Appl Environ Microbiol, 1990 Apr, 56(4), 977 - 83 Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments; Awong J et al.; Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms . In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system . The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment . Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C . The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions . In the presence of organisms indigenous to lake water, E . coli was eliminated after 20 days, whereas P . putida showed an initial decline but was able to stabilize its population after 5 days . A herbicide, Hydrothol-191, caused a significant decline in numbers of P . putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain . The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed. J Gen Microbiol, 1990 Apr, 136 ( Pt 4), 627 - 36 Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent; Saint CP et al.; The ability to degrade aromatic amines and m-toluate (Tdn+ phenotype), encoded by plasmid pTDN1, was lost from Pseudomonas putida hosts after subculture in benzoate, succinate, acetate and glucose minimal medium, the fastest rate of loss occurring where benzoate was the substrate . Tdn- cells had either lost the entire pTDN1 plasmid or suffered a recombinational deletion of a specific 26 kbp region . Proportional increase of Tdn- cells resulted from their growth-rate advantage, and additionally, where benzoate was the substrate, from its metabolism via the chromosomal ortho-cleavage pathway incorporating a short lag phase . The ratio of whole plasmid loss to deletion was substrate and pH dependent . Deletion of catabolic genes was not required for loss of pTDN1 but by comparison was a prerequisite for loss of TOL plasmid pWW0 . It appeared that m-toluate and benzoate were channelled via chromosomally encoded benzoate oxygenase and dihydroxycyclohexadiene carboxylate dehydrogenase prior to pTDN1 encoded meta-cleavage. J Gen Microbiol, 1990 Apr, 136 ( Pt 4), 615 - 25 Physical map of the aromatic amine and m-toluate catabolic plasmid pTDN1 in Pseudomonas putida: location of a unique meta-cleavage pathway; Saint CP et al.; A restriction endonuclease map was derived for the aromatic amine and m-toluate catabolic plasmid pTDN1 present in Pseudomonas putida UCC22, a derivative of P . putida mt-2 . The plasmid is 79 +/- 1 kbp in size and can be divided into a restriction-site-deficient region of 51 +/- 1 kbp and a restriction-site-profuse region of 28 kbp which begins and ends with directly repeating sequences of at least 2 kbp in length . A mutant plasmid isolated after growth of the host on benzoate had lost the restriction-profuse region by a straightforward recombinational loss retaining one copy of the direct repeat . Analysis of clones, deletion and Tn5 insertion mutants strongly suggested that the meta-cleavage pathway of pTDN1 was situated in the region readily deleted . The catechol 2,3-dioxygenase (C23O) gene of pTDN1 showed no hybridization or restriction homology to previously described C23O genes of TOL plasmids pWW0 and pWW15 . In addition, there was little homology between intact pTDN1, pWW0 and pWW15, suggesting the presence of a unique meta-cleavage pathway . We also demonstrated that pTDN1 did not originate from P . putida mt-2 chromosome. Appl Environ Microbiol, 1990 Apr, 56(4), 956 - 62 Identification and localization of 3-phenylcatechol dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase genes of Pseudomonas putida and expression in Escherichia coli; Khan AA et al.; The bphC and bphD genes of Pseudomonas putida involved in the catabolism of polychlorinated biphenyls or biphenyl were identified, localized, and studied for expression in Escherichia coli . This was achieved by cloning a 2.4-kilobase (kb) DNA fragment of recombinant cosmid pOH101 into HindIII site of pUC plasmids downstream of a lacZ promoter and measuring the enzyme activities of 3-phenylcatechol dioxygenase (3-PDase; a product of bphC) and the meta-cleavage product 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (a product of bphD) . The amount of 3-PDase produced in E . coli was about 20 times higher than that of the enzyme produced by the parent, P . putida . Determination of expression of the bphC and bphD genes through their own promoter sequences or by using the lacZ promoter of pUC plasmids was done by cloning the DNA that encodes bphC and bphD genes in a HindIII site of a promoter selection vector (pKK232-8) upstream of the gene for chloramphenicol acetyltransferase (CAT) . The recombinant plasmid (pAW787) constructed by inserting the 2.4-kb DNA in pKK232-8 expressed both 3-PDase and CAT activities . Another hybrid construct (pAW786) in which the DNA insert was cloned in the opposite orientation lacked CAT activity but produced normal amounts of 3-PDase activity . On the basis of these results, we suggest that the bphC and bphD genes were expressed by using promoter sequences that are independent of the promoter that expresses CAT activity in E . coli . The locations of the bphC and bphD genes were determined by insertional inactivation of the open reading frames of structural genes bphC and bphD by Tn5 mutagenesis.(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnol Appl Biochem, 1990 Apr, 12(2), 141 - 9 Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase; Ishiwata K et al.; Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied . The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor . Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E . coli and P . putida . After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively . Continuous production of L-tryptophan was also carried out using immobilized cells of E . coli and P . putida . The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days. Biochem Biophys Res Commun, 1990 Mar 30, 167(3), 891 - 7 Carbon catabolite regulation of phenylacetyl-CoA ligase from Pseudomonas putida; Martinez-Blanco H et al.; Phenylacetyl-CoA ligase (PA-CoA ligase) from P . putida U is a newly described enzyme involved in the aerobic catabolism of phenylacetic acid . The enzyme was specifically induced when P . putida was grown in a chemically defined medium containing phenylacetic acid as the sole carbon source . The induction of PA-CoA ligase was delayed by adding easily metabolizable carbon sources to the medium; the effect was more drastic in the presence of glucose . Glucose did not cause catabolic inactivation but rather catabolic repression, this effect being reversed by cAMP. Biochem J, 1990 Mar 1, 266(2), 605 - 9 Isolation and partial characterization of an extradiol non-haem iron dioxygenase which preferentially cleaves 3-methylcatechol; Wallis MG et al.; A purification procedure has been developed for an extradiol dioxygenase expressed in Escherichia coli, which was originally derived from a Pseudomonas putida strain able to grow on toluidine . Physical and kinetic properties of the enzyme have been investigated . The enzyme has a subunit Mr of 33,500 +/- 2000 by SDS/polyacrylamide-gel electrophoresis . Gel filtration indicates a molecular mass under non-denaturing conditions of 120,000 +/- 20,000 . The N-terminal sequence (35 residues) of the enzyme has been determined and exhibits 50% identity with other extradiol dioxygenases . Fe(II) is a cofactor of the enzyme, as it is for other extradiol dioxygenases . The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 3-methylcatechol cleaved at a higher rate than catechol or 4-methylcatechol . Km values for these substrates with this enzyme are all around 0.3 microM . The enzyme exhibits a bell-shaped pH profile with pKa values of 6.9 +/- 0.1 and 8.7 +/- 0.1 . These results are compared with those found for other extradiol dioxygenases. Mol Gen Genet, 1990 Mar, 221(1), 113 - 20 The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes; Harayama S et al.; The meta-cleavage operon of TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol . The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells . This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are xylX(57)-xylY(20)-xylZ(39)-xylL(28)-xylT(1 2)-xylE(36)-xylG(60)-xylF(34)- xylJ(28)-xylQ(42)-xylK(39)-xylI(29)-xylH(4 ) . The xylXYZ genes encode three subunits of toluate 1,2-dioxygenase . The xylL, xylE, xylG, xylF, xylJ, xylK, xylI, and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxaloccotonate tautomerase, respectively . The functions of xylT and xylQ are not known at present . The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xylX and xylH consists of coding sequences. Biochemistry, 1990 Feb 20, 29(7), 1749 - 56 Characterization of the multiple catalytic activities of tartrate dehydrogenase; Tipton PA et al.; Tartrate dehydrogenase (TDH) has been purified to apparent homogeneity from Pseudomonas putida and has been demonstrated to catalyze three different NAD(+)-dependent reactions . TDH catalyzes the oxidation of (+)-tartrate to form oxaloglycolate and the oxidative decarboxylation of D-malate to form pyruvate and CO2 . D-Glycerate and CO2 are formed from meso-tartrate in a reaction that is formally a decarboxylation with no net oxidation or reduction . The steady-state kinetics of the first two reactions have been investigated and found to follow primarily ordered mechanisms . The pH dependence of V and V/K was determined and indicates that catalysis requires that a base on the enzyme with a pK of 6.7 be unprotonated . TDH activity requires a divalent and a monovalent cation . Kinetic data suggest that the cations function in substrate binding and facilitation of the decarboxylation of beta-ketoacid intermediates. J Bacteriol, 1990 Feb, 172(2), 867 - 83 Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC; Essar DW et al.; Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway . All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome . This report describes the cloning and sequencing of P . putida trpE, trpG, trpD, and trpC . In P . putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC . In P . putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P . aeruginosa, they are separated by at least 25 kilobases (T . Shinomiya, S . Shiga, and M . Kageyama, Mol . Gen . Genet., 189:382-389, 1983) . The DNA sequence in P . putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized . Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan . The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study. Appl Environ Microbiol, 1990 Feb, 56(2), 569 - 71 Biotransformation of substituted benzoates to the corresponding cis-diols by an engineered strain of Pseudomonas oleovorans producing the TOL plasmid-specified enzyme toluate-1,2-dioxygenase; Wubbolts MG et al.; The conversion of substituted benzoates into 1,2-cis-dihydroxycyclohexa-3,5-diene carboxylic acids (cis-diols) was effected by using Escherichia coli and Pseudomonas recombinants carrying the xylXYZ genes originating from the Pseudomonas putida mt-2 TOL plasmid, thus producing toluate-1,2-dioxygenase . Pseudomonas oleovorans GPo12 recombinants readily produced meta- and para-substituted cis-diols, but were limited in their oxidation of ortho-substituted substrates. J Bacteriol, 1990 Feb, 172(2), 884 - 900 Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications; Essar DW et al.; Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa . They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P . aeruginosa, replacing the wild-type gene . One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG . The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism . trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida . The phenazine pathway genes phnA and phnB are cotranscribed . The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli . Homologous genes were not found in P . putida PPG1, a non-phenazine producer . Surprisingly, PhnA and PhnB are more closely related to E . coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E . coli PabB and PabA than to E . coli TrpE and TrpG . We replaced the wild-type trpE on the P . aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette . This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6) . The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB . phnA mutants retain a low level of pyocyanin production . Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis. Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1278 - 82 Pseudomonas chromosomal replication origins: a bacterial class distinct from Escherichia coli-type origins; Yee TW et al.; The bacterial origins of DNA replication have been isolated from Pseudomonas aeruginosa and Pseudomonas putida . These origins comprise a second class of bacterial origins distinct from enteric-type origins: both origins function in both Pseudomonas species, and neither functions in Escherichia coli; enteric origins do not function in either pseudomonad . Both cloned sequences hybridize to chromosomal fragments that show properties expected of replication origins . These origin plasmids are highly unstable, are present at low copy number, and show mutual incompatibility properties . DNA sequence analysis shows that both origins contain several 9-base-pair (bp) E . coli DnaA protein binding sites; four of these are conserved in position and orientation, two of which resemble the R1 and R4 sites of the E . coli origin . Conserved 13-bp direct repeats adjacent to the analogous R1 site are also found . No GATC sites are in the P . aeruginosa origin and only four are in the P . putida origin; no other 4-bp sequence is present in high abundance . Both origins are found between sequences similar to the E . coli and Bacillus subtilis dnaA, dnaN, rpmH, and rnpA genes, a gene organization identical to that for B . subtilis and unlike that of E . coli . A second autonomously replicating sequence was obtained from P . aeruginosa that has some properties of bacterial origins. J Bacteriol, 1990 Feb, 172(2), 1160 - 4 Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria; Hayase N et al.; We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715 . The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase. J Bacteriol, 1990 Feb, 172(2), 922 - 31 Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family; Rothmel RK et al.; Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source . This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon . The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies . However, the inducer is required for transcriptional activation of the catBC operon . The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240 . This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start . Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression . Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family . Striking homology was seen between CatR and a putative regulatory protein, TfdS. Gene, 1990 Jan 31, 86(1), 53 - 61 Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria; Ahmad D et al.; The genes of Pseudomonas testosteroni strain B-356, specifying the transformation of 4-chlorobiphenyl (4-CB) into 4-chlorobenzoic acid (4-CBA) were cloned into Pseudomonas putida KT2440 using a broad-host-range cosmid, pPSA842 . Of 10,000 clones tested, four were able to transform 4-CB . Gas chromatographic and mass spectrometric analysis of the catabolic products from two of the 4-CB-transforming clones carrying the hybrid plasmids, pDA1 and pDA2, demonstrated that pDA1 carried a complete set of structural genes involved in the transformation of 4-CB into 4-CBA, while pDA2 contained part of the pathway genes leading up to the meta-cleavage compound . Restriction endonuclease mapping and subcloning of pDA1 and pDA2 showed that the clones contained a common stretch of DNA of about 9.1 kb and that pDA2 carried gene(s) involved in regulation . Probing blots of genomic DNA from 13 different polychlorinated biphenyl(PCB)-degrading bacteria with radio-labelled pDA1 and pDA2, suggested that many PCB-degrading pathways have a common phylogenetic origin. J Biol Chem, 1990 Jan 25, 265(3), 1345 - 51 Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase; Ullah AJ et al.; The cytochrome P-450 heme-thiolate monooxygenases that hydroxylate monoterpene hydrocarbon groups are effective models for the cytochrome P-450 family . We have purified and characterized the three proteins from a P-450-dependent linalool 8-methyl hydroxylase in Pseudomonas putida (incognita) strain PpG777 . The proteins resemble the camphor 5-exohydroxylase components in chemical and physical properties; however, they show neither immunological cross-reactivity nor catalytic activity in heterogenous recombination . These two systems provide an excellent model to probe more deeply the heme-thiolate reaction center, molecular domains of substrate specificity, redox-pair interactions, and the regulation of the reaction cycle. Mikrobiologiia, 1990 Jan-Feb, 59(1), 163 - 5 {Peripheral metabolism of Pseudomonal putida transconjugants degrading chloro- and methylaromatic compounds}; Mal'tseva OV et al.; Peripheral metabolism was studied in the Pseudomonas putida 37cc transconjugant . In the strain grown on benzoate, pyrocatechase (PC) I with a low activity to chlorocatechols was induced, whereas PCII actively decomposing chlorocatechols was induced during its growth on 3-chlorobenzoic acid . The P . putida 37cc transconjugant grown on alpha-methylstyrene (MS) exerted the activity of both metapyrocatechase (MPC) and PC, whereas in the parent strain P . putida R-1 only MPC was involved in the degradation of alpha-MS . The substrate specificity of the enzymes involved in the ring cleavage by P . putida 37cc was compared to show that, apparently, MPC of the transconjugant was similar to this enzyme in the strain R-1 while PC decomposing chlorocatechols was similar to PC of the P . putida 87 donor . The regulation of the enzymes mediating the ring cleavage was studied in the parent strains and transconjugants. Nauchnye Doki Vyss Shkoly Biol Nauki, 1990, (3), 124 - 9 {The SOS-like reactions of Pseudomonas putida}; Broun II et al.; Under ultraviolet radiation of Pseudomonas putida 1087 the SOS-similar response which is expressed in the inhibition of cell respiration and cell division with the following filamentation is revealed . In the result of introduction of pPE24 and pMH21 plasmids into the cells of P . putida 1087 for inhibition of RecA-similar protein the SOS-similar response disappears and the basic cell mass dies. J Basic Microbiol, 1990, 30(1), 57 - 61 Electroporation and expression of plasmid pBR322 in Klebsiella aerogenes NCTC 418 and plasmid pRK2501 in Pseudomonas putida CYM 318; Trevors JT; Klebsiella aerogenes NCTC 418 and Pseudomonas putida CYM 318 were transformed via high-voltage electroporation with plasmids pBR322 and pRK2501, respectively . The number of transformants obtained was dependent on the applied voltage, capacitance, and cell recovery procedure . For example, 7.87 x 10(4) transformants/micrograms DNA were obtained at 2500 V, 25 muF when K . aerogenes cells were electroporated with pBR322 DNA . A lower voltage (1500) and capacitance (3 muF) yielded 2.4 x 10(3) transformants/micrograms DNA . P . putida CYM 318 required a 24 h outgrowth period to assist in the recovery of transformants containing pRK2501 . Electroporation may be a useful protocol to transform bacterial strains that are not easily transformed by traditional methods. Comp Biochem Physiol B, 1990, 97(4), 719 - 26 Phylogenetic comparisons of the branched-chain alpha-ketoacid dehydrogenase complex; Eisenstein RS et al.; 1 . Antibodies against the E1b and E2b components of bovine branched-chain alpha-ketoacid (BCKA) dehydrogenase (BCKAD) complex completely inhibited BCKA oxidation in mammalian and avian mitochondria . BCKA oxidation by salmonid mitochondria was less affected and the enzyme from Pseudomonas putida was unaffected . 2 . In rodents, anti-E1b E2b IgG inhibited oxidation of all three BCKA in a similar dose-dependent manner: oxidation of alpha-ketobutyrate and alpha-keto-y-methiolbutyrate was also partially inhibited . 3 . Except for the salmonid BCKAD, a similar Mr for the E2b and E1b alpha proteins was observed in these species . 4 . After digestion with V-8 protease similar immunoreactive peptides were observed for the human and rodent complex. Lab Delo, 1990, (8), 65 - 7 {Pseudomonas APS nutrient medium for the isolation and identification of Pseudomonas aeruginosa and Pseudomonas putida}; Sivolodskii EP; Pseudomonas APS selective medium has been developed on the basis of a newly detected selective antibacterial action of oxaphenamide (p-oxyphenylsalicylamide), a cholagogue . This medium permits a single-stage combined isolation and identification of P . aeruginosa after 16-24 hrs incubation of inoculated material at 42 degrees C . If the material is incubated at 35-37 degrees C, isolation of P . putida and P . aeruginosa is possible, that are differentiated by a nitroreductase microtest within 3 hrs . The sensitivity and selective ability of the developed medium are superior to other media manufactured in this country and commercial media with cetrimide and irgasan, produced abroad. Arch Microbiol, 1990, 154(5), 465 - 70 Microbial degradation of the morphine alkaloids: identification of morphine as an intermediate in the metabolism of morphine by Pseudomonas putida M10; Bruce NC et al.; A strain of Pseudomonas putida was isolated by selective enrichment with morphine that was capable of utilising morphine as a primary source of carbon and energy for growth . Experiments with whole cells showed that both morphine and codeine, but not thebaine, could be utilised . A novel NADP-dependent dehydrogenase, morphine dehydrogenase, was purified from crude cell extracts and was shown to be capable of oxidising morphine and codeine to morphinone and codeinone, respectively . This NADP-dependent morphine dehydrogenase was not observed in any other species of pseudomonads examined and was quite distinct from the beta-hydroxysteroid dehydrogenase found in Pseudomonas testosteroni, which had previously been shown to have activity against morphine. SAAS Bull Biochem Biotechnol, 1990 Jan, 3, 54 - 7 Molecular cloning of the xylL-xylE region from the P . putida TOL plasmid, pDK1; Voss JA et al.; A 5.2 kilobase EcoRI restriction fragment from the Pseudomonas putida HS1 TOL plasmid pDK1, encoding a portion of the lower toluene degradation pathway, was cloned into the E . coli plasmid pBR325 . A detailed map of the restriction endonuclease sites was constructed and the nucleotide sequence of three contiguous XhoI fragments, with a combined total length of approximately 3.9 kilobases, has been investigated . This region was determined to contain a total of four separate open reading frames, each preceded by an identical putative ribosome-binding site (nucleotide sequence of 5'-GAGGTG-3') . These open reading frames have been tentatively identified as encoding the lower pathway enzymes catechol 2,3-dioxygenase (C23O) and 1,2-dihydroxycyclohexa-3,5-diene carboxylate dehydrogenase (DHCDH) and a subunit of the toluate 1,2-dioxygenase complex (TO). Biochim Biophys Acta, 1989 Dec 22, 1009(3), 301 - 3 Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium; He JS et al.; The gene encoding barbiturate-inducible cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced . An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH2-terminal sequence of P-450BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an Mr of 47,439 . The sequence is most, but less than 27%, similar to that of P-450CAM from Pseudomonas putida, so that P-450BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH-P-450 reductase from the same strain of B . megaterium (Ruettinger, R.T., Wen, L.