of low concentrations of catechol 2,3-dioxygenase and 2-hydroxymuconic acid semialdehyde dehydrogenase, and NAH3 determines the constitutive production of high concentration of these. NAH2 and NAH3 gave identical fragments on digestion with BamHI or HindIII, but these were quite different from those of NAH. Nonetheless, NAH2 and NAH3 hybridized with NAH.     

Regulation of aryl hydrocarbon (benzo-(A)-pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity by N6,O2'-dibutyryl8 adenosine 3':5'-monophosphate and aminophylline.

Treatment of hamster BHK cells with N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), aminophylline, theophylline, or papaverine increased the level of aryl hydrocarbon (benzo(a)pyrene) hydrolxylase activity. The highese increase, 100-fold, was obtained with Bt2cAMP plus aminophylline or theophylline. N2,O2-Dibutyryl guanosine 3':5'-monophosphate gave a lower induction than Bt2cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt2cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2- to 8-fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt2cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolities with a fluorescence spectra similar to that of 3-hydroxybenzo(a)pyrene. These induced enzyme activities also showed a similar heat stability. Induction by Bt2cAMP and aminophylline, like induction by benz(a)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a Km of 4.3 muM, the hydroxylase induced by Bt2cAMP and aminophylline showed a Km of 0.14 muM, and was 100-fold more sensitive to inhibition by 7,8-benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(a)anthracene. The results indicate that aryl hydrocaarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3':5'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Regulation of the Pathway for the Degradation of Anthranilate in Aspergillus niger

Studies were carried out to determine the factors governing the induction of anthranilate hydroxylase and other enzymes in the pathway for the dissimilation of anthranilate by Aspergillus niger (UBC 814). The enzyme was induced by growth in the presence of tryptophan, kynurenine, anthranilate, and, surprisingly, by 3-hydroxyanthranilate, which was not an intermediate in the conversion of anthranilate to 2,3-dihydroxybenzoate. There was an initial lag in the synthesis of anthranilate hydroxylase when induced by tryptophan, anthranilate, and 3-hydroxyanthranilate. Cycloheximide inhibited the enzyme induction. Comparative studies on anthranilate hydroxylase, 2,3-dihydroxybenzoate carboxy-lyase, and catechol 1:2-oxygenase revealed that these enzymes were not coordinately induced by either anthranilate or 3-hydroxyanthranilate. Structural requirements for the induction of anthranilate hydroxylase were determined by using various analogues of anthranilate. The activity of the constitutive catechol oxygenase was increased threefold by exposure to anthranilate, 2,3-dihydroxybenzoate, or catechol. 3-Hydroxyanthranilate did not enhance the levels of catechol oxygenase activity.     

Suppression of fibroblast proliferation and lysyl hydroxylase activity by minoxidil

The effect of minoxidil on lysyl hydroxylase activity and proliferation of human skin fibroblasts in culture was examined. Exposure of cells to minoxidil resulted in a specific loss of lysyl hydroxylase activity, the extent of which was dependent on the concentration of minoxidil from 25 to 500 microM and the duration of the treatment from 6 to 48 h. This phenomenon was unaffected by culture conditions, i.e. ascorbic acid status, serum concentration, and cell density. Minoxidil added directly to cell extracts had no effect on lysyl hydroxylase activity, showing a requirement for intact cells. Mixing experiments with extracts of minoxidil-treated cells and controls gave additive results which rule out the possibility that a metabolite derived from minoxidil could be inhibiting the enzyme activity. The effect of minoxidil on fibroblast lysyl hydroxylase activity disappeared in the presence of cycloheximide, an inhibitor of protein synthesis. Moreover, the recovery of the enzyme activity that occurred after removal of minoxidil from the culture medium could be prevented by actinomycin D, an inhibitor of RNA synthesis. These results indicate that minoxidil may inhibit the synthesis of lysyl hydroxylase in the cell. In addition to suppressing fibroblast lysyl hydroxylase activity, minoxidil caused inhibition of cell growth within 48 h in a manner dependent on the concentration from 10 to 1000 microM, the latter resulting in almost complete cessation of cell proliferation. This effect was not accompanied by cytotoxicity as judged by the criteria of dye exclusion, plating efficiency, growth recovery, and protein synthesis. The inhibition of fibroblast proliferation by minoxidil appeared to be related to its ability to inhibit DNA synthesis measured by incorporation of tritiated thymidine into acid-precipitable material.     

Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba.

Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway. A strain of P. putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene. Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate). However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.     

Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region

Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.     

Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway.

From a soil isolate, Pseudomonas strain C18, we cloned and sequenced a 9.8-kb DNA fragment that encodes dibenzothiophene-degrading enzymes. Nine open reading frames were identified and designated doxABDEFGHIJ. Collectively, we refer to these genes as the DOX pathway. At the nucleotide level, doxABD are identical to the ndoABC genes that encode naphthalene dioxygenase of Pseudomonas putida. The DoxG protein is 97% identical to NahC (1,2-dihydroxynaphthalene dioxygenase) of P. putida. DoxE has 37% identity with cis-toluene dihydrodiol dehydrogenase. DoxF is similar to the aldehyde dehydrogenases of many organisms. The predicted DoxHIJ proteins have no obvious sequence similarities to known proteins. Gas chromatography with a flame ionization detector and mass spectroscopy confirmed that the DOX proteins convert naphthalene to salicylate and converting phenanthrene to 1-hydroxy-2-naphthoic acid. doxI mutants convert naphthalene to trans-o-hydroxybenzylidenepyruvate, indicating that the DoxI protein is similar to NahE (trans-o-hydroxybenzylidenepyruvate hydratase-aldolase). Comparison of the DOX sequence with restriction maps of cloned naphthalene catabolic pathway (NAH) genes revealed many conserved restriction sites. The DOX gene arrangement is identical to that proposed for NAH, except that the NAH equivalent of doxH has not been recognized. DoxH may be involved in the conversion of 2-hydroxy-4-(2'-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoat e to cis-o-hydroxybenzylidenepyruvate. doxJ encodes an enzyme similar to NahD (isomerase). Our findings indicate that a single genetic pathway controls the metabolism of dibenzothiophene, naphthalene, and phenanthrene in strain C18 and that the DOX sequence encodes a complete upper naphthalene catabolic pathway similar to NAH.     

The development of Alternaria alternata is prevented by chitinases and beta-1,3-glucanases from Citrus limon seedlings

In addition to phytoalexin synthesis, the defense response of intact Citrus limon seedlings against Alternaria alternata involves both constitutive and induced enzyme activities such as chitinases (Ch) and beta-1,3-glucanases (Glu). A alternata conidial germination was prevented by protein extracts from inoculated lemon seedlings, but also by extracts from mock-inoculated specimens. On the other hand, degradation of mycelia was accomplished only by protein extracts from inoculated seedlings. The presence of six Ch isoenzymes and of four Glu isoenzymes was detected in protein extracts from mock-inoculated seedlings. As a result of fungal inoculation, the isoenzyme pattern of Ch and Glu changed, making possible the detection of a new Ch isoenzyme and of three new Glu. Also, some constitutive Ch and Glu increased their enzyme activity, and those Ch that increased their activity also showed a broadening of their substrate specificity. These changes were prevented by alpha-amanitin and cycloheximide, suggesting that the presence of new Ch and Glu isoenzymes was due to de novo synthesis of proteins. Results suggest that constitutive Ch and Glu could act as pre-formed defense molecules in Citrus limon preventing A. alternata germination, while those induced after fungal inoculation of lemon seedlings could act along with the former, to produce lysis of fungal mycelia, resulting in a more efficient control of A. alternata development.     

Purification, composition, and some properties of rat liver carbamyl phosphate synthetase (ammonia).

Hepatic microsomes prepared from vitamin E deficient and supplemented rats were analyzed for cytochrome P-450 content and drug metabolizing activity. Reduced levels of benzo[α]pyrene hydroxylase and ethylmorphine N-demethylase activities were observed in microsomes derived from rats fed a diet deficient in vitamin E compared to those of control rats. NADPH-mediated destruction of P-450, and pentobarbital and zoxazolamine sleeping times were similar in the two groups. Induction with 3-methylcholanthrene raised the levels of benzo[α]pyrene hydroxylase activity of both supplemented and deficient rats to the same absolute levels. No differences were noted in cytochrome P-450 or P-448 content between control and tocopherol deficient rats, nor did the activity of liver catalase differ between the two dietary groups. Thus, these studies did not demonstrate any impairment of heme protein synthesis in vitamin E deficient rats.     

