Ps. putida

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 activity with catechol, 4-chlorocatechol, 4-methylcatechol, and 2,3-dihydroxybiphenyl.

J Gen Microbiol, 1988 Oct, 134 ( Pt 10), 2769 - 78
Comparison of the meta pathway operons on NAH plasmid pWW60-22 and TOL plasmid pWW53-4 and its evolutionary significance; Assinder SJ et al.; The regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pWW60-22 was cloned into the broad-host-range vector pKT230 on a 17.5 kbp BamHI fragment . The recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free Pseudomonas putida PaW130 . A detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in Escherichia coli and P . putida hosts . The existence of a regulatory gene was demonstrated by the induction of enzyme activities in the presence of salicylate . DNA-DNA hybridization indicated a high degree of structural homology between the pWW60-22 operon and the analogous meta pathway operon on TOL plasmid pWW53-4 . The data are consistent with the structural genes being arranged in an identical linear array and suggest an evolutionary link between the two catabolic systems.

J Bacteriol, 1988 Oct, 170(10), 4458 - 65
Cloning and expression of the catA and catBC gene clusters from Pseudomonas aeruginosa PAO; Kukor JJ et al.; A 9.9-kilobase (kb) BamHI restriction endonuclease fragment encoding the catA and catBC gene clusters was selected from a gene bank of the Pseudomonas aeruginosa PAO1c chromosome . The catA, catB, and catC genes encode enzymes that catalyze consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catA, catechol-1,2-dioxygenase (EC 1.13.11.1); catB, muconate lactonizing enzyme (EC 5.5.1.1); and catC, muconolactone isomerase (EC 5.3.3.4) . A recombinant plasmid, pRO1783, which contains the 9.9-kb BamHI restriction fragment complemented P . aeruginosa mutants with lesions in the catA, catB, or catC gene; however, this fragment of chromosomal DNA did not contain any other catabolic genes which had been placed near the catA or catBC cluster based on cotransducibility of the loci . Restriction mapping, deletion subcloning, and complementation analysis showed that the order of the genes on the cloned chromosomal DNA fragment is catA, catB, catC . The catBC genes are tightly linked and are transcribed from a single promoter that is on the 5' side of the catB gene . The catA gene is approximately 3 kb from the catBC genes . The cloned P . aeruginosa catA, catB, and catC genes were expressed at basal levels in blocked mutants of Pseudomonas putida and did not exhibit an inducible response . These observations suggest positive regulation of the P . aeruginosa catA and catBC cluster, the absence of a positive regulatory element from pRO1783, and the inability of the P . putida regulatory gene product to induce expression of the P . aeruginosa catA, catB, and catC genes.

J Bacteriol, 1988 Oct, 170(10), 4693 - 8
Siderophore-mediated uptake of Fe3+ by the plant growth-stimulating Pseudomonas putida strain WCS358 and by other rhizosphere microorganisms; de Weger LA et al.; Under iron-limited conditions, Pseudomonas putida WCS358 produces a siderophore, pseudobactin 358, which is essential for the plant growth-stimulating ability of this strain . Cells of strain WCS358, provided that they have been grown under Fe3+ limitation, take up 55Fe3+ from the 55Fe3+-labeled pseudobactin 358 complex with Km and Vmax values of 0.23 microM and 0.14 nmol/mg of cell dry weight per min, respectively . Uptake experiments with cells treated with various metabolic inhibitors showed that this Fe3+ uptake process was dependent on the proton motive force . Furthermore, strain WCS358 was shown to be able to take up Fe3+ complexed to the siderophore of another plant-beneficial P . fluorescens strain, WCS374 . The tested pathogenic rhizobacteria and rhizofungi were neither able to grow on Fe3+-deficient medium in the presence of pseudobactin 358 nor able to take up 55Fe3+ from 55Fe3+-pseudobactin 358 . The same applies for three cyanide-producing Pseudomonas strains which are supposed to be representatives of the minor pathogens . These results indicate that the extraordinary ability of strain WCS358 to compete efficiently for Fe3+ is based on the fact that the pathogenic and deleterious rhizosphere microorganisms, in contrast to strain WCS358 itself, are not able to take up Fe3+ from Fe3+-pseudobactin 358 complexes.

Eur J Biochem, 1988 Sep 15, 176(2), 311 - 7
Similarity of the E1 subunits of branched-chain-oxoacid dehydrogenase from Pseudomonas putida to the corresponding subunits of mammalian branched-chain-oxoacid and pyruvate dehydrogenases; Burns G et al.; The genes encoding proteins responsible for activity of the E1 component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida have been subcloned and the nucleotide sequence of this region determined . Open reading frames encoding E1 alpha (bkdA1, 1233 bp) and E1 beta (bkdA2, 1020 bp) were identified with the aid of the N-terminal sequence of the purified subunits . The Mr of E1 alpha was 45,158 and of E1 beta was 37,007, both calculated without N-terminal methionine . The deduced amino acid sequences of E1 alpha and E1 beta had no similarity to the published sequences of the E1 subunits of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli . However, there was substantial similarity between the E1 alpha subunits of Pseudomonas and rat liver branched-chain-oxoacid dehydrogenases . In particular, the region of the E1 alpha subunit of the mammalian branched-chain-oxoacid dehydrogenase which is phosphorylated, was found to be highly conserved in the Pseudomonas E1 alpha subunit . There was also considerable similarity between the E1 beta subunits of Pseudomonas branched-chain-oxoacid dehydrogenase and human pyruvate dehydrogenase.

J Biol Chem, 1988 Sep 15, 263(26), 13400 - 5
Alkane utilization in Pseudomonas oleovorans . Structure and function of the regulatory locus alkR; Eggink G et al.; The OCT plasmid-localized alkBAC operon encodes enzymes for alkane hydroxylation and alkanol dehydrogenation . The positively controlled expression of the operon is very efficient in both Pseudomonas putida and Escherichia coli . Two regulatory functions have been ascribed to the regulatory locus alkR: inducer recognition and transcriptional activation of the operon . We have cloned and localized the alkR locus on a 4.9-kilobase pair SalI fragment . The alkR region was analyzed for translation productions in E . coli minicells . Two proteins were identified: a 99- and a 48-kDa peptide . The positions of the cistrons encoding these proteins were established . Both cistrons were shown to be essential for an Alk phenotype . The first cistron (alkS), which encodes the 99-kDa protein, complemented alkR mutations affecting inducer specificity . Furthermore, we found that alkS is responsible for activation of expression of the alkBAC operon since it is required for the induction of the alkB gene product alkane hydroxylase . The second cistron (alkT), which encodes the 48-kDa protein, is required for reconstitution of an Alk phenotype but has no function in regulation of alkBAC expression . Thus, the expression of the alkBAC operon is regulated by a 99-kDa protein, whereas the 48-kDa protein is probably a component of the alkane hydroxylase complex.

Nucleic Acids Res, 1988 Sep 12, 16(17), 8603 - 17
Construction of an expression vector for the fission yeast Schizosaccharomyces pombe; Kudla B et al.; We have isolated and characterized a S . pombe promoter using a functional heterologous gene product assay . Random S . pombe genomic fragments were cloned upstream from the promoterless 'lacZ gene and tested in vivo for their efficiency to promote expression of the beta-galactosidase protein in the fission yeast . An efficient S . pombe promoter called 54/1 was isolated and shown to drive up to 5% of total protein synthesis as beta-galactosidase . The structure and nucleotide sequence of this promoter were determined, precise localization of its mRNA transcriptional start points established . Translational fusion of the Pseudomonas putida XylE gene with the 54/1 gene was shown to allow expression of catechol oxidase activity in S . pombe . An expression vector suitable for transcriptional fusions was then constructed from engineered 54/1 promoter sequences and used to drive expression of the E . coli Tn5 ble gene, thus confering resistance to the fission yeast against bleomycin and phleomycin antibiotics.

Biol Chem Hoppe Seyler, 1988 Sep, 369(9), 1031 - 43
Degradation of 1,4-naphthoquinones by Pseudomonas putida; Muller U et al.; Pseudomonas putida J1 and J2, enriched from soil with juglone, are capable of a total degradation of 1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, and 2-chloro-1,4-naphthoquinone . Naphthazerin and plumbagin are only converted into the hydroxyderivatives 2-hydroxynaphthazerin and 3-hydroxyplumbagin, respectively, whereas 2-amino-1,4-naphthoquinone is not attacked at all . The degradation of 1,4-naphthoquinone begins with a hydroxylation of the quinoid ring, yielding 2-hydroxy-1,4-naphthoquinone (lawsone) . Lawsone is reduced to 1,2,4-trihydroxynaphthalene with consumption of NADH . The fission product of the quinol could not be detected by direct means because of its instability . However, the presence of 2-chromonecarboxylic acid, a secondary product of lawsone degradation, leads to the conclusion, that the cleavage of the quinol takes place in the meta-position . The resulting ring fission product is converted into salicylic acid by removal of the side chain, presumably as pyruvate . Further degradation of salicyclic acid leads to the formation of catechol, which is then cleaved in the ortho-position and then metabolized via the 3-oxoadipate pathway . The initial steps in the degradation of 2-chloro-1,4-naphthoquinone, namely, the hydroxylation of the quinone to 2-chloro-3-hydroxy-1,4-naphthoquinone, followed by the elimination of the chlorine substituent lead to lawsone, which is further degraded through the pathway described . The degradation steps could be verified by the accumulation products of mutant strains blocked in different steps of lawsone metabolism . Generation of mutants was carried out by chemical and by transposon mutagenesis . The regulation of the first steps of the pathway catalysed by juglone hydroxylase and lawsone reductase, was investigated by induction experiments.

Mol Gen Genet, 1988 Sep, 214(1), 173 - 6
In vivo generation of R68.45-pPGH1 hybrid plasmids conferring a Phl+ (meta pathway) phenotype; Herrmann H et al.; Plasmid pPGH1 originating from Pseudomonas putida strain H carries all the genes required for the degradation of phenol (or cresols) via the meta cleavage pathway . Besides mobilization of pPGH1 by a plasmid of the incompatibility group P-1, hybrid plasmids conferring the Phl+ phenotype could be selected, when R68.45 was the conjugative plasmid . The hybrids contain the complete R68.45 and part of pPGH1 . Integration of Phl-DNA of pPGH1 into R68.45 occurred exclusively via the IS21 region of R68.45.

Bioorg Khim, 1988 Sep, 14(9), 1179 - 82
{Genes coding for RNA polymerase in bacteria . III . The use of modified Sanger's method for sequencing the C-terminal region of rpoB gene, N-terminal region of rpoC gene and intercistron region of RNA polymerase in Pseudomonas putida}; Borodin AM et al.; The Sanger method was modified and the primary structure of the SalI-C fragment of the Pseudomonas putida rpoBC operon was elucidated.

J Gen Microbiol, 1988 Sep, 134 ( Pt 9), 2463 - 74
Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1; Shaw LE et al.; Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1 . One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene . The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size . The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts . The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS . The insert in pDKT2 contained only the upper pathway operon and the regulatory region . Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies . However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4 . The significance of these findings to the evolution of TOL plasmids is discussed.

Genetika, 1988 Sep, 24(9), 1550 - 61
{Cloning and expression of Pseudomonas putida gene controlling the catechol-2,3-oxygenase activity in Escherichia coli cells}; Tsoi TV et al.; The genes nahC and nahD from Pseudomonas putida naphthalene degradation plasmid pBS286 were cloned on the vector pUC19 in Escherichia coli cells . The catechol-2,3-oxygenase activity observed in E . coli cells containing recombinant plasmid pBS955 demands the participation of 32 kD polypeptide which is apparently the product of the nahC gene . Second polypeptide of molecular weight 34.5 kD is synthesized in pBS955 containing E . coli minicells and perhaps it is a nahD gene product . The data obtained indicate that 1,2-dihydroxynaphthalene dioxygenase possesses a relaxed substrate specificity, at least, when cloned on a multicopy vector . The cloned DNA insert of pBS955 is not likely to contain its own promoter, so that expression of nahC and nahD genes from the nah1 operon is controlled by the vector lac promoter . The direction of transcription and localization of both polypeptides on the pBS955 map are determined.

Eur J Biochem, 1988 Sep 1, 176(1), 165 - 9
Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli; Burns G et al.; The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida has been determined and translated into its amino acid sequence . The start of bkdB was identified from the N-terminal sequence of E2b isolated from branched-chain-oxoacid dehydrogenase of the closely related species, P . aeruginosa . The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C . There was no intergenic space between bkdA2 and bkdB . No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage . The bkdB gene encoded 423 amino acids although the N-terminal methionine was absent from E2b prepared from P . aeruginosa . The relative molecular mass of the encoded protein was 45,134 (45,003 minus methionine) vs 47,000 obtained by SDS/polyacrylamide gel electrophoresis . There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E2o, the transacylases of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli respectively . There was significant similarity between the lipoyl domain of E2b and of E2p and E2o as well as between the E1-E2 binding domains of E2b, E2p and E2o . There was no similarity between the E3 binding domain of E2b to E2p and E2o which may reflect the uniqueness of the E3 component of branched-chain-oxoacid dehydrogenase of P . putida . The conclusions drawn from these comparisons are that the transacylases of prokaryotic pyruvate, 2-oxoglutarate and branched-chain-oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.