-P . and Fulco, A.J . (1989) J . Biol . Chem . 264, 10987-10995). Gene, 1989 Dec 21, 85(1), 145 - 52 Cloning and sequence analysis of the ntrA (rpoN) gene of Pseudomonas putida; Inouye S et al.; The gene encoding a sigma factor NtrA (RpoN) was cloned from Pseudomonas putida by cross-hybridization with a probe containing a part of the corresponding Escherichia coli gene . The cloned gene complemented an ntrA mutation of E . coli in activation of xyl genes on the TOL plasmid . The predicted amino acid (aa) sequence of P . putida NtrA (497 aa; Mr 56,215) is highly homologous to NtrA proteins from Azotobacter vinelandii (81.7%), Klebsiella pneumoniae (52.6%), and Rhizobium meliloti (36.1%) . There are two other open reading frames (ORF1, ORF3) upstream and downstream from the ntrA gene, respectively . These ntrA-flanking ORFs are also highly homologous to the corresponding ORFs of K . pneumoniae, A . vinelandii, and R . meliloti. Clin Chim Acta, 1989 Dec 15, 185(3), 241 - 52 Microbial enzymes for creatinine assay: a review; Shimizu S et al.; A novel metabolic pathway for the degradation of creatinine with N-methylhydantoin, N-carbamoylsarcosine and sarcosine as successive intermediates was found to operate in Pseudomonas putida 77 and many other microorganisms . Enzymes involved in this pathway were purified from cells of P . putida 77 and characterized . The first step, deimination of creatinine, is catalyzed by cytosine deaminase/creatinine deiminase . The following two steps, ring-opening of N-methylhydantoin and decarbamoylation of N-carbamoylsarcosine, are catalyzed by new enzymes, N-methylhydantoin amidohydrolase and N-carbamoylsarcosine amidohydrolase, respectively . The former requires ATP, Mg2+, and K+ for the hydrolysis and the reaction proceeds as follows: N-methylhydantoin + ATP + 2 H2O----N-carbamoylsarcosine + ADP + Pi . The latter catalyzes the following reaction; N-carbamoylsarcosine + H2O----sarcosine + NH3 + CO2 . Sarcosine dehydrogenase was found to be the responsible enzyme for the oxidation of sarcosine to glycine in P . putida 77, but sarcosine oxidase was also found to be involved in this oxidation in several microorganisms . These enzymes were found to be useful tools for determination of creatinine. Mol Microbiol, 1989 Dec, 3(12), 1765 - 75 Mutations in genes downstream of the rpoN gene (encoding sigma 54) of Klebsiella pneumoniae affect expression from sigma 54-dependent promoters; Merrick MJ et al.; Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniae sigma 54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12 kD and 16 kD, respectively . ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram-negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida . Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e . they cause an increase in the level of expression from sigma 54-dependent promoters . We propose that the products of both genes function to modulate the activity of E sigma 54, although a physiological role for these proteins has not yet been identified. Appl Environ Microbiol, 1989 Dec, 55(12), 3162 - 6 Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes; Zylstra GJ et al.; Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene . This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter . The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene . A linear rate of trichloroethylene degradation was observed with the E . coli recombinant strain . In contrast, P . putida F39/D, a mutant strain of P . putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time. J Bacteriol, 1989 Dec, 171(12), 6617 - 24 xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter; Ingram C et al.; We describe the development of a convenient and sensitive reporter gene system for Streptomyces spp . based on the use of a promoterless copy of the xylE gene of Pseudomonas putida . The xylE gene product is a catechol dioxygenase, which converts the colorless substrate catechol to an intensely yellow hydroxymuconic semialdehyde . A promoterless copy of xylE was placed under the transcriptional control of galP1, a glucose-repressed and galactose-induced promoter from Streptomyces lividans, and its expression was examined in bacterial colonies on agar plates or in liquid cultures grown in the presence of glucose or galactose as the sole carbon source . On plates, colonies of bacteria grown on galactose turned bright yellow within a few minutes of being sprayed with a solution of catechol, whereas colonies on glucose-containing plates remained white or only slightly colored, even after extensive incubation . Activity of galP1-xylE fusions was conveniently measured in crude cell extracts with a simple colorimetric assay and was shown to faithfully reflect intracellular RNA levels, as determined by quantitative dot blots . Moreover, differences in expression levels of xylE fusions driven by mutant galP1 promoters were readily apparent in color reactions on plates . The properties of xylE as a reporter gene thus make it suitable not only for quantitatively monitoring expression of regulated promoters in Streptomyces spp . but also for recovering mutations that alter the expression levels of promoters of interest. Appl Environ Microbiol, 1989 Dec, 55(12), 3243 - 6 Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere; Yeung KH et al.; The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined . Despite a high level of polygalacturonase production by genetically engineered P . putida and P . aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere. Eur J Biochem, 1989 Nov 20, 185(3), 615 - 9 The stoichiometry of the tightly bound NAD+ in urocanase . Separation and characterization of fully active and inhibited forms of the enzyme; Klepp J et al.; 1 . Urocanase, purified by classical methods {Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J . A . and Retey, J . (1979) J . Biol . Chem . 254, 843-851} from Pseudomonas putida was submitted to high-performance liquid chromatography on a TSK-DEAE column . The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively . 2 . The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm . 3 . Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively . Peak A could not be photoactivated . Rechromatography of the photoactivated peaks B and C on the TSK-DEAE column confirmed their partial transformation into peak A . 4 . Spectroscopic methods for quantitative protein determination were adapted to urocanase . The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+ released upon acid denaturation of the holoenzyme . A similar stoichiometry (1.8-1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of {7-14C}nicotinate into urocanase using a nicotinate auxotrophic mutant of P . putida . 5 . Form A of urocanase showed, after treatment with NaBH4 up to 50% inhibition, an elution pattern (TSK-DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1 . None of these forms could be photoactivated . 6 . We conclude that form A of the urocanase dimer contains two intact NAD+ molecules . In form B one of the two subunits contains an NAD+-nucleophile adduct which is present in both subunits of form C . Full urocanase activity requires intact NAD+ in both subunits . Intact NAD+ can be regenerated from the adduct but not from the reduced form by photolysis . The two subunits of urocanase are independent both in their catalytic activity and in modification reactions. J Bacteriol, 1989 Nov, 171(11), 5915 - 21 Genetic organization and sequence of the Pseudomonas cepacia genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase; Zylstra GJ et al.; The locations of the genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.11.3) on a 9.5-kilobase-pair PstI fragment cloned from the Pseudomonas cepacia DBO1 chromosome were determined . This was accomplished through the construction of several subclones into the broad-host-range cloning vectors pRO2317, pRO2320, and pRO2321 . The ability of each subclone to complement mutations in protocatechuate 3,4-dioxygenase (pcaA) was tested in mutant strains derived from P . cepacia, Pseudomonas aeruginosa, and Pseudomonas putida . These complementation studies also showed that the two subunits were expressed from the same promoter . The nucleotide sequence of the region encoding for protocatechuate 3,4-dioxygenase was determined . The deduced amino acid sequence matched that determined by N-terminal analysis of regions of the isolated enzyme . Although over 400 nucleotides were sequenced before the start of the genes, no homology to known promoters was found . However, a terminator stem-loop structure was found immediately after the genes . The deduced amino acid sequence showed extensive homology with the previously determined amino acid sequence of protocatechuate 3,4-dioxygenase from another Pseudomonas species. J Bacteriol, 1989 Nov, 171(11), 5907 - 14 Cloning, expression, and regulation of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase genes; Zylstra GJ et al.; The genes for the alpha and beta subunits of the enzyme protocatechuate 3,4-dioxygenase (EC 1.13.11.3) were cloned from the Pseudomonas cepacia DBO1 chromosome on a 9.5-kilobase-pair PstI fragment into the broad-host-range cloning vector pRO2317 . The resultant clone was able to complement protocatechuate 3,4-dioxugenase mutations in P . cepacia, Pseudomonas aeruginosa, and Pseudomonas putida . Expression studies showed that the genes were constitutively expressed and subject to catabolite repression in the heterologous host . Since the cloned genes exhibited normal induction patterns when present in P . cepacia DBO1, it was concluded that induction was subject to negative control . Regulatory studies with P . cepacia wild-type and mutant strains showed that protocatechuate 3,4-dioxygenase is induced either by protocatechuate or by beta-carboxymuconate . Further studies of P . cepacia DBO1 showed that p-hydroxybenzoate hydroxylase (EC 1.14.13.2), the preceding enzyme in the pathway, is induced by p-hydroxybenzoate and that beta-carboxymuconate lactonizing enzyme, which catalyzes the reaction following protocatechuate 3,4-dioxygenase, is induced by both p-hydroxybenzoate and beta-ketoadipate. J Bacteriol, 1989 Nov, 171(11), 6251 - 8 Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida; Harayama S et al.; The meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds . In this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route . In the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the actions of 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxovalerate aldolase . In this study, the isomerase, decarboxylase, and hydratase encoded in the TOL plasmid pWW0 of Pseudomonas putida mt-2 were purified and characterized . The 28-kilodalton isomerase was formed by association of extremely small identical protein subunits with an apparent molecular weight of 3,500 . The decarboxylase and the hydratase were 27- and 28-kilodalton polypeptides, respectively, and were copurified by high-performance-liquid chromatography with anion-exchange, hydrophobic interaction, and gel filtration columns . The structural genes for the decarboxylase (xylI) and the hydratase (xylJ) were cloned into Escherichia coli . The elution profile in anion-exchange chromatography of the decarboxylase and the hydratase isolated from E . coli XylI+XylJ- and XylI-XylJ+ clones, respectively, were different from those isolated from XylI+ XylJ+ bacteria . This suggests that the carboxylase and the hydratase form a complex in vivo . The keto but not the enol form of 4-oxalocrotonate was a substrate for the decarboxylase . The product of decarboxylation was 2-hydroxypent-2,4-dienoate rather than its keto form, 2-oxopent-4-enoate . The hydratase acts on the former but not the latter isomer . Because 2-hydroxypent-2,4-dienoate is chemically unstable, formation of a complex between the decarboxylase and the hydratase may assure efficient transformation of this unstable intermediate in vivo. Mol Gen Mikrobiol Virusol, 1989 Nov, (11), 32 - 7 {Plasmids for biphenyl, chlorobiphenyl and metatoluylate degradation from Pseudomonas putida}; Andreeva AL et al.; Pseudomonas putida strain SU83, harbors the pBS311 plasmid coding for the degradation of biphenyl, 2- and 4-chlorbiphenyl, meta- and paratoluylate . The insertional mutants of the plasmid obtained by the transposon Tn5 insertion were isolated . One of the mutants was used for cloning of the biphenyl degradation genes . The plasmid pBS311:: Tn5 DNA was inserted into the BamHI site of the plasmid pBR322 and cloned . 