Coordinated regulation of P-glycoprotein activity and cytochrome P-4501A induction in sublines of rat hepatoma McA RH7777 cells with different levels of colchicine resistance.

The constitutive and induced activities of cytochrome P-4501A isoforms in hepatoma McA 7777 sublines with different levels of colchicine (CH) resistance were studied. The higher CH resistance was associated with the elevated functional activity of P-glycoprotein (Pgp). The constitutive level of benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase (cytochrome P-4501A-dependent activities) were the same in sublines with different CH resistance levels. However, benzo(a)-anthracene, a cytochrome P-4501A inducing agent, more effectively induced benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase activities in sublines with elevated P-glycoprotein activity. The toxicity of benzo(a)pyrene, a compound which is simultaneously a cytochrome P-4501A-inducing agent and a toxic agent activated by cytochrome P-4501A, is more effective in sublines with elevated CH resistance. These results support the suggestion about the coordinated regulation of enzyme systems involved in the defence against various lipophilic xenobiotics. The possibility to overcome the Pgp-mediated MDR of some tumours by using a combination of some drugs including compounds which induce the cytochrome P-4501A isoforms and are activated by them is discussed.     

Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase.

Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.     

Regulation of choline sulphatase synthesis and activity in Aspergillus nidulans

1. Choline O-sulphate is taken up from the growth medium to the same extent by sulphur-deficient and sulphur-sufficient mycelia of Aspergillus nidulans, but hydrolysis of the transported sulphate ester in vivo only occurs in the sulphur-deficient mycelia. 2. Choline sulphatase activity could not be detected in vitro in sulphur-sufficient mycelia of wild-type and sulphur mutants of A. nidulans, but after sulphur starvation all strains showed appreciable activity of this enzyme. 3. Optimum activity of choline sulphatase in an ultrasonically treated preparation of sulphur-deficient mycelia was at pH7.5. The optimum substrate concentration was in excess of 25mm and K(m) was 0.035m. The enzyme was completely inhibited by 10mm-SO(3) (2-), PO(4) (3-), CN(-) and cysteine. 4. Growth of sulphur-deficient mycelia on various sulphur sources resulted in a decrease of choline sulphatase activity in vitro. The decrease appeared to be due to a repression of choline sulphatase synthesis rather than to inhibition of activity. De-repression by growth on a sulphur-deficient medium was prevented by cycloheximide. Unlike the choline sulphatase of bacteria the fungal enzyme did not need to be substrate-induced. 5. By using sulphur mutants the identity of the co-repressor was limited to S(2)O(3) (2-), cysteine-S-sulphonate, cysteine or compounds derived directly from them. Circumstantial evidence suggests that the co-repressor is cysteine. 6. Inhibition of choline sulphatase activity in vivo was demonstrated with cysteine as the sulphur source for growth.     

Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[¹⁴C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[¹⁴C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.     

Naphthalene oxidation by a Pseudomonas putida strain carrying a mutant plasmid

Naphthalene oxidation by a parent and a mutant strain of Pseudomonas putida was studied. The parent strain contained a plasmid NPL-1 which controlled oxidation of naphthalene to salicylic acid and was capable of oxidizing salicylate. The mutant strain did not oxidize salicylate because of a mutation in salicylate hydroxylase; it contained also a mutant plasmid NPL-41 which determined constitutive synthesis of naphthalene oxygenase. Salicylic acid which accumulated as a product of naphthalene catabolism in the cultural broth of the wild strain was found to undergo further oxidation by the population of growing cells. The content of salicylic acid in the cultural broth of the mutant strain reached maximum and then remained constant. An anion-exchange resin was tested in order to prevent the inhibition of naphthalene oxygenase by salicylate and to increase the yield of salicylic acid. The transmissible character of the mutant plasmid NPL-41 makes it possible, with the aid of conjugation, to construct Pseudomonas strains which would oxidize naphthalene to salicylic acid without further degradation of this compound.     