J Bacteriol, 1988 Sep, 170(9), 4272 - 9
Organization and multiple regulation of histidine utilization genes in Pseudomonas putida; Hu L et al.; The arrangement of the histidine utilization (hut) genes in Pseudomonas putida was established by examining the structure of a DNA segment that had been cloned into Escherichia coli via a cosmid vector . Southern blot analysis revealed that the restriction patterns of the hut genes cloned into E . coli and present in the P . putida genome were identical, indicating that no detectable DNA rearrangement took place during the cloning . Expression of the hut genes from a series of overlapping clones indicated the gene order to be hutG-hutI-hutH-hutU-hutC-hutF . The transcription directions of the different hut genes were determined by cloning the genes under control of the lambda pL promoter . This showed that hutF, encoding formiminoglutamate hydrolase, was transcribed in a direction opposite to that of the other genes . Inactivation of the cloned hut genes by Tn1000 insertion revealed that the hut genes were divided into three major transcriptional units (hutF, hutC {the repressor gene}, and hut UHIG), but hutG may also be independently transcribed . When cloned individually with hutC on the same vector, hutF and hutU (which encodes urocanase) expression was induced by urocanate, indicating that these two genes each possess an operator-promoter element . Tn1000 insertions (in the cloned genes) or Tn5 insertions (in the P . putida genome) affecting the hutI or hutH gene only partially eliminated hutG expression . Furthermore, hutG, which specifies N-formylglutamate amidohydrolase, was regulated by the hutC product when the two genes were cloned on the same vector and expressed in E . coli . Therefore, hutG can be expressed independently from its own promoter, in keeping with earlier observations that N-formylglutamate amidohydrolase synthesis is not coordinated with that of urocanase and histidase and can be induced by N-formylglutamate or urocanate.

J Biol Chem, 1988 Aug 25, 263(24), 11683 - 91
The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase; Vlahos CJ et al.; The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner . After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin . The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced . Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues . The primary structure of the molecule was determined to be as follows . (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E . coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively . No microheterogeneity is observed among the three subunits . The peptide containing the active-site arginine residue (Vlahos, C . J., Ghalambor, M . A., and Dekker, E . E . (1985) J . Biol . Chem . 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49 . The Schiff base-forming lysine residue (Vlahos, C . J., and Dekker, E . E . (1986) J . Biol . Chem . 261, 11049-11055) is located at position 133 . Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.

Biokhimiia, 1988 Aug, 53(8), 1256 - 64
{Isolation, purification and various properties of L-lysine-2-monooxygenase from Pseudomonas putida}; Khachatrian GE et al.; Isolation and purification of L-lysine-2- monooxygenase from the bacterium Pseudomonas species was carried out . The purification procedure included ammonium sulfate fractionation, acid treatment, gel filtration through Sephadex G-200 and ion-exchange chromatography on DEAE-Sephadex A-50 . Such treatment resulted in more than 220-fold purification and 22% yield; the specific activity of the enzyme is 14.6 U/mg . The enzyme spectrum is typical for flavoproteins, with peaks at 275, 386 and 462 nm . At 460 nm excitation, the enzyme fluorescence has an emission maximum at 530 nm, whereas at 360 nm extication--at about 520 nm . The molecular mass of L-lysine-2-monooxygenase as determined by SDS/PAAG electrophoresis is about 268 kD . The KM values for oxygen and lysine are equal to 6.5.10(-4) M and 2.3.10(-4) M, respectively . The curve for the dependence of the reaction rate on lysine concentration is sigmoidal . It was assumed that the electrophoretic behaviour of the enzyme confirms the hypothesis on the nature of allosteric regulation of the enzyme activity by alterations in the regulatory site charge.

Jpn J Antibiot, 1988 Aug, 41(8), 1002 - 7
{Clinical study of cefuzonam in the field of obstetrics and gynecology}; Naito H et al.; Cefuzonam (CZON, L-105) was used clinically for the treatment of obstetrical and gynecological infections at a dosage of 1 g once or twice daily by intravenous drip infusion . The results obtained are summarized as follows . 1 . Clinical effects of CZON were analyzed in 10 patients, including 5 patients with intrapelvic infections, 3 with intrauterine infections, and 1 each with adnexitis and an external genital infection . Excellent responses were observed in 1 patient (11.1%), good responses in 7 (77.8%), poor responses in 1 (11.1%), and one remaining case was unevaluable . The efficacy ratio was 88.9% . 2 . Upon the treatment, eradications of causative bacteria were observed in all 4 cases tested . Staphylococcus sp . (2 strains), Staphylococcus epidermidis (1 strain), Klebsiella oxytoca (1 strain) and Pseudomonas putida (1 strain) were all eradicated by the CZON treatment . 3 . The safety of the drug was analyzed in the 10 patients and rash occurred in 1 patient as a side effect . 4 . One patient showed eosinophilia after the CZON treatment . It appeared that CZON would be useful for the treatment of obstetrical and gynecological infections.

J Gen Microbiol, 1988 Aug, 134 ( Pt 8), 2325 - 31
Stability fluctuations of plasmid-bearing cells: immobilization effects; Nasri M et al.; The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pB lambda H3 in Escherichia coli hosts was studied in free and immobilized continuous cultures . pTG201, containing the strong lambda PR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter . The instability of pTG201 seems to be related to high expression of the cloned xylE genet . Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains . The loss of plasmid vectors from E . coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.

J Bacteriol, 1988 Aug, 170(8), 3774 - 7
Klebsiella pneumoniae origin of replication (oriC) is not active in Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides; O'Neill EA et al.; A DNA fragment carrying genes encoding the conjugal transfer system of the broad host range plasmid RK2 was inserted into a plasmid carrying the chromosomal origin of replication (oriC) from Klebsiella pneumoniae . The resulting plasmid, pEON1, was readily transferred between gram-negative bacteria and carried two potential origins of replication: oriC and the replication origin from pBR322 (oriPBR) . Although pEON1 could be transferred to Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides, pEON1 was not maintained in these strains . However, an oriC-containing plasmid was maintained in these nonenteric bacteria when an RK2 origin of replication was present on the plasmid . Thus, the inability of pEON1 to be established in a nonenteric bacterium represents a failure of oriC to function as an origin of replication rather than a toxic effect of oriC . The initiation potential of the chromosomal origin of replication from K . pneumoniae appears to be realized only in enteric bacteria.

J Bacteriol, 1988 Aug, 170(8), 3742 - 6
Benzoate-dependent induction from the OP2 operator-promoter region of the TOL plasmid pWWO in the absence of known plasmid regulatory genes; Cuskey SM et al.; Expression of the lower catabolic pathway of the TOL plasmid pWWO requires an aromatic acid inducer and the product of the xylS regulatory gene . Pseudomonas putida cells transformed with a plasmid containing the operator-promoter region of the lower pathway (OP2 {or Pm}), upstream from the catechol 2,3-dioxygenase structural gene, showed enzyme induction in the absence of known TOL plasmid regulatory genes . Induction was not seen in transformed Escherichia coli cells or in a P . putida mutant lacking chromosomally encoded benzoate catabolic functions.

Biochem Biophys Res Commun, 1988 Jul 29, 154(2), 537 - 43
Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from Pseudomonas putida; Raju SG et al.; 4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines . This enzyme was purified to homogeneity from Pseudomonas putida by affinity chromatography . The protein had a molecular weight of 91,000 and was a dimer of identical subunits . It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity . The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2 x 10(-4) M and 5.9 x 10(-5) M respectively . It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.

Appl Environ Microbiol, 1988 Jul, 54(7), 1703 - 8
Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1; Wackett LP et al.; Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b . With toluene-induced P . putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm) . At 80 microM (10.5 ppm) trichloroethylene and 30 degrees C, the initial rate was 1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time . A series of mutant strains derived from P . putida F1 that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo . A spontaneous revertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo, and to degrade trichloroethylene . The three isomeric dichloroethylenes were degraded by P . putida F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.

J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 2039 - 48
Loss of the toluene-xylene catabolic genes of TOL plasmid pWW0 during growth of Pseudomonas putida on benzoate is due to a selective growth advantage of 'cured' segregants; Williams PA et al.; During growth on benzoate-minimal medium Pseudomonas putida mt-2 (PaW1) segregates derivative ('cured') strains which have lost the ability to use the pathway encoded by its resident catabolic plasmid pWW0 . Experiments with two plasmids identical to pWW0 but each with an insert of Tn401, which confers resistance to carbenicillin, suggested that the 'benzoate curing' occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss . This effect was not pH-dependent, and was not produced during growth on other weak organic acids, such as succinate or propionate, or when benzoate was present in the medium with an alternative, preferentially used carbon source such as succinate . Growth on benzoate did not cause loss from strain PaW174 of the plasmid pWW0174, a derivative of pWW0 which has deleted the 39 kbp region but carries Tn401 . Similarly the naphthalene-catabolic plasmid pWW60-1, of the same incompatibility group as pWW0, was not lost from PaW701 during growth on benzoate . Competition between wild-type PaW1 and PaW174, which has the 'cured' phenotype, showed that the latter has a distinct growth advantage on benzoate over the wild-type even when initially present as only 1% of the population: when PaW174 was seeded at lower cell ratios, spontaneously 'cured' derivatives of PaW1 took over the culture after 60-80 generations, indicating that they are present in PaW1 cultures at frequencies between 10(-2) and 10(-3) . We conclude that the progressive takeover of populations of PaW1 only occurs when benzoate is present as the sole growth source and that neither benzoate, nor other weak acids, affect plasmid segregation or deletion events: a sufficient explanation is that the 'cured' segregants grow faster than the wild-type using the chromosomally determined beta-ketoadipate pathway.

Bioorg Khim, 1988 Jul, 14(7), 979 - 81
{Genes coding for RNA-polymerase in bacteria . II . Conservative sites in the central region of the beta-subunit of Pseudomonas putida RNA-polymerase}; Borodin AM et al.; SalI--L fragment of the P . putida rpoBC operon has been sequenced and conservative regions of the central part of the RNA-polymerase beta-subunit have been determined . Amino and acid residues interacting with Zn2+ are postulated.

Gene, 1988 Jun 30, 66(2), 301 - 6
Nucleotide sequence of the regulatory gene xylR of the TOL plasmid from Pseudomonas putida; Inouye S et al.; We have determined the nucleotide sequence of the xylR gene for a transcriptional activator for the degradative pathway of aromatic hydrocarbons on the TOL plasmid from Pseudomonas putida . The 1698-bp sequence for a 566-amino acid (aa) protein (Mr 63741) was identified as the XylR-encoding sequence . Three regions in XylR show homology to Klebsiella pneumoniae NtrC and NifA, both of which are transcriptional activators for the ntr and nif genes involved in the nitrogen metabolism . The central region of XylR (aa 234-473) corresponds to the region that was proposed to interact with RNA polymerase having a sigma factor, NtrA {Drummond et al., EMBO J . 5 (1986) 441-447} . The C-terminal region (aa 515-558) has a putative DNA-binding structure . A short segment proximal to the central region (aa 211-229) is thought to be an interdomain linker . No amino acid homology was found in the N-terminal regions among these proteins . These findings suggest the interaction of XylR with an NtrA in the transcriptional activation of the degradative pathway.

Appl Environ Microbiol, 1988 Jun, 54(6), 1399 - 404
Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp . strain JS6; Spain JC et al.; The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems . The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes . Pseudomonas sp . strain JS6 uses a similar system for growth on toluene or dichlorobenzenes . We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene . When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols . Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols . Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.

Biokhimiia, 1988 Jun, 53(6), 1040 - 7
{Purification and properties of two enzymes of meta-cleaving the aromatic ring controlled by the biphenyl biodegradation plasmid pBS 241 from Pseudomonas putida}; Selifonov SA et al.; It was shown that two metapyrocatechases (EC 1.13.11.2) function in Pseudomonas putida BS893 . Biphenyl degradative plasmid pBS241 carries the genes of these enzymes . The basic properties of the both enzymes, i . e., MPC1 and MPC2, were investigated . It was found that MPC1 is an enzyme with a molecular mass of 135 kD and has a heterotetrameric subunit structure (alpha 2 beta 2), being made up of two non-identical polypeptides with Mr of 34 and 22.5 kD; pI is 5.15, the pH optimum is at 8.0, a temperature optimum is at 54 degrees C . MPC2 has a molecular mass of 154 kD and possesses a homotetrameric subunit structure (alpha 4); it consists of identical polypeptides with Mr of 41 kD and has a pI of 4.95, a pH optimum at 7.5 and a temperature optimum at 60 degrees C . The substrate specificity of the enzymes was studied, and the Km and Vmax values for substituted catechols were determined . MPC1 shows a high affinity for 2.3-dihydroxybiphenyl and hydrolyzes 3-methylcatechol and catechol (but not 4-methylcatechol) at a low rate . MPC2 has a moderate affinity for catechol, 3- and 4-methylcatechols, but is incapable of cleaving 2.3-dihydroxybiphenyl . Both enzymes share in common some typical properties of metapyrocatechases . The different role of MPC1 and MPC2 in biphenyl catabolism is discussed.