11 recombinants of 354 tested were treated with 0.1% solution of 2,3-dioxybiphenyl . One of them has acquired the yellow colour testifying to conversion of 2,3-dioxyphenyl to "2-hydroxy-6-keto-6-phenylhexa-2,4-diene acid . The recombinant plasmid pBS312 from this clone is 10.5 kb in size, the size of the insert being 6.2 kb . Escherichia coli SU185 cells harbouring pBS312 are able to support metacleavage of 2,3-dioxybiphenyl, 3-methylcatechol and catechol, but not of 4-methylcatechol . The results suggest the cloned fragment to contain a gene for 2,3-dioxybiphenyl-1,2-dioxygenase, the third enzyme for biphenyl catabolism. J Biochem (Tokyo), 1989 Nov, 106(5), 831 - 6 Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida; Koga H et al.; Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source . Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism . We cloned from a HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of camA gene encoding NADH-putidaredoxin reductase and the entire camB gene encoding putidaredoxin . The 2.9 kbp CAM segment was adjacent to the 4.27 kbp HindIII CAM segment which has been previously cloned (Koga et al . (1986) J . Bacteriol . 166, 1089-1095) . Thus, the total 7.17 kbp HindIII CAM directed all the genes responsible for early steps of D-camphor degradation, i.e . 5-exo-hydroxycamphor dehydrogenase (camD gene), cytochrome P-450cam (camC), NADH-putidaredoxin reductase (camA), and putidaredoxin (camB) . These cam genes form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene . The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin. Biochem Biophys Res Commun, 1989 Oct 31, 164(2), 764 - 71 Isolation and characterization of altered plasmids in mutant strains of Pseudomonas putida NCIB 9816; Serdar CM et al.; The ability of P . putida NCIB 9816 to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid, pDTG1 . Derivatives of pDTG1 were obtained from cells after exposure to halogenated analogs of naphthalene or salicylate . The selection of mutants having a Nah-Sal- or a Nah-Sal+ phenotype could be enhanced by the addition of triphenyltetrazolium chloride to the indicator medium . Structurally modified plasmids were characterized by restriction endonuclease digestion and Southern hybridization experiments . The region of pDTG1 DNA that encodes the enzymes responsible for the conversion of naphthalene to salicylate was identified . The structural changes in mutant plasmids were correlated with the absence of essential enzymatic activities. Biochem J, 1989 Oct 15, 263(2), 431 - 7 The purification and characterization of 4-ethylphenol methylenehydroxylase, a flavocytochrome from Pseudomonas putida JD1; Reeve CD et al.; The enzyme 4-ethylphenol methylenehydroxylase was purified from Pseudomonas putida JD1 grown on 4-ethylphenol . It is a flavocytochrome c for which the Mr was found to be 120,000 by ultracentrifuging and 126,000 by gel filtration . The enzyme consists of two flavoprotein subunits each of Mr 50,000 and two cytochrome c subunits each of Mr 10,000 . The redox potential of the cytochrome is 240 mV . Hydroxylation proceeds by dehydrogenation and hydration to give 1-(4'-hydroxyphenyl)ethanol, which is also dehydrogenated by the same enzyme to 4-hydroxyacetophenone . The enzyme will hydroxylate p-cresol but is more active with alkylphenols with longer-chain alkyl groups . It is located in the periplasm of the bacterium. Biochemistry, 1989 Oct 3, 28(20), 8201 - 5 Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex; Stayton PS et al.; Cytochrome b5 has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450cam from Pseudomonas putida {Stayton, P . S., Fisher, M . T., & Sligar, S . G . (1988) J . Biol . Chem . 263, 13544-13548} . In the mutant cytochrome b5, threonine is replaced by a cysteine at position 65 (T65C) and has been labeled with the environmentally sensitive fluorophore acrylodan . In this paper, the physiological P-450cam reductant putidaredoxin, an Fe2S2.Cys4 iron-sulfur protein, is shown to competitively inhibit the cytochrome b5 association, suggesting that cytochrome b5 and putidaredoxin bind to a similar site on the cytochrome P-450cam surface . Since the crystal structures for both cytochrome b5 and cytochrome P-450cam have been solved to high resolution, the complex has been computer modeled, and a good fit was found on the proximal surface of nearest approach to the P-450cam heme prosthetic group . The proposed model includes electrostatic contacts between conserved cytochrome b5 carboxylates Glu-44, Glu-48, Asp-60, and the exposed heme propionate with cytochrome P-450cam basic residues Lys-344, Arg-72, Arg-112, and Arg-364, respectively . Putidaredoxin has similarly been shown to contain a carboxylate-based binding surface, and the current results suggest that if the model is correct, then it also interacts at the proposed site, probably utilizing similar P-450cam electrostatic contacts. Appl Environ Microbiol, 1989 Oct, 55(10), 2648 - 52 Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1; Spain JC et al.; Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen . Toluene-grown cells of P . putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism . Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol . Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol . Tests with specific mutants of P . putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system . 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water. J Biol Chem, 1989 Sep 15, 264(26), 15328 - 33 Bacterial aromatic ring-cleavage enzymes are classified into two different gene families; Harayama S et al.; Dioxygenases that catalyze the cleavage of the aromatic ring are classified into two groups according to their mode of ring fission . Substrates of ring-cleavage dioxygenases usually contain hydroxyl groups on adjacent aromatic carbons, and intradiol enzymes cleave the ring between these two hydroxyl groups . Extradiol enzymes in contrast cleave the ring between one hydroxylated carbon and its adjacent nonhydroxylated carbon . In this study, we determined the complete nucleotide sequence of nahC, the structural gene for 1,2-dihydroxynaphthalene dioxygenase encoded in the NAH7 plasmid of Pseudomonas putida . This enzyme is an extradiol ring-cleavage enzyme that cleaves the first ring of 1,2-dihydroxynaphthalene . The amino acid sequence of the dioxygenase deduced from the DNA sequence demonstrated that the molecular weight of the enzyme is 33,882 . This result was in agreement with those of maxicell analyses that showed that the nahC product was a 36-kDa protein . Interestingly, the amino acid sequence of 1,2-dihydroxynaphthalene dioxygenase was 50% homologous with that of 2,3-dihydroxybiphenyl dioxygenase, which catalyzes extradiol cleavage of the first ring of 2,3-dihydroxybiphenyl (Furukawa, K., Arimura, N., and Miyazaki, T . (1987) J . Bacteriol . 169, 427-429) . The amino acid sequence similarity of 1,2-dihydroxynaphthalene dioxygenase with catechol 2,3-dioxygenase, which is an authentic extradiol dioxygenase, was rather low (16%) . However, a statistical analysis by the method of S . B . Needleman and C . D . Wunsch {1970) J . Mol . Biol . 48, 443-453) clearly showed that these two dioxygenases are evolutionarily related . Therefore, these extradiol enzymes are considered as products of the same gene superfamily . From the significant sequence similarity between intradiol enzymes, it has been shown (Neidle, E . L., Harnett, C., Bonitz, S., and Ornston, L . N . (1988) J . Bacteriol . 170, 4874-4880) that intradiol enzymes evolved from a common ancestor . Comparison of the amino acid sequence of extradiol enzymes with those of intradiol dioxygenases did not show any significant global or localized similarity. J Biol Chem, 1989 Sep 5, 264(25), 14940 - 6 Toluene degradation by Pseudomonas putida F1 . Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli; Zylstra GJ et al.; The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined . The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE) . Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601}; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602}; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603} . The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The three E . coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively . The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P . putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K . (1987) J . Bacteriol . 169, 5174-5179) . In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T . (1987) J . Bacteriol . 169, 427-429). J Bacteriol, 1989 Sep, 171(9), 5111 - 6 Degradation of phenol and m-toluate in Pseudomonas sp . strain EST1001 and its Pseudomonas putida transconjugants is determined by a multiplasmid system; Kivisaar MA et al.; The utilization of phenol, m-toluate, and salicylate (Phe+, mTol+, and Sal+ characters, respectively) in Pseudomonas sp . strain EST1001 is determined by the coordinated expression of genes placed in different plasmids, i.e., by a multiplasmid system . The natural multiplasmid strain EST1001 is phenotypically unstable . In its Phe-, mTol-, and Sal- segregants, the plasmid DNA underwent structural rearrangements without a marked loss of plasmid DNA, and the majority of segregants gave revertants . The genes specifying the degradation of phenol and m-toluate were transferable to P . putida PaW340, and in this strain a new multiplasmid system with definite structural changes was formed . The 17-kilobase transposable element, a part of the TOL plasmid pWWO present in the chromosome of PaW340, was inserted into the plasmid DNA in transconjugants . In addition, transconjugant EST1020 shared pWWO-like structures . Enzyme assays demonstrated that ortho-fission reactions were used by bacteria that grew on phenol, whereas m-toluate was catabolized by a meta-fission reaction . Salicylate was a functional inducer of the enzymes of both pathways . The expression of silent metabolic pathways of phenol or m-toluate degradation has been observed in EST1001 Phe- mTol+ and Phe+ mTol- transconjugants . The switchover of phenol degradation from the ortho- to the meta-pathway in EST1033 also showed the flexibility of genetic material in EST1001 transconjugants. J Bacteriol, 1989 Sep, 171(9), 4603 - 8 A methyl-accepting protein is involved in benzoate taxis in Pseudomonas putida; Harwood CS; Pseudomonas putida is attracted to at least two groups of aromatic acids: a benzoate group and a benzoylformate group . Members of the benzoate