Ascorbate increases the synthesis of procollagen hydroxyproline by cultured fibroblasts from chick embryo tendons without activation of prolyl hydroxyla.

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 mug of enzyme protein per 10(8) cells and 40-50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein but only 15-20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and incultured tendon cells had the same apparent size and the same activity per mug of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme. When freshly isolated cells were incubated for 2 h in the presence of 40 mug per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 mug per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not icrease the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve "activation" of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.     

Characteristics of aryl hydrocarbon hydroxylase activity in rat mammary epithlial cells grown in primary culture.

Rat mammary epithelial cells grown in primary culture contain the microsomal enzyme, aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), which catalyses the oxidative conversion of polycyclic aromatic hydrocarbons (PAH) to more polar derivatives. Constitutive AHH activity, measured with an established fluorometric method, was 46 pmol/mg protein/h in homogenates of rat mammary epithelial cells after 5 days in culture. The addition of dimethylbenz[a]anthracene (DMBA), benz[a]anthracene (BA), or 3-methylcholanthrene (3-MC) to the cell culture medium increased AHH activity 5.3-, 4.7- and 2.4-fold, respectively. Kinetic studies revealed that maximal hydroxylase induction occurred 16 h after 1 micro M DMBA was added to the culture medium. The decay of the DMBA-induced hydroxylase was biphasic: one component had a t1/2 of 15--30 min and another a t1/2 of 4 h. Norepinephrine, 17 beta-estradiol and 5,6-benzoflavone also increased AHH activity in mammary epithelial cells in vitro, however, sodium phenobarbital had no effect. Fetal bovine serum (FBS), previously shown to be a potent in vitro inducer of AHH activity, had no effect on either constitutive or DMBA-induced mammary epithelial hydroxylase activities following treatment with 1% activated charcoal. Metyrapone and 7,8-benzoflavone, inhibitors of microsomal mixed function oxidase activity, reduced both constitutive and DMBA-induced AHH activities when added to homogenates of untreated and DMBA-treated mammary epithelial cells. The addition of 7,8-benzoflavone reduced both constitutive and DMBA-induced hydroxylase activities by approx. 80%, whereas metyrapone addition inhibited these activities by 20%. The study demonstrates several in vitro factors which alter AHH activity in primary cultures of rat mammary epithelial cells.     

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.     

A comparative study of the NAH and TOL catabolic plasmids in Pseudomonas putida

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives. The NAH and TOL plasmids were found to be incompatible. Under the conditions used in these experiments the TOL plasmid transferred into some strains into which NAH was unable to transfer. The use of mutants to remove certain catabolic activities encoded by the bacterial host cell facilitated the allocation of growth genotypes to the NAH and TOL plasmids. TOL encoded the degradation of benzoate, m-toluate and p-toluate, whereas NAH encoded the degradation of naphthalene and salicylate. The other plasmid-associated growth phenotypes were partly plasmid-specified and partly specified by the host cell. The pH optimum of the catechol 2,3-dioxygenase specified by the TOL plasmid was approximately 6.7, whereas that of the NAH-encoded enzyme was approximately 8.3.     

Metabolism of xenobiotic compounds by enzymes in cell extracts of the fungus Cunninghamella elegans.

Cell extracts of the filamentous fungus Cunninghamella elegans contain epoxide hydrolase (EC 3.3.2.3), glutathione S-transferase (EC 2.5.1.18) and UDP-glucuronosyltransferase (EC 2.4.1.17) activities. Epoxide hydrolase activity was determined with p-nitrostyrene oxide as substrate and was shown to be associated with the 100 000 g pellet obtained from disrupted mycelia. Glutathione S-transferase activity was demonstrated with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates. The presence of two or more glutathione S-transferase activities was indicated by different activity ratios for the two substrates in different extracts, and by distinct thermal denaturation curves. UDP-glucuronosyltransferase activity with 3-hydroxybenzo[a]pyrene as substrate was found only with the non-sedimentable fraction prepared from ruptured mycelia.