Mol Gen Mikrobiol Virusol, 1988 Jun, (6), 12 - 6
{Physical map of the DNA of bacteriophage tf of Pseudomonas putida}; Kulakov LA et al.; A physical map has been constructed for P . putida bacteriophage tf DNA containing single-strand breaks (nicks) . Localization of cleavage sites for EcoRI, HindIII, HpaI ClaI, BamHI, SalI, XbaI and XhoI restriction endonucleases was determined . Position of single-strand breaks was mapped by electrophoretic analysis of denatured tf DNA and electron microscopy of partially denatured DNA samples . The tf genome is characterized by the presence of two classes of nicks differing in the frequency of their presence in population of bacteriophage DNA molecules.

Genetika, 1988 Jun, 24(6), 980 - 92
{Cloning and localization of the replication and mobilization regions in the D-plasmid of Pseudomonas putida pBS286 (IncP-9) with a broad host range}; Tsoi TV et al.; Rep-mob loci of naphthalene degradative plasmid pBS286 (IncP-9) have been cloned on the Escherichia coli vectors pUC19 and pUBR322 . These loci confer to recombinant plasmids pBS952 and pBS953 the ability for effective mobilization by RP4 (IncP-1) and F plasmid, as well as constant maintenance in various gram-negative bacteria . Localization of cloned sequences in the restriction fragments of conservative part of the pBS286 genome was established . The data obtained correlate with the analysis of plasmids pBS950 and pBS951 which are spontaneous mini-derivatives of pBS286 and pBS292 (delta NPL1::Tn1/Tra+ Nah-) plasmids formed during transformation of E . coli HB101 cells . Plasmids pBS952 and pBS953 retain the incompatibility properties of parental IncP-9 replicon . These recombinant derivatives can be used for construction of bhr vectors with required properties and compatible with bhr vectors constructed on the basis of plasmids from the IncP-1 and IncP-4 groups.

Appl Environ Microbiol, 1988 Jun, 54(6), 1498 - 503
Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon; Zylstra GJ et al.; Pseudomonas putida PpF1 degrades toluene through cis-toluene dihydrodiol to 3-methylcatechol . The latter compound is metabolized through the well-established meta pathway for catechol degradation . The first four steps in the pathway involve the sequential action of toluene dioxygenase (todABC1C2), cis-toluene dihydrodiol dehydrogenase (todD), 3-methylcatechol 2,3-dioxygenase (todE), and 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase (todF) . The genes for these enzymes form part of the tod operon which is responsible for the degradation of toluene by this organism . A combination of transposon mutagenesis of the PpF1 chromosome, as well as analysis of cloned chromosomal fragments, was used to determine the physical order of the genes in the tod operon . The genes were determined to be transcribed in the order todF, todC1, todC2, todB, todA, todD, todE.

J Biol Chem, 1988 May 15, 263(14), 6495 - 501
Interaction of the protein components of 5-oxoprolinase . Substrate-dependent enzyme complex formation; Li LY et al.; 5-Oxo-L-prolinase from Pseudomonas putida is composed of two reversibly dissociable proteins: Component A catalyzes 5-oxoproline-dependent cleavage of ATP, but does not catalyze the decyclization of 5-oxoproline; Component B is required for the coupling of ATP cleavage to ring-opening of 5-oxoproline to form glutamate (Seddon, A . P., Li, L., and Meister, A . (1984) J . Biol . Chem . 259, 8091-8094) . We describe here the purifications of Components A and B to apparent homogeneity and the interactions between these two proteins . The cellular content of Component B activity is significantly greater than that of Component A . By gel filtration, Component A is a hexamer; but in the presence of substrates, it is a dimer . Component B can exist as an aggregate, an octamer, or a tetramer, depending upon the conditions used . Gel filtration of a mixture of Components A and B in the presence of substrates gives a unique protein species that exhibits 5-oxoprolinase activity . The Mr of this Component A-Component B complex indicates that it probably has an A2-B2 structure . The molar ratio of Component A to Component B in the complex was determined to be 1:1 by the continuous variation method (Job) . Titrations of each component by the other suggest that phosphorylated 5-oxoproline-bound Component A is the entity that interacts with Component B . These studies indicate that the binding of phosphorylated 5-oxoproline-bound Component A to Component B to form a complex proceeds by a cooperative type mechanism . This is supported by the observed shifts of the intersection points of the Job curves (see Appendix).

Mol Gen Mikrobiol Virusol, 1988 May, (5), 12 - 4
{Bacteriophages of Pseudomonas putida containing single-stranded canonical DNA breaks}; Kulakov LA et al.; It was shown that bacteriophage tf as well as bacteriophages phi p4/40, phi p25/42, phi p23/40 and phi p6/40, which are specific to different P . putida strains, contain the single strand breaks in their DNA . The breaks are localized in one strand of DNA molecules and are repairable with T4 DNA ligase . Bacteriophage tf has no detectable DNA homology with phi p4/40, phi p25/42, phi p23/40 and phi p6/40 bacteriophages . All the phages studied have no relation with other known Pseudomonas phages . Bacteriophages phi p4/40 and phi p25/42 share the extensive DNA homology.

Genetika, 1988 May, 24(5), 803 - 7
{Interaction of heterologous bacteriophages: growth suppression of temperate PP56 phage of Pseudomonas putida by the transposable phage D3112 of Pseudomonas aeruginosa}; Kopylova IuI et al.; We have found an inhibiting effect of hybrid RP4::D3112 plasmid (where D3112 is represented as genome of a transposable phage specific for Pseudomonas aeruginosa) on the development of temperate P . putida phage PP56 . The study of the effect has revealed a previously unknown locus (in the region 12-14.2 kb of the D3112 genome) which functions in the prophage state . The locus affects PP56 decreasing phage yield . Mutants of PP56 insensitive to inhibition were found.

FEBS Lett, 1988 Apr 25, 231(2), 336 - 40
Primary structure of protein B from Pseudomonas putida, member of a new class of 2Fe-2S ferredoxins; Morrice N et al.; The primary structure of the 2Fe-2S ferredoxin (protein B) from the benzene dioxygenase system of Pseudomonas putida strain NCIB 12190 was determined by gas-phase sequencing of the protein and its fragments . Fast atom bombardment mass spectrometry indicated a molecular mass of 11,860 Da . The sequence contained five cysteine residues, four of which would be required to coordinate the iron-sulphur cluster . The amino acid sequence determined in the present study is compared to that of a protein deduced from the DNA sequence from another strain of Pseudomonas putida . Little sequence homology was observed when protein B was compared to 2Fe-2S ferredoxins from plant and cyanobacterial sources . The novel sequence determined here suggests a new class of ferredoxin, which is consistent with the observed mid-point redox potential being significantly less negative (-155 mV) than those of the 2Fe-2S ferredoxins involved in photosynthesis (-310 to -455 mV).

Biochim Biophys Acta, 1988 Apr 14, 953(3), 249 - 57
Properties and functions of two succinic-semialdehyde dehydrogenases from Pseudomonas putida; Sanchez M et al.; Two forms of succinic-semialdehyde dehydrogenase have been isolated in Pseudomonas putida . The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx . 200,000 and 100,000 . The smaller enzyme, which is induced by growth on 4-hydroxyphenylacetate, has been purified to 88% homogeneity by anion-exchange and affinity chromatography . Electrophoresis in sodium dodecyl sulphate gave rise to a molecular weight of 53,000, indicating that the native enzyme is dimeric . Under standard assay conditions this enzyme acts preferentially with NAD but reduces NADP at 9% of the rate observed for NAD . The large enzyme, which is dependent on NADP, is induced by growth on putrescine and its induction is highly coordinated with putrescine: 2-oxoglutarate transaminase, gamma-amino-butyraldehyde dehydrogenase and gamma-aminobutyrate: 2-oxoglutarate transaminase activities . Activity and stability conditions and true Km values for substrate and cosubstrates of the two enzymes were determined.

Nucleic Acids Res, 1988 Apr 11, 16(7), 3075 - 89
Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1; Schnier J et al.; A 7 kb chromosomal DNA fragment from R . melilotii was cloned, which complemented temperature-sensitivity of an E . coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1) . From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1) . DNA sequence analysis of the R . melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E . coli . The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1 . Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene . Binding studies with RNA polymerase from E . coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream . One possible promoter was also identified by DNA sequence comparison with the corresponding E . coli promoter.

J Bacteriol, 1988 Apr, 170(4), 1789 - 94
Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite; Zeyer J et al.; A nitrophenol oxygenase which stoichiometrically converted ortho-nitrophenol (ONP) to catechol and nitrite was isolated from Pseudomonas putida B2 and purified . The substrate specificity of the enzyme was broad and included several halogen- and alkyl-substituted ONPs . The oxygenase consisted of a single polypeptide chain with a molecular weight of 58,000 (determined by gel filtration) or 65,000 (determined on a sodium dodecyl sulfate-polyacrylamide gel) . The enzymatic reaction was NADPH dependent, and one molecule of oxygen was consumed per molecule of ONP converted . Enzymatic activity was stimulated by magnesium or manganese ions, whereas the addition of flavin adenine dinucleotide, flavin mononucleotide, or reducing agents had no effect . The apparent Kms for ONP and NADPH were 8 and 140 microM, respectively . 2,4-Dinitrophenol competitively (Ki = 0.5 microM) inhibited ONP turnover . The optimal pH for enzyme stability and activity was in the range of 7.5 to 8.0 . At 40 degrees C, the enzyme was totally inactivated within 2 min; however, in the presence of 1 mM ONP, 40% of the activity was recovered, even after 10 min . Enzymatic activity was best preserved at -20 degrees C in the presence of 50% glycerol.

Bioorg Khim, 1988 Apr, 14(4), 545 - 7
{Genes encoding bacterial RNA-polymerase . I . Cloning of rpoBC-operon of Pseudomonas putida and its physical map}; Borodin AM et al.; The P . putida rpoBC operon, coding for beta and beta' subunits of RNA polymerase, was cloned and its physical map constructed.

J Bacteriol, 1988 Apr, 170(4), 1812 - 9
Genetic organization and transcriptional analysis of a major gene cluster involved in siderophore biosynthesis in Pseudomonas putida WCS358; Marugg JD et al.; In iron-limited environments, the plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358 . The transcriptional organization and the iron-regulated expression of a major gene cluster involved in the biosynthesis and transport of pseudobactin 358 were analyzed in detail . The cluster comprises a region with a minimum length of 33.5 kilobases and contains at least five transcriptional units, of which some are relatively large . The directions of transcription of four transcriptional units were determined by RNA-RNA hybridization and by analysis in Escherichia coli minicells . The latter also demonstrated that large polypeptides were encoded by these transcriptional units . The results allowed us to localize several promoter regions on the DNA . The iron-dependent expression of at least two genes within this cluster appears to be regulated at the transcriptional level.

Biochemistry, 1988 Mar 22, 27(6), 2197 - 205
Kinetics and mechanism of benzoylformate decarboxylase using 13C and solvent deuterium isotope effects on benzoylformate and benzoylformate analogues; Weiss PM et al.; Benzoylformate decarboxylase (benzoylformate carboxy-lyase, BFD; EC 4.1.1.7) from Pseudomonas putida is a thiamine pyrophosphate (TPP) dependent enzyme which converts benzoylformate to benzaldehyde and carbon dioxide . The kinetics and mechanism of the benzoylformate decarboxylase reaction were studied by solvent deuterium and 13C kinetic isotope effects with benzoylformate and a series of substituted benzoylformates (pCH3O, pCH3, pCl, and mF) . The reaction was found to have two partially rate-determining steps: initial tetrahedral adduct formation (D2O sensitive) and decarboxylation (13C sensitive) . Solvent deuterium and 13C isotope effects indicate that electron-withdrawing substituents (pCl and mF) reduce the rate dependence upon decarboxylation such that decreased 13(V/K) effects are observed . Conversely, electron-donating substituents increase the rate dependence upon decarboxylation such that a larger 13(V/K) is seen while the D2O effects on V and V/K are not dramatically different from those for benzoylformate . All of the data are consistent with substituent stabilization or destabilization of the carbanionic intermediate (or carbanion-like transition state) formed during decarboxylation . Additional information regarding the mechanism of the enzymic reaction was obtained from pH studies on the reaction of benzoylformate and the binding of competitive inhibitors . These studies suggest that two enzymic bases are required to be in the correct protonation state (one protonated and one unprotonated) for optimal binding of substrate (or inhibitors).