group of chemoattractants stimulated the methylation of a P . putida polypeptide with an apparent molecular weight of 60,000 in sodium dodecyl sulfate-polyacrylamide gels . This polypeptide is presumed to be a methyl-accepting chemotaxis protein for several reasons: its molecular weight is similar to the molecular weights of Escherichia coli methyl-accepting chemotaxis proteins, the amount of time required to attain maximal methylation correlated with the time needed for behavioral adaptation of P . putida cells to benzoate, and methylation was stimulated by benzoate only in cells induced for chemotaxis to benzoate . Also, a mutant specifically defective in benzoate taxis failed to show any stimulation of methylation upon addition of benzoate . Benzoylformate did not stimulate protein methylation in cells induced for benzoylformate chemotaxis, suggesting that sensory input from this second group of aromatic-acid attractants is processed through a different kind of chemosensory pathway . The chemotactic responses of P . putida cells to benzoate and benzoylformate were not sensitive to external pH over a range (6.2 to 7.7) which would vary the protonated forms of these weak acids by a factor of about 30 . This indicates that detection of cytoplasmic pH is not the basis for aromatic-acid taxis in P . putida. J Bacteriol, 1989 Sep, 171(9), 4930 - 7 Transcription initiation at multiple promoters of the pfl gene by E sigma 70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo; Sawers G et al.; In vitro transcription experiments were used to provide further evidence that the gene encoding pyruvate formate-lyase (EC 2.3.1.54) from Escherichia coli is transcribed from seven promoters which cover a region of 1.2 kilobase pairs of DNA (G . Sawers and A . Bock, J . Bacteriol., 171:2485-2498, 1989) . The results demonstrated that all promoters were recognized by the major RNA polymerase holoenzyme species E sigma 70 in vitro . Further corroboration for multiple functional promoters came from heterologous expression of the pfl operon in the obligate aerobe Pseudomonas putida . An immunological analysis indicated that the pyruvate formate-lyase protein was synthesized from a multicopy plasmid in P . putida, and S1 nuclease protection of RNA transcripts confirmed that all the pfl promoters on the plasmid were recognized by the host RNA polymerase . Transcription initiated at the same sites in P . putida and in E . coli for all the transcripts that were analyzed. Genetika, 1989 Sep, 25(9), 1559 - 70 {Study of morphology and genome structure of Pseudomonas putida bacteriophages for their classification}; Krylov VN et al.; A group of 27 bacteriophages specific for Pseudomonas putida strains PpG1 and PpN has been isolated . The phages were characterized and compared with the previously described virulent (pf 16, af, tf and PMW) and temperate (PP56 and PP71) phages . The new phages belong to B1 and C1 morphotypes, according to Ackerman's classification . Phage DNAs were digested with several endonucleases; the molecular weights and homology of the DNAs were determined . All phages of P . putida isolated up to now were distributed into 10 species (groups), on the basis of particle morphology, genome size and the results of homology studies . Recombination processes are believed to participate in formation of phages belonging to certain species. J Bacteriol, 1989 Sep, 171(9), 5048 - 55 Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products; Harayama S et al.; The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates . The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells . This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN . Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified . In contrast, most of the DNA between xylC and xylN consists of coding sequences . The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase . The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase . The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide . The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function. Mol Gen Mikrobiol Virusol, 1989 Sep, (9), 33 - 8 {Expression of the phospholipase C gene of Pseudomonas aeruginosa in Escherichia coli and Pseudomonas}; Zinov'eva VN et al.; The plc gene for phospholipase of Pseudomonas aeruginosa, able to be transcribed only from its own promoter, has been introduced into Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida cells in the recombinant plasmid pPMS21 of a wide host range . The expression of plc gene in all recipient cells has been shown to be phosphate regulated . The fact emphasizes the identity of pho-regulation systems in Escherichia coli and Pseudomonas cells . The level of phospholipase activity is similar in Pseudomonas putida and Pseudomonas aeruginosa under the conditions of the gene derepression, while in Escherichia coli cells the level does not exceed 10% of activity registered in Pseudomonas cells. J Bacteriol, 1989 Sep, 171(9), 5155 - 61 Plasmid control of the Pseudomonas aeruginosa and Pseudomonas putida phenotypes and of linalool and p-cymene oxidation; de Smet MJ et al.; Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida) . Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P . aeruginosa phenotype . Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P . aeruginosa phenotype characters . The plasmid can be transferred by PpG777 to both P . putida and P . aeruginosa strains . Surprisingly, the latter assume the P . putida phenotype . We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed . Similarly, this observation implies that support of linalool oxidation effectively conceals the P . aeruginosa character. Mol Gen Genet, 1989 Aug, 218(2), 266 - 71 Identification of nucleotides critical for activity of the Pseudomonas putida catBC promoter; Aldrich TL et al.; Pseudomonas putida utilizes the catBC operon, which encodes cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1) and muconolactone isomerase (MI; EC 5.3.3.4), for growth on benzoate as a sole carbon source . This operon is positively regulated, and the promoter is located 64 bp upstream of the catB translational start site . Using site-specific mutagenesis, we identified nucleotides that influenced the induction of this promoter . Promoter activity was monitored with the promoter probe vector pKT240 . Transcription of mRNA from mutant promoters was determined by primer extension mapping . Comparison of the initiation start site of mutant promoters with that of the wild-type promoter identified a single functional promoter. J Bacteriol, 1989 Aug, 171(8), 4326 - 33 Involvement of Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions; Kohler T et al.; The RpoN protein was originally identified in Escherichia coli as a sigma (sigma) factor essential for the expression of nitrogen regulons . In the present study we cloned the Pseudomonas putida rpoN gene and identified its gene product as a protein with an apparent molecular weight of 78,000 . A mutant rpoN gene was constructed by in vitro insertion mutagenesis with a kanamycin cassette . A P . putida rpoN mutant was then isolated by replacement of the intact chromosomal rpoN gene by the mutant rpoN gene through homologous recombination . Examination of the phenotypes of the P . putida rpoN mutant thus obtained allowed the identification of a series of metabolic functions whose expression depends upon the RpoN sigma factor . The rpoN mutation in P . putida affected the utilization by this organism of nitrate, urea, and uncharged amino acids, namely, alanine, glycine, isoleucine, leucine, and serine, as nitrogen sources . The mutation also affected the utilization of the above-mentioned amino acids, as well as lysine, C4-dicarboxylates (succinate, fumarate), and alpha-ketoglutarate, as carbon sources . In contrast to the P . putida wild-type strain, the rpoN mutant was nonmotile . The colony morphology of the mutant strain was different from that of the wild-type strain . Studies on the expression of the TOL plasmid catabolic operons in the mutant strain demonstrated that transcription from the upper-operon promoter and from the xylS gene promoter requires the RpoN sigma factor. J Bacteriol, 1989 Aug, 171(8), 4189 - 95 Identification of multiple repressor recognition sites in the hut system of Pseudomonas putida; Hu L et al.; The hutC gene in Pseudomonas putida encodes a repressor protein that negatively regulates the expression of all hut genes . We have overexpressed this cloned hutC gene in Escherichia coli to identify P . putida hut regions that could specifically bind the repressor . Ten restriction fragments, some of which were partially overlapping and spanned the coding portions of the P . putida hut region, were labeled and tested for their ability to recognize repressor in a filter binding assay . This procedure identified three binding sites, thus supporting previous indications that there were multiple operons . A 1.0-kilobase-pair SalI restriction fragment contained the operator region for the hutUHIG operon, whereas a 1.9-kilobase-pair SmaI fragment contained the hutF operator . A 2.9-kilobase-pair XhoI segment appeared to contain the third operator, corresponding to a separate and perhaps little used control region for hutG expression only . The addition of urocanate, the normal inducer, caused dissociation of all operator-repressor complexes, whereas N-formylglutamate, capable of specifically inducing expression of the hutG gene, inhibited binding only of repressor to fragments containing that gene . Formylglutamate did not affect the action of urocanate on the repressor-hutUHIG operator complex, indicating that it binds to a site separate from urocanate on the repressor . DNA footprinting and gel retardation analyses were used to locate more precisely the operator for the hutUHIG operon . A roughly 40-base-pair portion was identified which contained a 16-base-pair region of dyad symmetry located near the transcription initiation site for this operon. FEMS Microbiol Lett, 1989 Jul 15, 51(1), 143 - 7 Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250; Engesser KH et al.; Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy . Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step . After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway . After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts . Other ortho-substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route. Med Parazitol (Mosk) . 