A Spot Test for Catechol 2:3‐Oxygenase in Bacteria

Pankhurst, Eileen S.

doi:10.1111/j.1365-2672.1965.tb02158.x

Cited by 11 publications

(8 citation statements)

References 13 publications

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“…Small-scale recombinant plasmid preparations were made by the method of Holmes and Quigley (12) to permit rapid screening of plasmids, and stocks of recombinant plasmid DNA for subcloning, mutagenesis, and restriction mapping were prepared by a cleared lysate procedure (10) followed by CsCl-ethidium bromide density gradient centrifugation. PaWl + + + + + pWWO 117 10-2_10-3 + Japan 34 MT5 + + + + + pWW5 160 <10-7 + Wales 7, 35 MT5-S3 -----NDd _ Cured derivative of This study MT5 MT14-26 + + + + + pWW14 270 <10-7 + Wales 27 MT14-263 -----ND -Cured derivative of This study MT14-26 MT74 + + + + + pWW74 105 <10-7 + Shetland Islands, This study Scotland MT74-1 -----ND -Cured derivative of This study MT74 MT84 + + -+ + pWW84 180 <10- (8,25). The vector plasmids pKT230, pBR322, and pBR328 have been described previously (2,4,30).…”

Section: Methodsmentioning

confidence: 99%

“…All Pseudomonas strains were cultured at 30°C. E. coli strains were routinely grown at 37°C in Luria broth supplemented with the appropriate antibiotic at the following concentrations (in micrograms per milliliter): streptomycin, 15; kanamycin, 20; ampicillin, 25; and tetracycline, 7.5. Bacterial matings. Conjugation experiments were carried out with broth cultures containing donor strains mixed with the streptomycin-resistant recipient PaW340 in a 1:10 ratio.…”

Section: Methodsmentioning

confidence: 99%

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Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes

Chatfield¹,

Williams²

1986

J Bacteriol

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Structural genes for catechol 2,3-oxygenase (C230) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated fro*n Pseudomonas strains of diverse geographical origins. Each pKT230-based C230+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xyLE gene from the archetype TOL plasmid pWWO. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C230 structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xyLE of pWWO by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C230 gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C230 gene related to the second C230 gene (C2301) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248&255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.The degradation of toluene and some substituted toluenes via the meta pathway by Pseudomonas spp. is often plasmid mediated (7,15,19,26,34,35,39). TOL plasmids from different strains are diverse with regard to molecular sizes, fragmentation patterns by restriction endonucleases, selftransmissibility, rate of dissimilation ofp-methyl-substituted substrates, and formation of different classes of deletion mutants after growth on benzoate (7,17,27,35,37,38).Although the archetype TOL plasmid pWWO (34) has a single xylE gene coding for the ring cleavage dioxygenase catechol 2,3-oxygenase (C230), it was recently demonstrated in this laboratory that the 250-kilobase (kb) plasmid pWW15 carries two nonhomologous genes which specify the apparently unrelated enzymes C2301 and C2301I (16). To investigate the evolutionary relationship between the catabolic genes on different TOL plasmids, we cloned xylE genes from five TOL plasmids found in Pseudomonas strains isolated from soil samples of diverse geographical origins. Like pWW15, two of the plasmids reported here also carried two nonhomologous C230 genes. Unexpectedly, the other plasmids in this study each possessed two closely related C230 genes which exhibited strong homology with each other and with the xylE gene of pWWO. MATERIALS AND METHODSBacterial strains and plasmids. Pseudomonas strains and plasmids used in this study are described in Table 1. Strains MT74, MT84, and MT88 were isolated by selective enrichment on m-toluate minimal medium during this study; MT5 and MT14-26 have been described previously (7,26,35).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes

Chatfield¹,

Williams²

1986

J Bacteriol

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“…The spot test for detection of catechol 2,3-dioxygenase (C23O) activity [26] revealed bright yellow M213 colonies grown on naphthalene vapors, indicative of C230 activity. The speci¢c activity of C23O in cell free lysates of M213 was somewhat lower (6.9 units mg 31 protein) than was observed for G7 (7.8 units mg 31 protein).…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…Spot test for catechol 2,3-dioxygenase (C23O) activity The rapid spot test described by Pankhurst [26] was used to assess catechol 2,3-dioxygenase (C230) activity. Catechol dissolved in diethylether was sprayed onto 1 or 2 days old M213 and P. putida G7 colonies grown on naphthalene vapor plates.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Characterization of the naphthalene-degrading bacterium, Rhodococcus opacus M213

2000

FEMS Microbiology Letters

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Bacterial strain M213 was isolated from a fuel oil-contaminated soil in Idaho, USA, by growth on naphthalene as a sole source of carbon, and was identified as Rhodococcus opacus M213 by 16S rDNA sequence analysis and growth on substrates characteristic of this species. M213 was screened for growth on a variety of aromatic hydrocarbons, and growth was observed only on simple 1 and 2 ring compounds. No growth or poor growth was observed with chlorinated aromatic compounds such as 2,4-dichlorophenol and chlorobenzoates. No growth was observed by M213 on salicylate, and M213 resting cells grown on naphthalene did not attack salicylate. In addition, no salicylate hydroxylase activity was detected in cell free lysates, suggesting a pathway for naphthalene catabolism that does not pass through salicylate. Enzyme assays indicated induction of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase on different substrates. Total DNA from M213 was screened for hybridization with a variety of genes encoding catechol dioxygenases, but hybridization was observed only with catA (encoding catechol 1,2-dioxygenase) from R. opacus 1CP and edoD (encoding catechol 2,3-dioxygenase) from Rhodococcus sp. I1. Plasmid analysis indicated the presence of two plasmids (pNUO1 and pNUO2). edoD hybridized to pNUO1, a very large (V750 kb) linear plasmid. ß

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“…The rapid spot test described by Pankhurst [6] was carried out to assess C23O activity. Catechol or 3-methylcatechol dissolved in acetone was sprayed onto 1-day-old T-57 colonies grown on toluene vapor plates.…”

Section: Characterization Of Toluene Pathway In P Putida T-57mentioning

confidence: 99%

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Faizal

Dozen

Hong

et al. 2005

J IND MICROBIOL BIOTECHNOL

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Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.

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A Spot Test for Catechol 2:3‐Oxygenase in Bacteria

Cited by 11 publications

References 13 publications

Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes

Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes

Characterization of the naphthalene-degrading bacterium, Rhodococcus opacus M213

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

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