Biochemistry, 1988 Mar 8, 27(5), 1587 - 91
Specific labeling of the essential cysteine residue of L-methionine gamma-lyase with a cofactor analogue, N-(bromoacetyl)pyridoxamine phosphate; Nakayama T et al.; L-Methionine gamma-lyase from Pseudomonas putida is composed of four identical polypeptide chains and contains four cysteinyl residues per subunit . We have found one of them catalytically essential by its specific cyanylation with 2-nitro-5-thiocyanobenzoic acid . We have shown its essentiality also with N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP), which is a cofactor analogue and also an affinity-labeling agent . The kinetic data show that the apoenzyme forms a binary complex with BAPMP prior to covalent binding . The stoichiometry of inactivation was 1 mol of BAPMP per subunit . We have shown that the cysteine residue modified with BAPMP is identical with that labeled specifically with {14C}iodoacetic acid . The amino acid sequences of the peptides containing the essential cysteine residue and the lysine residue to which pyridoxal 5'-phosphate is bound were determined by automated Edman degradation.

Appl Environ Microbiol, 1988 Mar, 54(3), 638 - 41
Transfer and expression of mesophilic plasmid-mediated degradative capacity in a psychrotrophic bacterium; Kolenc RJ et al.; A psychrotrophic bacterium, originally isolated from a natural aquatic environment, was characterized and identified as Pseudomonas putida Q5 for use as a representative recipient for biodegradative genes from a mesophilic microorganism . The TOL plasmid pWWO of the mesophile P . putida PaW1 was successfully transferred by conjugation to the naturally isolated psychrotroph P . putida Q5, as shown by plasmid analysis by agarose gel electrophoresis . Expression of the genes encoded by the mesophilic TOL plasmid in the psychrotroph was shown by the fact that the transconjugant (designated P . putida Q5T) had the capacity to degrade and utilize toluate (1,000 mg/liter) as a sole source of carbon at temperatures as low as 0 degrees C . Comparison of growth rates over a wide temperature range (0 to 30 degrees C) indicated that the physiological activity of the transconjugant was not reduced and that the plasmid DNA from the mesophile and its encoded enzymes functioned effectively in the psychrotroph at temperatures well below those at which the mesophile could grow . The production and demonstrated functioning of P . putida Q5T illustrates the possibility of developing specific degradative capacities in bacteria which can readily function at low temperatures in chemically contaminated environments or in industrial wastewater treatment systems.

J Gen Microbiol, 1988 Mar, 134 ( Pt 3), 605 - 9
Construction of a shuttle vector for inducible gene expression in Escherichia coli and Bacillus subtilis; Leonhardt H et al.; The construction of a shuttle vector for inducible gene expression allowing fast and easy cloning in Escherichia coli and subsequent transformation of Bacillus subtilis is presented . The expression is based on the regulation of the tac promoter by the Lac repressor which was assayed with the xylE gene from Pseudomonas putida as a marker gene . The lacIq gene, transcribed by the strong spo promoter, allowed full repression of the weak tac promoter.

J Bacteriol, 1988 Mar, 170(3), 1297 - 304
Transcriptional regulation, nucleotide sequence, and localization of the promoter of the catBC operon in Pseudomonas putida; Aldrich TL et al.; The catB and catC genes encode cis,cis-muconate lactonizing enzyme I (EC 5.5.1.1) and muconolactone isomerase (EC 5.3.3.4), respectively . These enzymes are required for the dissimilation of benzoate to beta-ketoadipate by Pseudomonas putida and are under coordinate transcriptional regulation . By deletion analysis and the use of pKT240 as a promoter probe vector, we located a single promoter region for the catBC operon upstream of catB . RNA-DNA hybridization studies, together with reverse transcriptase mapping, demonstrated that this promoter must be activated in the presence of an inducer molecule for effective transcription of the operon . In addition, the transcription initiation site was located 64 base pairs upstream of the catB initiation codon, and sequences upstream of -43 were required for promoter function . The catBC promoter was compared with other positively regulated procaryotic promoters to identify possible consensus sequences.

Biochemistry, 1988 Feb 23, 27(4), 1360 - 7
Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida; Wackett LP et al.; Toluene dioxygenase, a multicomponent enzyme system known to oxidize mononuclear aromatic hydrocarbons to cis-dihydrodiols, oxidized indene and indan to 1-indenol and 1-indanol, respectively . In addition, the enzyme catalyzed dioxygen addition to the nonaromatic double bond of indene to form cis-1,2-indandiol . The oxygen atoms in 1-indenol and cis-1,2-indandiol were shown to be derived from molecular oxygen, whereas 70% of the oxygen in 1-indanol was derived from water . All of the isolated products were optically active as demonstrated by 19F NMR and HPLC discrimination of diastereomeric esters and by chiroptic methods . The high optical purity of (-)-(1R)-indanol (84% enantiomeric excess) and the failure of scavengers of reactive oxygen species to inhibit the monooxygenation reaction supported the contention that the monooxygen insertion is mediated by an active-site process . Experiments with 3-{2H}indene indicated that equilibration between C-1 and C-3 occurred prior to the formation of the carbon-oxygen bond to yield 1-indenol . Naphthalene dioxygenase also oxidized indan to 1-indanol, which suggested that benzylic monoxygenation may be typical of this group of dioxygenases.

Appl Environ Microbiol, 1988 Feb, 54(2), 604 - 6
Trichloroethylene metabolism by microorganisms that degrade aromatic compounds; Nelson MJ et al.; Trichloroethylene (TCE) was metabolized by the natural microflora of three different environmental water samples when stimulated by the addition of either toluene or phenol . Two different strains of Pseudomonas putida that degrade toluene by a pathway containing a toluene dioxygenase also metabolized TCE . A mutant of one of these strains lacking an active toluene dioxygenase could not degrade TCE, but spontaneous revertants for toluene degradation also regained TCE-degradative ability . The results implicate toluene dioxygenase in TCE metabolism.

Genetika, 1988 Feb, 24(2), 239 - 49
{Isolation and comparative characteristics of 2 unrelated temperate phages of Pseudomonas putida PpG1}; Krylov VN et al.; Two temperate bacteriophages, PP56 and PP71, specific for bacteria of Pseudomonas putida strain PpG1 have been isolated for the first time . Characterization of the phages was performed . Both of them accomplish stable lysogenization of P . putida PpG1 cells . The phages are inducible . Several groups of clear plaque (c) mutants of PP56 and PP71 with altered processes of establishment and maintenance of lysogenic state have been isolated, according to complementation test . The phages differ in following characters: 1) in morphology of mature phage particles (C and B types, according to Bradley classification); 2) in sizes and genome organizations: DNA of PP56 is permuted (up to 30%), its size is 45 kb, the size of terminal redundancy being 2.5 kb; there is no permutation in PP71 DNA of 48 kb and it has "cohesive" ends; 3) no DNA/DNA homology is shown in tests between PP56 and PP71 DNAs . All the data obtained permitted to consider the phages PP56 and PP71 to belong to different unrelated species (families).

Pharm Res, 1988 Feb, 5(2), 67 - 75
Cytochrome P450: molecular architecture, mechanism, and prospects for rational inhibitor design; Poulos TL; Cytochromes P450 catalyze the insertion of one O2-derived oxygen atom into an aliphatic or aromatic molecule . P450s are best known for the metabolism of xenobiotic molecules, where hydroxylation renders insoluble hydrocarbons more soluble for easier elimination . In addition to this important catabolic function, P450s catalyze key steps in steroid and plant growth regulator metabolism . A variety of therapeutic, fungicidal, and agochemical agents that perturb these metabolic pathways very likely operate by binding in the lipophilic P450 active site and coordinating with the heme iron atom . Recent determination of a bacterial P450 crystal structure, P450cam from Pseudomonas putida, in addition to the crystal structure of four inhibited complexes, has provided some insight into the potential use of P450 as a model system for the rational design of therapeutic agents . The crystal structure has also shed light on the P450 catalytic mechanism . P450cam operates differently from peroxidase or catalase in cleaving the O-O bond, since unlike these other enzymes, P450 contains no acid-base catalytic groups near the oxygen binding site . Instead, the O2 pocket is lined with aliphatic and aromatic residues . This strongly suggests that the catalytic push required to cleave the O-O bond resides with the ability of the Cys heme ligand to donate electron density to the heme-oxy system . A comparison of the substrate-free and -bound P450cam crystal structures has revealed some interesting aspects regarding the dynamics of substrate binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1988 Feb, 54(2), 446 - 53
Enumeration of Tn5 mutant bacteria in soil by using a most- probable-number-DNA hybridization procedure and antibiotic resistance; Fredrickson JK et al.; Investigations were made into the utility of DNA hybridization in conjunction with a microdilution most-probable-number procedure for the enumeration of Rhizobium spp . and Pseudomonas putida in soil . Isolates of Rhizobium spp . and P . putida carrying the transposon Tn5 were added to sterile and nonsterile Burbank sandy loam soil and enumerated over time . Soil populations of rhizobia were enumerated by colony hybridization, most-probable-number-DNA hybridization procedure, plate counts, plant infectivity most probable number, and fluorescent antibody counts . Population values compared well for all methods at 5 and 30 days after the addition of cells, although the fluorescent antibody method tended to overestimate the viable population . In nonsterile soil, most-probable-number-DNA hybridization procedure enumerated as few as 10 P . putida Tn5 cells g of soil-1 and 100 R . leguminosarum bv . phaseoli Tn5 cells g of soil-1 and should have utility for following the fate of genetically engineered microorganisms released to the environment . Among the Kmr isolates containing Tn5, approximately 5% gave a dark, more intense autoradiograph when probed with 32P-labeled pGS9 DNA, which facilitated their detection in soil . Hybridization with a pCU101 probe (pGS9 without Tn5) indicated that donor plasmid sequences were being maintained in the bacterial chromosome . Transposon-associated antibiotic resistance was also utilized as a phenotypic marker . Tn5 vector-integrate mutants were successfully enumerated at low populations (10 to 100 cells g of soil-1) in soil by both phenotypic (Kmr) and genotypic (DNA probe) analysis . However, determination of the stability of Tn5 or Tn5 and vector sequences in the bacteria is necessary.

Biochemistry, 1988 Jan 26, 27(2), 540 - 5
Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida; Ransom SC et al.; The gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) was cloned in Pseudomonas aeruginosa (ATCC 15692) . The selection for the cloned gene was based upon the inability of P . aeruginosa to grow on (R)-mandelate as sole carbon source by virtue of the absence of mandelate racemase in its mandelate pathway . Fragments of P . putida DNA obtained by digestion of chromosomal DNA with Sau3A were ligated into the BamHI site of the Gram-negative vector pKT230 and transformed into the P . aeruginosa host . A transformant able to utilize (R)-mandelate as sole carbon source was characterized, and the plasmid was found to contain approximately five kilobase pairs of P . putida DNA . Subcloning of this DNA revealed the position of the gene for the racemase within the cloned DNA from P . putida . The dideoxy-DNA sequencing procedure was used to determine the sequence of the gene and its translated sequence . The amino acid sequence and molecular weight for mandelate racemase deduced from the gene sequence (38 570) are in excellent agreement with amino acid composition and molecular weight data for the polypeptide recently determined with enzyme isolated from P . putida; these recent determinations of the polypeptide molecular weight differ significantly from the originally reported value of 69,500 {Fee, Judith A., Hegeman, G.D., & Kenyon, G.L . (1974) Biochemistry 13,2528}, which was used to demonstrate that alpha-phenylglycidate, an active site directed irreversible inhibitor, binds to the enzyme with a stoichiometry of 1:1.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Microbiol, 1988 Jan, 149(3), 198 - 206
Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 4-trifluoromethyl(TFM)-benzoate by 4-isopropylbenzoate grown Pseudomonas putida JT strains; Engesser KH et al.; Enzymes of the p-cymene pathway in Pseudomonas putida strains cometabolized the intermediate analogue 4-trifluoromethyl(TFM)benzoate . Three products, 4-TFM-2,3-dihydro-2,3-dihydroxybenzoate, 4-TFM-2,3-dihydroxybenzoate and 2-hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties . Certain TFM-substituted analogue metabolites of the p-cymene pathway were transformed at drastically reduced rates . Hammett type analysis of ring cleavage reactions of 4-substituted 2,3-dihydroxybenzoates revealed the negative inductive and especially mesomeric effect of substituents to be rate determining . Whereas decarboxylation of 3-carboxy-7-TFHOD was not affected by fluorine substitution the subsequent hydrolysis of 7-TFHOD proceeded very slowly . The negative inductive effect of the TFM-group probably inhibited heterolysis of the carbon bond between C5 and C6 of 7-TFHOD.