1989 Jul-Aug;(4):90. {Preservation of the viability of Opisthorchis eggs by joint cultivation with Pseudomonas putida}; Mefod'ev VV et al.; The effect of Pseudomonas putida on Opisthorchis' ova was studied with a view to assess the feasibility of using bacteria as biological agents against opisthorchiasis . Experiments on mixed culture of the above-mentioned bacteria and helminths' ova demonstrated the lack of ovicidal effect of Pseudomonas putida on the ova. J Bacteriol, 1989 Jul, 171(7), 4063 - 6 Flagellation of Pseudomonas putida and analysis of its motile behavior; Harwood CS et al.; Pseudomonas putida flagella were examined . Also, changes in motile behavior in response to chemoattractants were analyzed quantitatively by computer . Reversals in the rotation direction of bundles of polar flagella resulted in changes in swimming direction . Cells swimming in buffer changed direction once every 2 s on average, whereas cells exposed to the attractant benzoate changed direction an average of once every 10 s . The findings show that P . putida responds to temporal gradients of chemoattractant by suppressing changes in the direction of rotation of flagella. J Photochem Photobiol B, 1989 Jun, 3(3), 429 - 35 Thermal activation of photoactivatable urocanase from Pseudomonas putida; O'Donnell PS et al.; The dark inactivation of urocanase from Pseudomonas putida is caused by the formation of a sulfite adduct of the tightly bound coenzyme, nicotinamide adenine dinucleotide . Photodissociation of this adduct by UV radiation restores the enzyme activity . Based on cold exhaustive dialysis the modification reaction appeared to be irreversible . However, we now report that sulfite modification of urocanase is reversible at higher temperatures . An Arrhenius plot of the thermal activation is linear (20-38 degrees C) . The activation energy for the enzyme activation is 114 kJ mol-1 . The substance that is photodissociated from inactive urocanase reacts with urocanase to reform the modified enzyme indicating that sulfite is not oxidized, or otherwise changed through these processes . Nucleophiles (sulfite, hydroxylamine, hydride, cyanide) are known to inhibit urocanase by forming adducts with nicotinamide adenine dinucleotide . Urocanase inactivated by hydride or cyanide is not reactivated thermally or photochemically . Urocanase inactivated by hydroxylamine and by glycylglycine can be reactivated by a thermal reaction . In conclusion, sulfite-modified urocanase, which is formed in cells, can be reactivated not only by sunlight but also at physiological temperatures. Gene, 1989 May 15, 78(1), 19 - 27 A simple procedure for transferring genes cloned in Escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK; Harayama S et al.; A simple method to transfer non-conjugative Escherichia coli plasmids to other Gram-negative bacteria and their maintenance is described . This method involves generation of inverse transposition-mediated cointegrates of the non-conjugative E . coli plasmid with a conjugative IncW broad-host-range plasmid, R388, carrying Tn10 . Isolation of such cointegrates was readily effected by conjugal transfer from an E . coli donor containing the two plasmids to an E . coli recipient, with selection for transconjugants expressing a marker of the E . coli plasmid . This method is particularly useful when large series of E . coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination . Utility of the method is shown by a functional analysis in Pseudomonas putida of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0. J Biol Chem, 1989 May 5, 264(13), 7742 - 6 Construction and nucleotide sequence of a cDNA encoding the full-length preprotein for human branched chain acyltransferase; Danner DJ et al.; A cDNA (1.6 kilobases) for branched chain acyltransferase (E2b) isolated from a human liver library encoded only the amino-terminal half of the protein (Hummel, K . B., Litwer, S., Bradford, A . P., Aitken, A., Danner, D . J., and Yeaman, S . J . (1988) J . Biol . Chem . 263, 6165-6168) . Here we report the isolation of other cDNAs which encode the carboxyl-terminal half of E2b and the construction of a cDNA which encodes the entire pre-E2b . cDNA from the original clone encoding the leader sequence, lipoate binding domain, and E3 binding domain was ligated to the cDNA from a clone which by restriction maps contained an additional 3' sequence . Both cDNAs used in the construct made a fusion protein in their original phage isolate recognized by antibodies to E2b . The nucleotide sequence of the constructed cDNA was determined, and the 1431 base pairs in the open reading frame encoded a protein of 477 amino acids . In vitro transcription and translation of this cDNA produced a 57-kDa protein recognized by E2b-specific antibodies . Mouse liver mitochondria imported and processed the 57-kDa protein to a 52-kDa antigenic protein which co-migrated with E2b isolated from tissue . Comparing the protein structure of this human pre-E2b protein with that for other acyltransferase proteins showed a similarity in structure throughout all the proteins suggesting evolutionary conservation . Branched chain acyltransferase from Pseudomonas putida showed the most similarity to human E2b. Pept Res, 1989 May-Jun, 2(3), 240 - 5 Specific inhibition of bacterial and bovine urocanases by glycylglycine; Hunter JK et al.; Urocanase (EC 4.2.1.49) purified from Pseudomonas putida was unexpectedly inhibited by the dipeptide glycylglycine . Using a spectrophotometric assay for urocanase activity, we characterized the inhibition . The inhibition was temperature-, concentration-, and time-dependent; 0.1, 0.5 and 1.0 mM glycylglycine inhibited the enzyme by 20%, 50% and 78%, respectively, in 60 min at 30 degrees C . Dithiothreitol and reduced glutathione did not prevent the process . The inhibition was a pseudo first-order reaction . Three ligands that bind to the active site, urocanate, imidazole-propionate (a competitive inhibitor) and sulfite, protected the enzyme from glycylglycine inhibition . The inhibition was very specific for glycylglycine, because fifteen related biochemicals, including glycine, triglycine, and tetraglycine, were not effective . Ethylenediaminetetraacetic acid and other chelators did not inhibit urocanase . Bovine liver urocanase was also inhibited by this peptide . The characteristics of this inhibition suggest that glycylglycine acts at the active site, does not function by metal binding and that minor alterations in the glycylglycine molecule preclude the inhibition . A specific inhibition of urocanases by glycylglycine has been observed. Biochim Biophys Acta, 1989 Apr 12, 1007(3), 301 - 8 Analysis of nonpolar insertion mutations in the trfA gene of IncP plasmid RK2 which affect its broad-host-range property; Shingler V et al.; Replication of broad-host-range plasmid RK2 requires the protein product(s) of the plasmid-encoded trfA gene to initiate replication at oriV, the vegetative replication origin . The trfA gene contains two translational starts which direct translation of two polypeptides, of 382 and 285 amino acids, which differ by the 97 amino acids at their N-terminus . Nonpolar insertions which abolish expression of the larger TrfA polypeptide but otherwise retain the trfA gene's normal expression signals severely reduce plasmid replication efficiency in Pseudomonas aeruginosa and to a lesser extent in Pseudomonas putida, but have very little effect in Escherichia coli . This indicates that the organization of the trfA gene, producing two polypeptides products, plays an important part in the broad-host-range of plasmid RK2 by providing a degree of flexibility in the way the plasmid's replication system interacts with host biochemistry. Bioorg Khim, 1989 Apr, 15(4), 560 - 1 {Nucleotide sequence of the rplL gene coding for ribosomal protein L7/L12 of Pseudomonas putida}; Borodin AM et al.; The Pseudomonas putida rpl L gene coding for ribosomal protein L7/L12 was cloned and sequenced . Although Asp55 residue in L7/L12 was previously shown to be conservative in ten different organisms, the Pseudomonas putida L7/L12 proved to contain Asn55, thus showing that Asp55 is not invariant. Appl Environ Microbiol, 1989 Apr, 55(4), 798 - 805 Cloning of bacterial genes specifying degradation of 4-chlorobiphenyl from Pseudomonas putida OU83; Khan A et al.; Genes capable of 4-chlorobiphenyl (4-CBP) degradation were cloned from 4-CBP-degrading Pseudomonas putida OU83 by using a genomic library which was constructed in the broad-host-range cosmid vector pCP13 . P . putida AC812 containing chimeric cosmid-expressing enzymes involved in the 4-CBP degradation pathway were identified by detecting 3-phenylcatechol dioxygenase activity (3-PDA) . Chimeric cosmid clones pOH83, pOH84, pOH85, pOH87, and pOH88 positive for 3-PDA grew in synthetic basal medium containing 4-CBP (5 mM) as a carbon source . Restriction digestion analysis of recombinant cosmids showed DNA inserts ranging from 6 to 30 kilobase pairs . Southern hybridization data revealed that the cloned DNA inserts originated from strain OU83 . Gas chromatography-mass spectrometry analysis of the metabolites of P . putida AC812(pOH88) incubated with 4-CBP and 4'-chloro-3-phenylcatechol showed the formation of 4-chlorobenzoic acid and benzoic acid . These results demonstrate that the cloned DNA fragments contain genes encoding for chlorobiphenyl dioxygenase (cbpA), dihydrodiol dehydrogenase (cbpB), 4'-chloro-3-phenylcatechol dioxygenase (cbpC), a meta-cleavage compound (a chloro derivative of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate) hydrolase (cbpD), and a new dechlorinating activity (dcpE) . The location of the cbpC gene specifying 3-PDA was determined by subcloning an EcoRI DNA fragment (9.8 kilobase pairs) of pOH88 in plasmid vector pUC19 . The cloned gene encoding 3-PDA was expressed in Escherichia coli HB101 and had substrate specificity only for 3-phenylcatechol and 4'-chloro-3-phenylcatechol. J Bacteriol, 1989 Apr, 171(4), 1952 - 9 Evidence that the transcription activator encoded by the Pseudomonas putida nahR gene is evolutionarily related to the transcription activators encoded by the Rhizobium nodD genes; Schell MA et al.; The nahR gene of the 83-kilobase naphthalene degradation plasmid NAH7 of Pseudomonas putida encodes a 34-kilodalton polypeptide which binds to the nah and sal promoters to activate transcription of the degradation genes in response to the inducer salicylate . The DNA sequence of the nahR gene was determined, and a derived amino acid sequence of the NahR protein was obtained . A computer search for homologous proteins showed that within the first 124 amino-terminal residues, NahR has approximately 35% identity with the transcriptional activator proteins encoded by the nodD genes of Rhizobium species . Allowing for ultraconservative amino acid substitutions, greater than 47% overall similarity was found between NahR and NodD, while 32% similarity was found between NahR and another transcription activator, LysR of Escherichia coli . The region of greatest similarity among all three proteins contained a probable helix-turn-helix DNA-binding motif as suggested by homology with the proposed consensus sequence for Cro-like DNA-binding domains . The high level of amino acid identity between NahR and NodD, in conjunction with the observations that nahR and nodD are 45% homologous in DNA sequence, are divergently transcribed from homologous promoters near the structural genes they control, and have similar DNA-binding sites, strongly suggests that these two genes evolved from a common ancestor. Can J Microbiol, 1989 Apr, 35(4), 423 - 31 2-Oxoaldehyde metabolism in microorganisms; Murata K et al.; The properties of methylglyoxal-metabolizing enzymes in prokaryotic and eukaryotic microorganisms were studied systematically and compared with those of mammalian enzymes . The enzymes constitute a glycolytic bypass and convert methylglyoxal into pyruvate via lactate . The first step in this conversion is catalyzed by glyoxalase I, methylglyoxal reductase, or methylglyoxal dehydrogenase . The regulation of the yeast glyoxalase system was analyzed . The system was closely related to the proliferative states of yeast cells, the activity of the system being high in dividing cells and low in nondividing ones . The gene for the glyoxalase I of Pseudomonas putida and the genes responsible for the activity of glyoxalase I and methylglyoxal reductase in Saccharomyces cerevisiae were cloned and their structural and phenotypic characters studied. Biochimie, 1989 Apr, 71(4), 521 - 31 DNA sequence of the tryptophan synthase genes of Pseudomonas putida; Crawford IP et al.; Genes encoding the 2 subunits of tryptophan synthase in Pseudomonas putida have been identified and cloned by their similarity to the corresponding genes in Pseudomonas aeruginosa . The deduced amino acid sequences were confirmed by comparison with regions ascertained earlier