Arch Microbiol, 1988 Jan, 149(3), 188 - 97
Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 3-trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus N657; Engesser KH et al.; The TOL plasmid-encoded enzymes of the methylbenzoate pathway in Pseudomonas putida mt-2 cometabolized 3-trifluoromethyl (TFM)-benzoate . Two products, 3-TFM-1,2-dihydroxy-2-hydrobenzoate (3-TFM-DHB) and 2-hydroxy-6-oxo-7,7,7-trifluoro-hepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties . TFM-substituted analogues of the metabolites of the methylbenzoate pathway were generally converted at drastically reduced rates . The catechol-2,3-dioxygenase from Pseudomonas putida showed moderate turnover rates with 3-TFM-catechol . The catechol-1,2-dioxygenase of Rhodococcus rubropertinctus N657 was totally inhibited by 3-TFM-catechol and did not cleave this substrate . Hammett-type analysis showed the catechol-1,2-dioxygenase reaction to be strongly dependent on the electronic nature of the substituents . Electronegative substituents strongly inhibited catechol cleavage . The catechol-2,3-dioxygenase reaction, however, was only moderately sensitive to electronegative substituents.

Arch Microbiol, 1988, 149(5), 413 - 6
Physiological comparison of D-cysteine desulfhydrase of Escherichia coli with 3-chloro-D-alanine dehydrochlorinase of Pseudomonas putida CR 1-1; Nagasawa T et al.; D-Cysteine desulfhydrase of Escherichia coli W3110 delta trpED102/F' delta trpED102 was physiologically characterized . It was found to be located in the cytosolic fraction, as 3-chloro-D-alanine dehydrochlorinase is . D-Cysteine desulfhydrase catalyzed not only the alpha, beta-elimination reaction of O-acetyl-D-serine to form pyruvate, acetic acid and ammonia, but also the beta-replacement reaction of O-acetyl-D-serine with sulfide to form D-cysteine . However, these reactions appeared not to proceed in vivo . No other activity of D-cysteine synthesis from O-acetyl-D-serine and sulfide was detected in a crude cell extract of E . coli which was immunotitrated with antibodies raised against the purified D-cysteine desulfhydrase . Although D-cysteine desulfhydrase catalyzes the degradation (alpha, beta-elimination reaction) of 3-chloro-D-alanine, which is an effective antibacterial agent, E . coli W3110 delta trpED102/F' delta trpED102 did not show resistance against 3-chloro-D-alanine . Therefore, D-cysteine desulfhydrase does not contribute to 3-chloro-D-alanine detoxification in vivo.

Gene, 1988, 62(2), 219 - 27
Transcription of the fimbrial subunit gene and an associated transfer RNA gene of Pseudomonas aeruginosa; Hobbs M et al.; Gene fimA encoding the structural subunit of the fimbriae of Pseudomonas aeruginosa PAK is located in the centre of a 1.2-kb HindIII genomic DNA fragment {see also Sastry et al., J . Bacteriol . 164 (1985) 571-577}, which in turn is located within a 6.2-kb EcoRI fragment . Immediately downstream from fimA is a putative threonine tRNA gene {Dalrymple and Mattick, Biochem . Int . 13 (1986) 547-553} . Northern blotting experiments showed that fimA is transcribed to an mRNA of approx . 650 nucleotides, which also includes the threonine tRNA sequence but no other protein-coding region . There was no indication that this mRNA is processed to release the tRNA sequence . However, the tRNA did appear to be expressed independently from its own promoter in the region 3' to fimA . When these sequences were introduced into Pseudomonas putida, we found that the level of expression of fimA from the cloned 6.2-kb EcoRI fragment was approx . 30-fold greater than that from the smaller HindIII fragment, whereas that of the specific tRNA species was unaltered . The size of the fimA transcript was also unaltered . These results provide evidence that the fimA gene is subject to specific transcriptional activation in vivo and that this activation involves sequences flanking the 1.2-kb HindIII fragment.

J Biol Chem, 1987 Dec 25, 262(36), 17712 - 8
Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli; Eggink G et al.; The OCT plasmid encodes enzymes for alkane hydroxylation and alkanol dehydrogenation . Structural components are encoded on the 7.5-kilobase pair alkBAC operon, whereas positive regulatory components are encoded by alkR . We have constructed plasmids containing fusions of cloned alkBAC and alkR DNA and used these fusion plasmids to study the functional expression of the alkBAC operon and the regulatory locus alkR in Pseudomonas putida and in Escherichia coli . Growth on alkanes requires a functional chromosomally encoded fatty acid degradation system in addition to the plasmid-borne alk system . While such a system is active in P . putida, it is active in E . coli only in fadR mutants in which fatty acid degradation enzymes are expressed constitutively . Using such mutants, we found that E . coli as well as P . putida grew on octane as the sole source of carbon and energy when they were supplied with the cloned complete alk system . The alkR locus was strictly necessary in E . coli as well as in P . putida for expression of the alkBAC operon . The alkBAC operon could, however, be further reduced to a 5-kilobase pair operon without affecting the Alk phenotype in either species to a significant extent . Although with this reduction the plasmid-encoded alkanol dehydrogenase activity was lost, chromosomally encoded alkanol dehydrogenases in P . putida and E . coli compensated for this loss . The induction kinetics of the alk system was studied in detail in P . putida and E . coli . We used specific antibodies raised against alkane hydroxylase to follow the appearance of this protein following induction with octane . We found the induction kinetics of alkane hydroxylase to be similar in both species . A steady-state level was reached after about 2 h of induction in which time the alkane hydroxylase accounted for about 1.5% of total newly synthesized protein . Thus, alkBAC expression is very efficient and strictly regulated to both P . putida and E . coli.

J Bacteriol, 1987 Nov, 169(11), 5174 - 9
Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida; Irie S et al.; The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined . Five open reading frames were found in the sequence . Four corresponding protein molecules were detected by a DNA-directed in vitro translation system . Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol . The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed.

Mol Microbiol, 1987 Nov, 1(3), 293 - 300
Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas; Ramos JL et al.; TOL plasmid pWWO of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons . The 'upper' operon encodes enzymes for the oxidation of toluene to benzoate and xylenes to toluates, whereas the meta-cleavage operon specifies the further oxidation of benzoate and toluates . Transcription of the upper pathway operon is positively regulated by the XylR protein, which is activated by toluene/xylenes and their alcohol catabolic products, in combination with the NtrA protein, a sigma factor . Expression of the meta-operon is positively controlled by the XylS protein which is activated by meta-pathway substrates, and is independent of NtrA protein . Expression of the meta pathway is also induced by toluene/xylene-activated XylR protein via a cascade regulatory system in which this protein in combination with NtrA protein stimulates transcription from the xylS gene promoter . Hyper-production of XylS protein in turn provokes high level expression of the meta-operon, which is independent of meta-pathway substrates . The two promoters, which are activated by the XylR and NtrA proteins, the upper pathway promoter and the xylS gene promoter, exhibit three regions of homology centred at -12(5'-TTGCATG-3'), -24(5'-TGGCPuT-3') and -45(5'-TAAAATAAGPuPuCGPuTC-3'), with respect to their principal transcription initiation points . The possible physiological significance of activated XylR-protein-induced expression of the meta-operon through amplification of XylS protein levels is considered.

Genetika, 1987 Oct, 23(10), 1823 - 31
{Molecular genetic organization and origin of plasmid pBS52 with a broad range of bacterial hosts}; Polevoda BV et al.; The data are presented on the localization of genetic determinants of resistance to streptomycin, ampicillin and sulfanilamides on the physical map of conjugative R plasmid pBS52 of 38,000 bp which has a broad bacterial host range and belongs to a new incompatibility group . The plasmid has a natural "polylinker" site (less than 200 bp) containing (in the order of arrangement) the recognition sites for restriction enzymes: BamHI-EcoRI-PstI-EcoRV-BglII (PvuII) . The comparative analysis shows that pBS52 contains a segment homologous to DNA of plasmid RSE1010 (IncP-4) . The evolutionary origin of plasmid pBS52 is discussed . The recA-independent formation of the mini-derivatives of pBS373 and pBS374 types during the transformation of Escherichia coli with pBS52 plasmid DNA has been shown . Plasmids pBS373 and yBS374 are capable of autonomous replication in Pseudomonas putida and P . aeruginosa cells, which is provided by the rep system of IncP-4 replicon.

J Bacteriol, 1987 Oct, 169(10), 4696 - 702
Purification and properties of formylglutamate amidohydrolase from Pseudomonas putida; Hu L et al.; Formylglutamate amidohydrolase (FGase) catalyzes the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida . By this action, N-formyl-L-glutamate (FG) is hydrolyzed to produce L-glutamate plus formate . Urocanate, the first product in the pathway, induced all five enzymes, but FG was able to induce FGase alone, although less efficiently than urocanate did . This induction by FG resulted in the formation of an FGase with electrophoretic mobility identical to that of the FGase induced by urocanate . A 9.6-kilobase-pair HindIII DNA fragment containing the P . putida FGase gene was cloned into the corresponding site on plasmid pBEU1 maintained in Escherichia coli . Insertion of the fragment in either orientation on the vector resulted in expression, but a higher level was noted in one direction, suggesting that the FGase gene can be expressed from either of two vector promoters with different efficiencies or from a single vector promoter in addition to a less efficient Pseudomonas promoter . FGase was purified 1,110-fold from the higher-expression clone in a yield of 10% through six steps . Divalent metal ions stimulated activity, and among those tested (Co, Fe, Zn, Ca, Ni, Cd, Mn, and Mg), Co(II) was the best activator, followed by Fe(II) . FGase exhibited a Km of 14 mM for FG and a specific activity of 100 mumol/min per mg of protein in the presence of 5 mM substrate and 0.8 mM CoCl2 at 30 degrees C . The enzyme was maximally active in the range of pH 7 to 8 . FGase was found to be a monomer of molecular weight 50,000 . N-Acetyl-L-glutamate was not a substrate for the enzyme, but both it and N-formyl-L-aspartate were competitive inhibitors of formylglutamate hydrolysis, exhibiting Ki values of 6 and 9 mM, respectively . The absence of FGase activity as an integral part of histidine breakdown in most other organisms and the somewhat uncoordinated regulation of FGase synthesis with that of the other hut enzymes in Pseudomonas suggest that the gene encoding its synthesis may have evolved separately from the remaining hut genes.

Biochem Biophys Res Commun, 1987 Sep 15, 147(2), 831 - 8
Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli; Rhee H et al.; The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318 . The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423 . The P . putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E . coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E . coli cells without pGI 423 . The E . coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E . coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.

J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2563 - 6
Qualitative evidence for expression of Klebsiella pneumoniae nif in Pseudomonas putida; Postgate JR et al.; Pseudomonas putida MT20-3 carrying the Klebsiella pneumoniae nif plasmids pRD1 or pMF250 showed highly O2-sensitive aerobic acetylene reduction on low-N pyruvate or glucose agar . This finding confirms unequivocally that K . pneumoniae nif can be expressed in an obligate aerobe.

J Biol Chem, 1987 Aug 15, 262(23), 11020 - 5
18O studies on the 5-oxoprolinase reaction . Evidence for a phosphorylated tetrahedral intermediate; Li LY et al.; 5-Oxoprolinase catalyzes a reaction in which the cleavage of ATP to ADP and Pi and the decyclization of 5-oxoproline to form glutamate are coupled . When the reaction catalyzed by 5-oxoprolinase of Pseudomonas putida was carried out to 90% completion in H2(18)O, the residual 5-oxoproline was found to contain 18O in the amide carbonyl oxygen atom . Such isotopic incorporation was not observed in similar studies with a subunit of the enzyme which catalyzes 5-oxoproline-dependent ATPase and formation of a phosphorylated 5-oxoproline intermediate (Seddon, A.P., and Meister, A . (1986) J . Biol . Chem . 261, 11538-11543) . When the complete reaction was carried out in H2(18)O, the products glutamate (gamma-carboxyl) and inorganic phosphate were mono- and di-labeled with 18O . Studies with 5-{18O}oxo-L-proline confirmed such replacement of the oxygen atoms of the gamma-carboxyl group of glutamate and the carbonyl oxygen of 5-oxoproline . Oxygen was not transferred from 5-oxoproline to inorganic phosphate . Studies with analogs of 5-oxoproline showed that di-labeling of inorganic phosphate occurred only when ATP hydrolysis was coupled or partially coupled with the decyclization of the substrate . Studies with 5-oxoprolinase from rat kidney gave similar results . These observations are in accord with the view that the reaction involves enzyme-bound phosphorylated intermediates and provide evidence for a phosphorylated tetrahedral intermediate, whose formation is required for coupling.

J Bacteriol, 1987 Aug, 169(8), 3587 - 92
Overproduction of the xylS gene product and activation of the xylDLEGF operon on the TOL plasmid; Inouye S et al.; The effect of high-level expression of the regulatory gene xylS of the Pseudomonas putida TOL plasmid on the activation of the xylDLEGF operon was investigated in Escherichia coli . The xylS gene was placed downstream from the tac promoter, and the resultant fusion was cloned in cis to the xylDLEGF operon . The expression of the operon was monitored by the level of catechol 2,3-dioxygenase, whose structural gene xylE was placed directly after the operator-promoter region of xylDLEGF . xylS transcription was also determined by reverse transcriptase mapping of mRNA . Overproduction of the xylS gene product elicited constitutive high expression of the xylDLEGF operon even in the absence of the inducer for the operon . The results were consistent with a cascade model for the positive control of the xylDLEGF operon by the xylR and xylS genes (S . Inouye, A . Nakazawa, and T . Nakazawa, Proc . Natl . Acad . Sci . USA, in press): m-xylene, a substrate of the degradative pathway, binds to the xylR gene product; the m-xylene-xylR product complex activates the xylS gene; and the xylS product thus synthesized de novo activates the xylDLEGF operon.

J Bacteriol, 1987 Aug, 169(8), 3581 - 6
Activation of the xylDLEGF promoter of the TOL toluene-xylene degradation pathway by overproduction of the xylS regulatory gene product; Spooner RA et al.; The xylS regulatory gene of the Pseudomonas putida TOL plasmid (pWWO) has been cloned under the transcriptional control of the Escherichia coli tac promoter in a broad-host-range controlled-expression vector . Induction with isopropylthiogalactoside allowed overproduction and characterization of the xylS product by specific interaction with the TOL meta-cleavage pathway operator-promoter region (OP2) in vivo in E . coli . Examination of plasmid-specified polypeptides in E . coli maxicells led to identification of the xylS product as a 36-kilodalton polypeptide . The operator sequences required for xylS interactions lay upstream of the OP2 transcriptional start, and the xylS gene product recognized this region even in the absence of known coinducers.

Mol Gen Mikrobiol Virusol, 1987 Aug, (8), 36 - 41
{Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida}; Chistoserdov AIu et al.; The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter . A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E . coli trp-operon intercystronic region . Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l . This plasmid was cloned in broad-host-range vector plasmid pAYC31 . The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6 . Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E . coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.

Proc Natl Acad Sci U S A, 1987 Aug, 84(15), 5182 - 6
Expression of the regulatory gene xylS on the TOL plasmid is positively controlled by the xylR gene product; Inouye S et al.; The regulatory gene xylS on the TOL plasmid of Pseudomonas putida activates the transcription of the xylDLEGF operon for the m-toluate-degrading pathway in the presence of m-toluate . The gene also activates the transcription of the same operon in the presence of m-xylene or m-methylbenzyl alcohol, but for this activation another regulatory gene, xylR, is required . In this study we examined the xylS expression by determining the mRNA by reverse transcriptase mapping and by monitoring the enzyme activity of the xylE gene product, which was expressed under the control of the xylS promoter . The results of the above experiments provide evidence that xylR positively controls the transcription of xylS in the presence of m-xylene or m-methylbenzyl alcohol . The xylS product thus amplified may in turn activate the xylDLEGF operon . The nucleotide sequence of the xylS promoter resembles that of the promoter of the xylCAB operon for the m-xylene-degrading pathway, which is also activated by xylR in the presence of m-xylene or m-methylbenzyl alcohol . In addition, we have demonstrated that the expression of xylR is negatively controlled by its own product . On the basis of these findings, we propose a revised model for the regulation of expression of xyl genes on the TOL plasmid.

Biochemistry, 1987 Jul 14, 26(14), 4527 - 34
Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues; Tsubaki M et al.; Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique . Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes . Upon addition of substrates {such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol}, the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry . Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum . The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes) . These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.

J Gen Microbiol, 1987 Jul, 133 ( Pt 7), 1891 - 9
The effect of lipophilic weak acids on the segregational stability of TOL plasmids in Pseudomonas putida; Stephens GM et al.; The effect of various lipophilic weak acids on the stability of certain TOL plasmids was investigated . Benzoate induced deletion of TOL plasmid DNA in Pseudomonas putida MT15, followed by loss of the plasmid; this effect was pH- and concentration-dependent, suggesting that undissociated benzoic acid was a more effective curing agent than the benzoate anion . Plasmid loss always approached a frequency of 100% after a lag and apparently depended on the prior occurrence of deletions, although deleted plasmid was stably maintained in the absence of the acid . m-Toluate, acetate and butyrate also induced deletions and plasmid loss at high frequencies, although these acids were less effective than benzoate . Benzoate inhibited the growth of plasmid-containing cells rather than permitting faster growth of cured cells on benzoate . Similar results were obtained with P . putida strains MT20 and MT84, which contain different TOL plasmids . We suggest that lipophilic weak acids induced deletions, possibly by excision of a transposon-like region, and disrupted the segregation of deleted plasmid.

J Bacteriol, 1987 Jul, 169(7), 2962 - 6
Expression of degradative genes of Pseudomonas putida in Caulobacter crescentus; Chatterjee DK et al.; The recombinant plasmid RP4-TOL was transferred into Caulobacter crescentus at a high frequency, and the plasmid was maintained for at least 50 generations . C . crescentus cells which contained RP4-TOL grew on all the aromatic compounds that the plasmid normally allowed Pseudomonas putida to grow on . Reciprocal transfers from C . crescentus donor to P . putida or Escherichia coli recipients were less efficient and occurred at frequencies of approximately 10(-3) . Some representative TOL-specified enzymes in cell-free extracts of C . crescentus(RP4-TOL) were inducible, and their levels were similar to those of P . putida . The amounts of mRNA from induced cells of C . crescentus(RP4-TOL) and P . putida(RP4-TOL) were also similar . Moreover, the restriction enzyme digestion maps of RP4-TOL from both C . crescentus and P . putida were the same, indicating that the expression of the TOL genes occurred without any apparent alteration of the gene structure . This suggest that the degradative genes of Pseudomonas spp . can be transferred, maintained, and expressed efficiently in C . crescentus and that the mechanism of transcriptional activation of TOL genes observed in C . crescentus is similar to that of Pseudomonas spp.

J Gen Microbiol, 1987 Jul, 133 ( Pt 7), 1901 - 8
Effect of temperature on the stability of plasmid pTG201 and productivity of xylE gene product in recombinant Escherichia coli: development of a two-stage chemostat with free and immobilized cells; Sayadi S et al.; The effect of temperature on the stability of pTG201, a plasmid carrying the xylE gene (which encodes catechol 2,3-dioxygenase from Pseudomonas putida), and the production of catechol 2,3-dioxygenase in free and immobilized Escherichia coli during continuous culture have been studied at various temperatures . Immobilization of cells increased the stability of pTG201 considerably, even under conditions when expression of the xylE product was enhanced . Since xylE transcription was controlled by the lambda PR promoter and cI857 repressor, increasing derepression temperatures increased catechol 2,3-dioxygenase productivity and decreased pTG201 stability . A two-stage continuous culture system to overcome the impact of the high-level expression of the xylE gene on the stability of pTG201 is described . In the first stage, immobilized cells were grown in the repressed state in order to prevent loss of pTG201, whereas in the second stage, cultures were maintained in the derepressed state.

Proc Natl Acad Sci U S A, 1987 Jul, 84(13), 4460 - 4
Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation; Frantz B et al.; Three critical enzymes catechol oxygenase II (chlorocatechol dioxygenase), muconate cycloisomerase II, and dienelactone hydrolase, are involved in the degradation of chlorocatechols, which are obligatory intermediates in the catabolism of chlorinated aromatic compounds . The organization and complete nucleotide sequence of the genes for these enzymes have been determined on a 4.2-kilobase-pair (kbp) Bgl II fragment cloned from the plasmid pAC27, based on the agreement of open reading frame lengths with apparent mobilities of polypeptides expressed in Escherichia coli maxicells, predicted N-terminal amino acid sequences with those of the purified proteins, and predicted total amino acid compositions with those of the purified proteins . The 4.2-kbp fragment contains the three genes and ribosome binding sites for those genes but no promoter . When placed downstream of the tac promoter in the broad-host-range plasmid pMMB22, this fragment directs the synthesis of all three enzymes in both E . coli and Pseudomonas putida only on induction with isopropyl beta-D-thiogalactopyranoside, suggesting that the gene cluster is regulated as a single unit under the control of a single promoter . Endogenous transcription initiation of the gene cluster on pAC27, however, occurs from a site present within a 386-bp Bgl II fragment upstream of the 4.2-kbp fragment, and sequences 5' to that site are similar to the sequences of other positively controlled Pseudomonas promoters occurring on the TOL and NAH plasmids.

Mikrobiologiia, 1987 Jul-Aug, 56(4), 608 - 12
{Bacterial destruction of anionic sulfoethoxylate surfactants}; Stavskaia SS et al.; A Pseudomonas putida strain G was isolated and its activity in the destruction of sulfoethoxylates (surfactants) was studied as a function of the cultivation conditions . Ultrastructural changes were found in the cells utilizing sulfoethoxylates . Sulfoethoxylates were found to be decomposed by P . putida G via two pathways: (a) desulfation of the molecule and (b) cleavage of the alkyl ester bond . The intermediate products were not toxic and did not pollute the habitat . The strain can be used for microbiological purification of sewage.

Appl Environ Microbiol, 1987 Jul, 53(7), 1649 - 54
In vivo formation of gene fusions in Pseudomonas putida and construction of versatile broad-host-range vectors for direct subcloning of Mu d1 and Mu d2 fusions; Simon V et al.; The Mu d1 and Mu d2 prophages were integrated into the conjugative broad-host-range plasmid R751 . The two plasmids were then transferred into Pseudomonas putida, and derivatives carrying intact Mu prophages were recovered . After induction of Mu at 42 degrees C, both operon and gene fusions were observed on 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) plates . Broad-host-range vectors were constructed which allow direct cloning of both operon or gene fusions and their analysis in Escherichia coli and P . putida . By using one of these vectors, two operon fusions were isolated from the P . putida chromosome and comparatively analyzed in E . coli and P . putida.

Biochem J, 1987 Jun 15, 244(3), 611 - 6
Benzene dioxygenase in Pseudomonas putida . Subunit composition and immuno-cross-reactivity with other aromatic dioxygenases; Zamanian M et al.; The terminal oxygenase component of benzene dioxygenase from Pseudomonas putida strain ML2 was shown to contain two subunits, of Mr 54,500 and 23,500, by SDS/polyacrylamide-gel electrophoresis . The native Mr of the terminal oxygenase was estimated to be 168,000 +/- 4000 . Polyclonal antibodies raised against each of the subunits cross-reacted with two polypeptides in cell-free extracts from toluene-grown Pseudomonas putida strain N.C.I.B . 11767 . The Mr values of these polypeptides were similar to those reported for the subunits from the terminal dioxygenase component of toluene dioxygenase . These polypeptides were present only when this strain was grown on toluene . No cross-reactivity was observed with subunits of the naphthalene dioxygenase or benzoate dioxygenase systems.

J Mol Biol, 1987 Jun 5, 195(3), 687 - 700
High-resolution crystal structure of cytochrome P450cam; Poulos TL et al.; The crystal structure of Pseudomonas putida cytochrome P450cam with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 A . While the 1.63 A model confirms our initial analysis based on the 2.6 A model, the higher resolution structure has revealed important new details . These include a more precise assignment of sequence to secondary structure, the identification of three cis-proline residues, and a more detailed picture of substrate-protein interactions . In addition, 204 ordered solvent molecules have been found, one of which appears to be a cation . The cation stabilizes an unfavorable polypeptide conformation involved in forming part of the active site pocket, suggesting that the cation may be the metal ion binding site associated with the well-known ability of metal ions to enhance formation of the enzyme-substrate complex . Another unusual polypeptide conformation forms the proposed oxygen-binding pocket . A localized distortion and widening of the distal helix provides a pocket for molecular oxygen . An intricate system of side-chain to backbone hydrogen bonds aids in stabilizing the required local disruption in helical geometry . Sequence homologies strongly suggest a common oxygen-binding pocket in all P450 species . Further sequence comparisons between P450 species indicate common three-dimensional structures with changes focused in a region of the molecule postulated to be associated with the control of substrate specificity.