by protein sequencing . The Pseudomonas amino acid sequences are 85% identical for the beta subunit and 70% identical for the alpha subunit . These sequences are compared to those of Salmonella typhimurium, where the structure is known from X-ray crystallography . Although amino acid conservation drops to 54% and 36% for the beta and alpha subunits, only 3 single residue gaps are required to maintain alignment throughout and most of the residues identified as important for catalysis or cofactor binding are conserved . The 23 residues surrounding the beta chain lysine that enters into a Schiff base linkage with the pyridoxal phosphate cofactor are compared in 13 species, including representatives from the eukaryotic and both prokaryotic kingdoms; appreciable conservation is apparent . The approximately 100 base pairs separating the trpB gene from its divergently transcribed activator gene are similar in the 2 pseudomonads, but do not resemble those of any other bacterium or fungus studied to date. Biochemistry, 1989 Mar 7, 28(5), 2160 - 8 In vivo enzymology: a deuterium NMR study of formaldehyde dismutase in Pseudomonas putida F61a and Staphylococcus aureus; Mason RP et al.; High-resolution deuterium NMR spectroscopy has been used to follow the detoxifying metabolism of {D2}formaldehyde in vivo in several bacterial species . Production of {D2}methanol in Escherichia coli confirms that the oxidation and reduction pathways of metabolism are independent in this organism . Efficient production of equimolar quantities of {D}formate and {D3}methanol in Pseudomonas putida F61a and Staphylococcus aureus implicates a formaldehyde dismutase, or "cannizzarase", activity . These observations imply that the unusual formaldehyde resistance in P . putida F61a is a direct result of efficient dismutation acting as a route for detoxification . Cross-dismutation experiments yield an enzymic kinetic isotope effect of ca . 4 for H vs D transfer and a similar spectrum of substrate specificity to the isolated enzyme . {D}benzyl alcohol produced by cross-dismutation of {D2}formaldehyde and benzaldehyde in P . putida is demonstrated to have the R configuration by a novel deuterium NMR assay . Additionally, S . aureus produces methyl formate as a product of formaldehyde detoxification, apparently by oxidizing the methanol hemiacetal of formaldehyde. Mikrobiologiia, 1989 Mar-Apr, 58(2), 298 - 304 {Mutants of the plasmid for biodegradation of naphthalene, determining catechol oxidation via the meta-pathway}; Kulakova AN et al.; Most of the known naphthalene biodegradation plasmids determine the process of naphthalene degradation via salicylate and catechol using the meta pathway of catechol degradation . However, Pseudomonas putida strains with plasmids pBS2, pBS216, pBS217 and NPL-1 exert no activity of the enzymes involved in the meta pathway of catechol degradation . When 2-methylnaphthalene was added to the medium as a sole carbon source, mutants growing on this compound were isolated in the strains with the studied plasmids . Plasmid localization of the mutations was established using conjugation transfer as well as by obtaining spontaneous variants that had lost the ability to grow on 2-methylnaphthalene; the respective plasmid mutants were referred to as pBS101, pBS102, pBS103 and pBS105 . The strains with the mutant plasmids were tested for the activity of the key enzymes involved in naphthalene catabolism and the activity of catechol-2,3-dioxygenase was found . The data allow one to arrive at the conclusion that plasmids pBS2, pBS216, pBS217 and NPL-1 contain silent genes for the meta pathway of catechol degradation, which are activated by the respective mutations. J Biol Chem, 1989 Feb 25, 264(6), 3096 - 101 Study of the 5-oxoprolinase reaction by 13C NMR; Li LY et al.; 5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate . Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A . P., and Meister, A . (1986) J . Biol . Chem . 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A . P., and Meister, A . (1987) J . Biol . Chem . 262, 11020-11025) . A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here . The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds . Using 5-{18O}oxo{5-13C}proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney . This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction . The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively . The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate . Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis. Arch Biochem Biophys, 1989 Feb 15, 269(1), 295 - 304 Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida; Kim Y et al.; The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively . Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1 . The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase . The pTK1- or pTK3-transformed P . aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates . In wild-type P . cepacia as well as in pTK1- or pTK3-transformed P . aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P . cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P . putida, appears to be encoded on chromosome . As revealed by DNA cross hybridizations, the P . cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P . putida but the P . cepacia sal was not homologous to the P . putida sal . Furthermore, polyclonal antibodies developed against purified P . cepacia salicylate hydroxylase inactivated the cloned P . cepacia salicylate hydroxylase but not the cloned P . putida salicylate hydroxylase in P . aeruginosa PAO1 . It appears that P . cepacia and P . putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution. Biochemistry, 1989 Feb 7, 28(3), 969 - 75 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; Tsou AY et al.; 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) J Mol Biol, 1989 Feb 5, 205(3), 557 - 71 Crystal structure of muconolactone isomerase at 3.3 A resolution; Katti SK et al.; The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 A resolution . The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map . The backbone trace is complete and confirmed by the amino acid sequence fit . Each subunit is composed of a body with two alpha-helices and an antiparallel twisted beta-sheet of four strands, and an extended arm . The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket . The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket . The decameric protein is almost spherical, with the helices forming the external coat . There is a large hydrophilic cavity in the center with open ends along the 5-fold axis . Molecular interactions between subunits are extensive . Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization. J Bacteriol, 1989 Feb, 171(2), 975 - 82 Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light; McBeth DL; The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes . CAM-OCT substantially increased both survival and mutagenesis in the two species . P . aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P . putida strains . UV-induced mutagenesis of plasmid-free P . putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P . aeruginosa without CAM-OCT was seen only in the forward mutation assay . These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT . A number of P . putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined . All mutations producing a UV-sensitive phenotype in P . putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others . On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded. J Bacteriol, 1989 Feb, 171(2), 837 - 46 Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters; Schell MA et al.; The nahR gene of plasmid NAH7 of Pseudomonas putida encodes a 36-kilodalton polypeptide which activates transcription of the nah and sal operons in response to the inducer salicylate . A gel mobility shift assay was used to identify a DNA-binding activity which was present only in extracts from either P . putida or Escherichia coli containing a functional nahR gene . The binding activity was highly specific for DNA containing the nah or sal promoters, but the apparent affinity for the promoters was not altered by the presence of salicylate . DNase I protection experiments with a partially purified NahR protein preparation showed that NahR protects both nah and sal promoter sequences between -82 and -47 . The location and amount of protection were not dramatically altered by the presence of salicylate . In vitro mutagenesis was used to make mutations in the protected region of the sal promoter . Analysis of the mutants showed that binding of NahR is required for transcription activation and identified two nucleotides in the protected region that are essential for binding and activation by NahR. J Bacteriol, 1989 Feb, 171(2), 665 - 8 Isolation of a third lipoamide dehydrogenase from Pseudomonas putida; Burns G et al.; Pseudomonads are the only organisms so far known to produce two lipoamide dehydrogenases (LPDs), LPD-Val and LPD-Glc . LPD-Val is the specific E3 component of branched-chain oxoacid dehydrogenase, and LPD-Glc is the E3 component of 2-ketoglutarate and possibly pyruvate dehydrogenases and the L-factor of the glycine oxidation system . Three mutants of Pseudomonas putida, JS348, JS350, and JS351, affected in lpdG, the gene encoding LPD-Glc, have been isolated; all lacked 2-ketoglutarate dehydrogenase, but two, JS348 and JS351, had normal pyruvate dehydrogenase activity . The pyruvate and 2-ketoglutarate dehydrogenases of the wild-type strain of P . putida were both inhibited by anti-LPD-Glc, but the pyruvate dehydrogenase of the lpdG mutants was not inhibited, suggesting that the mutant pyruvate dehydrogenase E3 component was different from that of the wild type . The lipoamide dehydrogenase present in one of the lpdG mutants, JS348, was isolated and characterized . This lipoamide dehydrogenase, provisionally named LPD-3, differed in molecular weight, amino acid composition, and N-terminal amino acid sequence from LPD-Glc and LPD-Val . LPD-3 was clearly a lipoamide dehydrogenase as opposed to a mercuric reductase or glutathione reductase . LPD-3 was about 60% as effective as LPD-Glc in restoring 2-ketoglutarate dehydrogenase activity and completely restored pyruvate dehydrogenase activity in JS350 . These results suggest that LPD-3 is a lipoamide dehydrogenase associated with an unknown multienzyme complex which can replace LPD-Glc as the E3 component of pyruvate and 2-ketoglutarate dehydrogenases in lpdG mutants. Genetika, 1989 Feb, 25(2), 226 - 37 {Cloning of Pseudomonas putida genes responsible for the primary stages of oxidation of naphthalene in Escherichia coli cells}; Boronin AM et al.; Data on cloning Pseudomonas putida D-plasmid pBS286 (IncP-9) genes which are responsible for primary stages of naphthalene oxidation as well as data on the expression of these genes in Escherichia coli cells are presented . Recombinant plasmid pBS959 containing the whole constitutive nahA locus encoding naphthalene dioxygenase, a key enzyme of the naphthalene oxidation pathway, has been constructed on the basis of the pUC19 vector . An evidence has been obtained that at least a portion of the sequence of structural nahB gene is cloned in the recombinant pBS959 plasmid . Constitutive expression of the nahA gene is controlled by its own promoter and leads to conversion of indole to indigo in E . coli cells, strain HB101 . Plasmid pBS959 is characterized by high structural stability; some instability observed is due to segregation. FEMS Microbiol Lett, 1989 Feb, 48(3), 307 - 11 5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases of Escherichia coli C and Klebsiella pneumoniae M5a1 show very