Am J Med, 1987 Jun, 82(6), 1191 - 4
Pseudomonas putida . Newly recognized pathogen in patients with cancer; Anaissie E et al.; Pseudomonas putida was recovered from blood culture specimens between 1980 and 1985 in 15 patients with cancer . No isolates were found in specimens obtained before 1980 . Eight patients were considered to have septicemia (more than one positive blood culture result plus clinical signs of infection) . Septicemia was monomicrobial in three of those eight patients and polymicrobial in five . Of these eight patients, one had pneumonia and three had phlebitis, cellulitis, or both at the site of the venous catheter . The infection appeared to be catheter-related in these three patients, with response to catheter removal in one patient, response to catheter removal and antibiotics in one patient, and response to antibiotics alone in one patient . P . putida was isolated from the site of insertion and the tip of the catheter in one of these three patients . Following therapy, all patients had a rapid recovery from their infection . In vitro susceptibility testing revealed that 90 percent of the isolates were susceptible to piperacillin, ceftazidime, imipenem, and ciprofloxacin.

J Bacteriol, 1987 Jun, 169(6), 2889 - 92
Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba; Durham DR et al.; Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway . A strain of P . putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene . Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate) . However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.

FEBS Lett, 1987 May 11, 215(2), 285 - 90
The amino acid sequence of the aliphatic amidase from Pseudomonas aeruginosa; Ambler RP et al.; Amino acid sequence studies show that the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa PAC142 consists of a single polypeptide chain of 346 residues, giving an Mr of 38,400 . The evidence from the amino acid studies is in complete agreement with that deduced from the DNA sequence of the amiE gene . Studies of the protein from Pseudomonas putida A87 show that it differs from the Ps . aeruginosa protein by about 30 amino acid substitutions . It now becomes possible to relate changes in the enzyme which result in altered specificity to structural changes in the protein.

J Biol Chem, 1987 May 5, 262(13), 6400 - 6
Structure of the Pseudomonas putida alkBAC operon . Identification of transcription and translation products; Eggink G et al.; The structural genes of the Pseudomonas oleovorans alk (alkane utilization) system, which are localized on the alkBAC operon, were cloned as a 16.9-kilobase pair EcoRI fragment . We have measured the length and determined the position of the alkBAC operon on this fragment by electron microscopy of R-loops . Furthermore, the 7.3-kilobase pair long alkBAC operon was analyzed for translation products in Escherichia coli minicells . Using a spectrum of overlapping subclones, six different proteins were identified . Starting from the alkBAC promotor, these polypeptides had molecular masses of 41, 15, 49, 58, 59, and 20 kDa, respectively . The 41-kDa protein was identified as alkane hydroxylase by reaction with a specific antibody . The 15- and 49-kDa peptides are soluble components of the alkane hydroxylase complex . The 58-kDa protein is most likely involved in alkanol dehydrogenase activity.

J Dairy Res, 1987 May, 54(2), 267 - 73
Comparison of a gelation and a chromogenic Limulus (LAL) assay for the detection of gram-negative bacteria, and the application of the latter assay to milk; Svensson A et al.; When a chromogenic Limulus Amoebocyte Lysate (LAL) assay and a tube gelation LAL assay were compared for the detection of Gram-negative bacteria using a strain of Pseudomonas putida, the detection level (approximately 10(3) cfu/ml) and cost of the assays were approximately the same for both assays but the reading was more precise for the chromogenic substrate assay . A modified chromogenic assay was devised for detection of Ps . putida in milk.

Mol Gen Genet, 1987 May, 207(2-3), 349 - 54
The xylS gene positive regulator of TOL plasmid pWWO: identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon; Mermod N et al.; The Pseudomonas putida TOL plasmid pWWO carries an operon that specifies a meta-cleavage pathway for the catabolism of benzoate and toluates whose transcription is positively regulated by the xylS gene product . Stimulation of transcription of the operon is thought to result from activation of this protein by pathway substrates/effectors . In the present study, overexpression of the xylS gene has led to identification of the regulator as a 33 kDa protein . Overexpression of xylS also resulted in partially constitutive, i.e . effector-independent expression of the meta-cleavage operon . Determination of the polynucleotide sequence of the xylS gene revealed amino acid sequence homology with several DNA binding proteins, particularly with the araC products of Escherichia coli and Salmonella typhimurium and with the nifA and ntrC products of Klebsiella pneumoniae . Homologous sequences were mainly located in an alpha-helix-turn-alpha-helix domain of the polypeptide . Interestingly, amino acid sequence homology was also found with sigma factors of E . coli (ntrA and htpR products) and Bacillus subtilis (spoIIG and phage SPOI Gp34 products) and other RNA polymerase core-interacting proteins, such as the E . coli nusA product.

J Gen Microbiol, 1987 May, 133 ( Pt 5), 1149 - 58
Molecular analysis of regulatory and structural xyl genes of the TOL plasmid pWW53-4; Keil H et al.; pWW53-4 is a cointegrate between RP4 and the catabolic plasmid pWW53 from Pseudomonas putida MT53, which contains 36 kbp of pWW53 DNA inserted close to the oriV gene of RP4; it encodes the ability to grow on toluene and the xylenes, characteristic of pWW53, as well as resistance to tetracycline, kanamycin and carbenicillin, characteristic of RP4 . A physical map of the 36 kbp insert of pWW53 DNA for 11 restriction enzymes is presented, showing that the relative positions of the two xyl operons are different from those on the archetypal TOL plasmid pWW0 . The location of the genes for 4-oxalocrotonate decarboxylase (xylI) and 4-oxalocrotonate tautomerase (xylH) were shown by subcloning and enzyme assay to lie at the distal end of the meta pathway operon . Although 2-oxopent-4-enoate hydratase (xylJ) and 4-hydroxy-2-oxovalerate aldolase (xylK) could be detected on a large cloned HindIII fragment, they could not be accurately located on smaller subcloned DNA, but the only credible position for them is between xylF and xylI . The gene order in the meta pathway operon is therefore xylDLEGF(J,K)IH . The regulatory genes xylS and xylR were located close to and downstream of the meta pathway operon, and the restriction map of the DNA in this region, as has previously been shown for the two operons carrying the structural genes, shows similarities with the corresponding region on pWW0 . Evidence is also presented for the existence of two promoters, termed P3 and P4, internal to the meta pathway operon which support low constitutive expression of the structural genes downstream in Pseudomonas hosts but not in E . coli.

Appl Environ Microbiol, 1987 May, 53(5), 996 - 1002
Maintenance and stability of introduced genotypes in groundwater aquifer material; Jain RK et al.; Three indigenous groundwater bacterial strains and Pseudomonas putida harboring plasmids TOL (pWWO) and RK2 were introduced into experimentally contaminated groundwater aquifer microcosms . Maintenance of the introduced genotypes was measured over time by colony hybridization with gene probes of various specificity . On the basis of the results of colony hybridization quantitation of the introduced organisms and genes, all introduced genotypes were stably maintained at approximately 10(5) positive hybrid colonies g-1 of aquifer microcosm material throughout an 8-week incubation period . Concomitant removal of the environmental contaminants, viz., toluene, chlorobenzene, and styrene, in both natural (uninoculated) and inoculated aquifer microcosms was also demonstrated . The results indicate that introduced catabolic plasmids, as well as indigenous organisms, can be stably maintained in groundwater aquifer material without specific selective pressure for the introduced genotypes . These results have positive implications for in situ treatment and biodegradation in contaminated aerobic groundwater aquifers.

Plasmid, 1987 May, 17(3), 222 - 32
Analysis of transcription from the trfA promoter of broad host range plasmid RK2 in Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa; Pinkney M et al.; Reverse transcriptase mapping has been used to analyze transcription from the trfA promoter of broad host range plasmid RK2 . The results show that trfA operon mRNA has the same 5' end in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli . The strengths of wild-type and mutant trfA promoters, which differ by defined base substitutions, have been compared and the positions of their transcriptional start sites determined . While these base substitutions do not alter the transcriptional start site, they do have marked effects on promoter strength which are broadly similar in each of the host species . A single base pair substitution, which lies in the region corresponding to the E . coli promoter consensus, brings about a large reduction in gene expression while the introduction of a second mutation, at a locus outside this region, has no further effect on promoter strength . The results indicate that these Pseudomonas species possess an RNA polymerase which recognizes the same region of the trfA promoter as that utilized by E . coli RNA polymerase . Within the limits of these observations it is clear that the trfA operon is transcribed from a single promoter which can function efficiently in diverse species, a property which may be important for its broad host range.

Biochim Biophys Acta, 1987 Apr 8, 912(2), 167 - 77
Hydrodynamic characterization of the size and shape of atropinesterase from Pseudomonas putida; van der Drift AC et al.; Atropinesterase from Pseudomonas putida has been investigated by means of different ultracentrifugation methods under native and denaturing conditions . The following quantities were determined: sedimentation coefficient, translational diffusion and friction coefficient, partial specific volume and molecular weight . From these data the size, shape and hydration of the enzyme molecule in solution were estimated . The results suggest that atropinesterase is a globular protein which consists of a single polypeptide chain with a molecular weight of about 30,000 . In solution under non-denaturing conditions, it occurs mainly as a dimer which hydrodynamically behaves as a rigid impenetrable particle . Calculations based on the spheroid model indicate that this particle resembles a hydrated sphere with a diameter of 6.1 +/- 0.2 nm and a hydration of 0.4 +/- 0.1 g of H2O/g of protein rather than a significantly less hydrated ellipsoid of revolution . Under denaturing conditions dissociation into monomers takes place . The effects of sodium dodecyl sulphate (SDS) on size and shape suggest that dimerization results from side-by-side association of two elongated monomers rather than from end-to-end association . Approximately 57 molecules of SDS are bound per dimer before dissociation occurs concomitant with the additional binding of about 19 molecules of detergent.

J Bacteriol, 1987 Apr, 169(4), 1780 - 3
Characterization of a novel TOL-like plasmid from Pseudomonas putida involved in 1,2,4-trimethylbenzene degradation; Bestetti G et al.; A strain of Pseudomonas putida (TMB) was found to resemble P . putida mt-2 (PaW1) in its ability to degrade 1,2,4-trimethylbenzene, toluene, m-xylene, and p-xylene via oxidation of a methyl substituent and reaction of the meta fission pathway, but a different regulatory model is suggested . The ability of P . putida TMB to degrade these substrates was encoded by plasmid pGB (85 kilobase pairs), which showed considerable differences in size, restriction patterns, and DNA sequence from those of plasmid pWWO of strain PaW1.

J Bacteriol, 1987 Apr, 169(4), 1619 - 25
Molecular cloning of genes encoding branched-chain keto acid dehydrogenase of Pseudomonas putida; Sykes PJ et al.; We cloned the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 7.8-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230 . A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P . putida branched-chain keto acid dehydrogenase mutants . The recombinant plasmid, pSS1-1, increased expression of branched-chain keto acid dehydrogenase up to five times in wild-type P . putida . The complex was expressed constitutively in P . putida(pSS1-1) but was inducible in Escherichia coli HB101(pSS1-1) by high valine . E . coli minicells transformed with pSS1-1 produced three polypeptides which did not match the four polypeptides of the purified complex . To resolve this problem, we inserted P . putida DNA from pSS1-1 into pUC18 and pUC19 . The pUC-derived plasmids were used as DNA templates in an E . coli transcription-translation system . Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned . Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established . The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping.

Prikl Biokhim Mikrobiol, 1987 Mar-Apr, 23(2), 199 - 203
{Effect of the medium composition on the accumulation of 2-keto-D-gluconic acid in Pseudomonas putida cultures}; Voloshenko MI et al.; The effect of the composition of the culture medium and the age of the culture on the activities of the enzymes involved in accumulation of 2-ketogluconic acid by Pseudomonas putida was studied . The activities of glucose and gluconate dehydrogenases that are responsible for direct oxidation of glucose to 2-ketogluconic acid, were 2-3 times higher during the active growth of the culture than in the stationary phase . The activities of 2-ketogluconokinase and 2-keto-6-phosphogluconate reductase, enzymes converting 2-ketogluconic acid, increased 2-4-fold on the glucose exhausting . The latter enzymes were not active when the culture was grown on nitrogen or phosphorus deficient media, and in this case 2-ketogluconic acid was accumulated in the medium.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 683 - 90
Isolation and characterization of Pseudomonas putida R-prime plasmids; Bray R et al.; A number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient . For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved . Physical analysis of one R-prime showed that 3.5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.

Biochem Biophys Res Commun, 1987 Feb 13, 142(3), 1006 - 12
Amidohydrolysis of N-methylhydantoin coupled with ATP hydrolysis; Kim JM et al.; A new enzyme, N-methylhydantoin amidohydrolase, was highly purified from Pseudomonas putida 77: it catalyzes the hydrolysis of N-methylhydantoin to N-carbamoylsarcosine with the concomitant stoichiometric cleavage of ATP to ADP and orthophosphate . The enzyme absolutely requires ATP, MG2+ and K+ for the N-methylhydantoin hydrolysis . The rapid and complete degradation of N-methylhydantoin during the cultivation of P . putida 77, which rapidly degrades creatinine via only N-methylhydantoin and which shows high activities of the enzymes involved in creatinine degradation (Yamada et al . (1985) FEMS Microbiol . Lett . 30, 337-340), seems to be due to the continuous ATP-generation during cultivation.