high N-terminal sequence homology; Fawcett T et al.; 5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) dehydrogenase from Escherichia coli C and Klebsiella pneumoniae M5a1 have been purified and some of their properties studied . The apparent Km values for NAD and CHMS were 11.7 +/- 1.5 microM and 5.2 +/- 1.9 microM, respectively, for the K . pneumoniae enzyme, and 19.5 +/- 2.7 microM and 9.2 +/- 1.4 microM, respectively, for the E . coli enzyme . Both enzymes were optimally active at pH 7.5 in sodium phosphate buffer . They had subunit molecular weights of 52,000 (+/- 1000) and the native enzymes appeared to be dimers of identical subunits . The first 20 residues of their N-terminal amino acid sequences were 90% homologous . A degenerate oligonucleotide probe constructed to a six amino acid sequence common to both enzymes gave strong hybridization with DNA from E . coli strains B and W as well as with E . coli C and K . pneumoniae but little or no hybridization to DNA from E . coli K12 or Pseudomonas putida. FEMS Microbiol Lett, 1989 Feb, 48(3), 295 - 300 Cloning and expression in Escherichia coli of the toluene dioxygenase gene from Pseudomonas putida NCIB11767; Stephens GM et al.; The genes encoding toluene dioxygenase, toluene cis-glycol dehydrogenase and catechol 2.3-oxygenase from Pseudomonas putida NCIB 11767 were cloned and expressed in Escherichia coli HB101 on a 20 kb fragment . The recombinant strain produced indigo and a variety of other coloured products . Although the enzymes were expressed in the absence of inducers, further induction was observed in the presence of toluene or benzene, implying the presence of regulatory elements on the 20 kb insert. J Gen Microbiol, 1989 Feb, 135 ( Pt 2), 409 - 24 Factors affecting conjugal transfer of plasmids encoding mercury resistance from pure cultures and mixed natural suspensions of epilithic bacteria; Rochelle PA et al.; Sixty-five pure cultures of epilithic bacteria were examined for their ability to transfer mercury resistance to Pseudomonas aeruginosa; five isolates transferred plasmids encoding mercury resistance with frequencies ranging from 8.4 x 10(-8) to 2.8 x 10(-3) per recipient . Two of the plasmids, pQM3 and pQM4, encoded narrow-spectrum mercury resistance, pQM3 also encoded streptomycin resistance, and both plasmids were broad host range . Maximum transfer frequencies of epilithic plasmids from pure cultures occurred over the range 10-25 degrees C at 3.5 g C l-1 and with donor to recipient ratios of 0.4-30 . Transfer occurred over a range of pH values (pH 5.0-8.0) but the effect of pH was most significant at non-optimal temperature . Anaerobiosis inhibited transfer of one epilithic plasmid, pQM1, but not that of pQM3 . Plasmids encoding mercury resistance were also transferred from mixed natural suspensions of epilithic bacteria (MNS) to Pseudomonas spp . on agar in the laboratory . Transfer from MNS occurred over a wide range of environmentally relevant conditions with maximum frequencies (2 x 10(-5) per recipient) after 24 h, at 25 degrees C, pH 5.5-8.0 and on a medium containing 10 g C l-1 . The optimal initial cell density of MNS and recipient was 1.7 x 10(5) c.f.u . cm-2 and highest frequencies were obtained with donor to recipient ratios ranging from 1.2 x 10(-1) to 1.7 x 10(-3) . Most of the plasmids (54%) from MNS transferred from their original P . aeruginosa transconjugants to a Pseudomonas putida strain, with frequencies ranging from 1.1 x 10(-6) to greater than 1.0 x 10(-1) per recipient . The majority (80%) of the plasmids were larger than 300 kb and all of these large plasmids encoded UV resistance in addition to mercury resistance . Twenty-one plasmids greater than 300 kb were analysed by restriction digests and were shown to be similar, with only minor structural alterations . One of these alterations was associated with the acquisition of streptomycin resistance . Overall, these results suggest that the epilithic bacteria examined possess the potential to transfer mercury resistance within the epilithon under a wide range of environmentally relevant conditions. Eur J Biochem, 1989 Jan 15, 179(1), 61 - 9 Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched-chain-oxoacid dehydrogenase of Pseudomonas putida; Burns G et al.; The production of two lipoamide dehydrogenases by Pseudomonas is so far unique . One, LPD-val, is the specific E3 component of the branched-chain-oxoacid dehydrogenase and the second, LPD-glc, is the E3 component of 2-oxoglutarate dehydrogenase and the L-factor of the glycine oxidation system . The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD-val in order to compare its deduced amino acid structure with that of other redox-active disulfide flavoproteins . Northern blots using mRNA isolated from P . putida grown in media with branched-chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex . The nucleotide sequence of the structural gene for LPD-val, lpdV, was determined and consists of 459 codons plus the stop codon . The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G + C . Codon usage is characteristic of moderately strongly expressed genes . There is a ribosome-binding site preceding the ATG start codon and a strong candidate for a rho-independent terminator at the 3' end of the reading frame . The Mr of the protein encoded is 48,164 and when the Mr of FAD is added, the total Mr is 48,949, which is very close to the value of 49,000 obtained by SDS-polyacrylamide gel electrophoresis . Similarity comparisons of LPD-val with sequences of three other lipoamide dehydrogenases showed that LPD-val was somewhat more distantly related . It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein. Gene, 1988 Dec 20, 73(2), 355 - 62 Cloning, nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816; Kurkela S et al.; We have cloned the naphthalene dioxygenase(ND)-coding genes from Pseudomonas putida strain NCIB9816 based on their ability to convert indole to indigo . The region coding for this enzyme activity was sequenced and three successive open reading frames were found . The corresponding gene products were identified using the T7 polymerase/promoter system . All of them are necessary for the ND activity . A comparison of the ND-coding genes with the ones coding for benzene dioxygenase revealed significant homology which was more pronounced at the nucleotide level than at the amino acid level. Biochim Biophys Acta, 1988 Dec 2, 957(3), 335 - 9 Purification and properties of carnitine dehydrogenase from Pseudomonas putida; Goulas P; Carnitine dehydrogenase (carnitine:NAD+ oxidoreductase, EC 1.1.1.108) from Pseudomonas putida IFP 206 catalyzes the oxidation of L-carnitine to 3-dehydrocarnitine . The enzyme was purified 72-fold to homogeneity as judged by polyacrylamide gel electrophoresis . The molecular mass of this enzyme is 62 kDa and consists of two identical subunits . The isoelectric point was found to be 4.7 . the carnitine dehydrogenase is specific for L-carnitine and NAD+ . The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0 and 7.0 in the reduction reaction . The optimal temperature is 30 degrees C . The Km values for substrates were determined. Arch Biochem Biophys, 1988 Dec, 267(2), 701 - 13 Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1; Nakai C et al.; Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2 . Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 {Y . Kojima et al . (1967) J . Biol . Chem . 242, 3270-3278} . These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants . The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively . The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000 . The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation . During the degradation, a single amino acid was released at each step . The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine . The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis . These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta {C . Nakai et al . (1979) Arch . Biochem . Biophys . 195, 12-22}, although these two enzymes have very similar properties. Appl Environ Microbiol, 1988 Dec, 54(12), 3183 - 4 Mineralization of the dibenzothiophene biodegradation products 3-hydroxy-2-formyl benzothiophene and dibenzothiophene sulfone; Mormile MR et al.; Dibenzothiophene is degraded to 3-hydroxy-2-formyl benzothiophene by various bacteria, including a strain of Pseudomonas putida that also forms dibenzothiophene sulfone via an alternate pathway . By using these end products as substrates, mixed enrichment cultures that could degrade 3-hydroxy-2-formyl benzothiophene and dibenzothiophene sulfone with the formation of CO2 were established. J Mol Biol, 1988 Nov 20, 204(2), 417 - 33 Crystal structure determination, refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida; Hoeffken HW et al.; The three-dimensional crystal structure of creatine amidinohydrolase (creatinase EC 3.5.3.3) from Pseudomonas putida, a dimeric enzyme with a molecular weight of 97,000, has been determined by multiple isomorphous replacement, averaging over the local dyad and restrained crystallographic refinement at 1.9 A with a crystallographic R-value of 17.7% . The asymmetric unit contains a dimer . The two chemically identical subunits consist of 403 residues each . A subunit is built up of two domains, a small N-terminal and a larger C-terminal domain . The small domain has a central seven-stranded beta pleated sheet with short helices on the outside . The large domain forms a six-stranded antiparallel beta half-barrel with helices on the outside . The two domains are connected by a segment that links two helices . The binding site of the competitive inhibitor carbamoyl sarcosine, a close analog of the substrate creatine, is located in the center of the large domain and partly covered by the small domain of the other subunit . The carbamoyl group is tightly co-ordinated to a water molecule, which presumably represents the nucleophile involved in hydrolysis of creatine . A catalytic mechanism is proposed on the basis of this structure. Prikl Biokhim Mikrobiol, 1988 Nov-Dec, 24(6), 779 - 83 {A comparative study of the formation of 2-keto-D-gluconic acid by free and immobilized cells of Pseudomonas putida}; Voloshenko MI et al.; The effect of glucose, oxygen and 2-keto-D-gluconic acid (2KG) concentrations on the 2KG production by free and immobilized cells of Pseudomonas putida was studied . The effect of these factors was found to be similar in case of both free and immobilized cells, but the rate of the 2KG production by the free cells was a little higher as compared to the immobilized cells. Appl Environ Microbiol, 1988 Nov, 54(11), 2664 - 71 Molecular cloning of 3-phenylcatechol dioxygenase involved in the catabolic pathway of chlorinated biphenyl from Pseudomonas putida and its expression in Escherichia coli; Khan A et al.; Genes encoding 3-phenylcatechol dioxygenases were cloned from the chlorobiphenyl-degrading Pseudomonas putida strain OU83, using broad-host-range cosmid vector pCP13 . Restriction enzyme analysis of DNA from 2,3-dioxygenase-positive chimeric cosmids showed DNA inserts ranging in size from 6.0 to 30 kilobases . The origin of the DNA insert in hybrid clones was established by using 32P-labeled hybrid clones (pOH101 and pOH810) . A 2.3-kilobase HindIII fragment was common to two clones . The 2,3-dioxygenase from the parent P . putida strain, OU83, and the recombinant clones (pOH101 and pOH8101) showed similar characteristics as determined by isoelectric focusing and polyacrylamide gel electrophoresis . The 2,3-dioxygenase from the Escherichia coli recombinant cosmid showed a pI of 5.0, a Km of 14 microM, and broad substrate act