J Bacteriol, 1987 Feb, 169(2), 704 - 9
Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp . strain B13; Frantz B et al.; The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate . The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27 . A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27 . In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence . The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp . strain B13 plasmid pWR1 . A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.

J Bacteriol, 1987 Feb, 169(2), 764 - 70
Gene organization of the first catabolic operon of TOL plasmid pWW53: production of indigo by the xylA gene product; Keil H et al.; The entire operon coding for the enzymes responsible for conversion of toluenes to benzoates has been cloned from TOL plasmid pWW53 and the position of the genes accurately located . The coding region was 7.4 kilobase pairs (kbp) long, and the gene order was operator-promoter region (OP1)-a small open reading frame-xylC (1.6 kbp)-xylA (2.9 kbp)-xylB (1.8 kbp) . Within the coding region there was considerable homology with the isofunctional region of the archetypal TOL plasmid pWW0 . A central region of 2.9 kbp complemented an xylA (for xylene oxygenase) mutant of Pseudomonas putida mt-2 and was also capable of conferring the ability to convert indole to indigo on strains of Escherichia coli and P . putida . This reaction has been reported previously only for dioxygenases involved in aromatic catabolism but not for monooxygenases . It is proposed that the region encodes xylene oxygenase activity capable of direct monohydroxylation of indole to 3-hydroxyindole (oxindole), which then spontaneously dimerizes to form indigo.

J Basic Microbiol, 1987, 27(2), 83 - 9
Use of salicylate to estimate the "threshold" inducer level for de novo synthesis of the phenol-degrading enzymes in Pseudomonas putida strain H; Janke D; A special approach was used to elucidate the "threshold" inducer concentration for coordinative de novo synthesis of phenol hydroxylase(s), catechol 2,3-dioxygenase and the 2-hydroxymuconic semialdehyde-metabolizing enzymes which initiate phenol catabolism in Pseudomonas putida strain H . It is based on cell-precultivation with glucose (as the carbon and energy source) in the presence of different concentrations of sodium salicylate which proved to be a potent non-metabolizable inducer in strain H of these enzymes . Subsequent estimation of the activity status of resting cell suspensions and cell-free extracts, respectively, prepared from those strain H cultures clearly revealed failing de novo synthesis of the mentioned phenol-degrading enzymes at salicylate concentrations lower than 0.2 mg/l.

Gene, 1987, 55(1), 19 - 28
Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL pWWO; Ghosal D et al.; The enzyme catechol 2,3-dioxygenase (C23O) encoded by the nahH gene of plasmid NAH7 converts catechol to alpha-hydroxymuconic epsilon-semialdehyde in Pseudomonas putida . We have cloned this structural gene into vectors pUC18 and pKT240, determined the nucleotide sequence and deduced the amino acid sequence . In comparison to the gene xylE of the TOL plasmid pWWO which encodes a similar C230 enzyme {Nakai et al . J . Biol . Chem . (1983b), 2923-2928}, the respective G + C contents were 55% and 57%, the nucleotide sequences 81% homologous, and the amino acid homology 85% . The flanking sequences of the two C23O-coding genes also show homology . Clones of the nahH gene in Escherichia coli overproduce the protein product at least ten fold and the gene product was identified by polyacrylamide gel electrophoresis.

Gene, 1987, 52(2-3), 185 - 95
Cloning and complete nucleotide sequence determination of the catB gene encoding cis,cis-muconate lactonizing enzyme; Aldrich TL et al.; The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a key role in the beta-ketoadipate pathway of benzoate degradation . A 10.2-kb EcoRI fragment isolated from a Pseudomonas putida genomic library complemented a mutant deficient in this enzyme . The MLEI coding gene, catB, was localized to a 1.6-kb fragment which was sequenced by the dideoxy chain termination method . MLEI was purified 25-fold from crude extracts of benzoate-grown P . putida PRS2015 harboring the cloned catB gene . Purified MLEI was greater than 95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The subunit Mr was 40,000 which was in close agreement with the nucleotide sequence data . N-terminal sequence analysis of purified MLEI protein agreed with the N terminus predicted by the nucleotide sequence . Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene (K.L . Ngai, B.F., D.K . Chatterjee, L.N . Ornston, and A.M.C., unpublished), whose gene product catalyzes the analogous reaction in 3-chlorobenzoate degradation, showed significant homology . These results suggest that catB and clcB have diverged from a common ancestral gene.

Gene, 1987, 57(1), 61 - 72
A set of cassettes and improved vectors for genetic and biochemical characterization of Pseudomonas genes; Deretic V et al.; A set of broad-host-range vectors allowing direct selection of recombinant DNA molecules to facilitate subcloning and expression analyses of Pseudomonas genes was constructed using Bg/II lacZ alpha cassette . Controlled expression vectors pVDtac39 and pVDtac24 were shown to be useful for determination of enzymatic activities encoded by the cloned DNA fragments and Mr determination of the corresponding polypeptides . A set of Pseudomonas putida xylE gene cassettes truncated at the 5' end was constructed for translational (protein) fusion studies . A protein fusion of the Pseudomonas aeruginosa algD gene, coding for GDPmannose dehydrogenase, and the truncated xylE gene cassette was used to verify the putative coding region and translational signals predicted from the algD nucleotide sequence.

Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 993 - 9
Purification and characterization of glyoxalase I from Pseudomonas putida; Rhee H et al.; Glyoxalase I was purified to apparent homogeneity from Pseudomonas putida . The enzyme was a monomer with a molecular weight of 20,000 . The enzyme was most active at pH 8.0 . The Km values for methylglyoxal and 4,5-dioxovale-rate are 3.5 mM and 1.2 mM, respectively . Contrary to the case of eukaryotic enzymes, chelating agents showed little inhibitory effects on the enzyme activity . Among the metal ions tested, Zn++ specifically and completely inhibited the activity of the enzyme at a millimolar level . The properties of bacterial glyoxalase I were quite different from mammalian and yeast enzymes.

J Bacteriol, 1986 Dec, 168(3), 1302 - 8
Chromosomal location of TOL plasmid DNA in Pseudomonas putida; Sinclair MI et al.; The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid . By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB . A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18 . Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.

Steroids, 1986 Nov-Dec, 48(5-6), 439 - 50
A catecholic 9,10-seco steroid as a product of aerobic catabolism of cholic acid by a Pseudomonas sp; Park RJ et al.; A mutant of the efficient bile acid-utilizing Pseudomonas putida ATCC 31752 was found to accumulate three major catabolites on aerobic growth on cholic acid . One of these catabolites was isolated and identified as 3,4,7,12 beta-tetrahydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (2) . This is the first catecholic 9,10-secosteroid isolated from the microbial degradation of bile acids or sterols and confirms the role of such secosteroids in the microbial degradative pathway for steroids.

Biochem Cell Biol, 1986 Nov, 64(11), 1190 - 4
L-malate transport and proton symport in vesicles prepared from Pseudomonas putida; Agbanyo FR et al.; In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion . L-Malate also acts as an electron donor and generates a proton motive force (delta p) of 129 mV which is composed of a membrane potential (delta psi) of 60 mV and a delta pH of 69 mV . In contrast, vesicles from succinate-grown cells transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol X mg protein-1 X min-1, generate no delta psi, delta pH, or delta p when L-malate is the electron donor, and produce an extravesicular alkaline pH during the transport of L-malate . A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+ X mg protein-1 X min-1 . This corresponds to a H+/L-malate ratio of 2.1 . The failure to generate a delta p in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.

Genetika, 1986 Nov, 22(11), 2702 - 12
{Nah-genes of Pseudomonas putida: molecular genetic analysis of the plasmid pBS286}; Tsoi TV et al.; The hybridization and restriction analysis of the plasmid pBS286 (73 Kb, the P-9 Inc group) as well as parental plasmids NPL-1, NPL-41 demonstrated that pBS286 plasmid (delta NPL-41::TnA) with the constitutive synthesis of naphthalene dioxygenase carried genes for naphthalene oxidation to salicylate and those participating in degradation of catechol . Restriction map of pBS286 using XhoI restriction endonuclease and that of the nah region using EcoRI, BamHI, SalI and XhoI were established . Structural peculiarities of nah genes from pBS286 are compared with previously described NAH7 . Some nah genes were localized . An inverted DNA segment involved in nah gene regulation was shown to be closely linked to a proximal part of the nah1 operon or overlapped . Possible occurrence of a regulatory R locus in this region is suggested.

Biochemistry, 1986 Oct 7, 25(20), 5975 - 81
Amino acid and sequence analysis of the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase; McIntire W et al.; The flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida has been reported to have a Mr of 114,000 and to consist of two subunits, a flavoprotein and a cytochrome c, each with a Mr of 58,000 . Recent X-ray crystallographic data from our laboratories {Shamala, N., Lim, L . W., Mathews, F . S., McIntire, W., Singer, T . P., & Hopper, D . J . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 4626-4630}, however, indicate an alpha 2 beta 2 structure and a much lower molecular mass (approximately 8000) for the cytochrome subunit . In this paper we report data confirming the conclusions of X-ray crystallographic analysis . From quantitative amino acid analysis, the molecular mass of the flavoprotein monomer is shown to be 48,600 +/- 2200 and that of the cytochrome 8780 +/- 250 . These values have been confirmed by gel electrophoresis under denaturing conditions . Gel chromatography under nondenaturing conditions shows that the isolated flavoprotein exists as a dimer, whereas the isolated cytochrome is a monomer . The complete amino acid sequence of the cytochrome c subunit is presented and is shown to have regions of homology to other bacterial c-type cytochromes . The partial N-terminal amino acid sequence (56 amino acids) of the flavoprotein subunit is also reported . The implications of the now established tetrameric structure of the flavocytochrome on data in the literature regarding the redox and association properties of the subunits are examined.

Appl Environ Microbiol, 1986 Oct, 52(4), 930 - 4
Comparative analysis of different Pseudomonas strains that degrade cinnamic acid; Andreoni V et al.; Strains of Pseudomonas stutzeri (CINNS) and Pseudomonas putida (CINNP and CINNW) isolated from soil with cinnamic acid as the sole carbon source were found to be simultaneously adapted to grow on phenylpropionic and p-hydroxybenzoic acids . In cinnamic acid-grown cultures, phenylpropionic acid was isolated . A catabolic plasmid of approximately equal to 75 kilobase pairs encoding the metabolism of cinnamic acid was found in strains CINNP and CINNS.

J Bacteriol, 1986 Oct, 168(1), 81 - 5
Cloning of Pseudomonas sp . strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate; Savard P et al.; The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp . strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway . The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp . strain CBS3 in Pseudomonas putida . Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp . strain CBS3 . This plasmid confers on P . putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon . However, pPSA843 did not complement mutants of P . putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned . Subcloning of Pseudomonas sp . strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.

Genetika, 1986 Oct, 22(10), 2389 - 97
{Comparative analysis of the organization of the NPL-1 plasmid controlling naphthalene oxidation in Pseudomonas putida and its derivatives}; Kosheleva IA et al.; The paper contains the data on the structure of NPL-1 plasmid controlling naphthalene biodegradation . The plasmid which pertains to the P-9 incompatibility group is transferrable conjugatively and is maintained stably within a wide range of gram-negative bacteria . The analysis of mutants and transposon derivatives of the plasmid made it possible to localize nah-genes in a DNA fragment, 23 kb in size . An inverted DNA segment of 4.2 kb was discovered which may participate in the regulation of nah-genes expression . The other peculiar features of NPL-1 were found distinguishing it from NAH and NAH7 plasmids described in literature.

J Bacteriol, 1986 Oct, 168(1), 428 - 30
Spontaneous deletion of a 20-kilobase DNA segment carrying genes specifying isopropylbenzene metabolism in Pseudomonas putida RE204; Eaton RW et al.; The genes encoding isopropylbenzene metabolism in Pseudomonas putida RE204 are readily lost in two ways: by loss (curing) of plasmid pRE4 which specifies the catabolic pathway and by deletion from pRE4 of an approximately 20-kilobase segment of DNA carrying the catabolic genes . The presence of DNA sequences at the ends of the catabolic gene region sharing homology with one another suggests that the deletions result from recombination events between these homologous sequences.

J Bacteriol, 1986 Oct, 168(1), 123 - 31
Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204; Eaton RW et al.; A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated . Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism . These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts . Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates . Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis . Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4 . DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments . These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.

Biochemistry, 1986 Sep 9, 25(18), 5314 - 22
Crystal structure of substrate-free Pseudomonas putida cytochrome P-450; Poulos TL et al.; The crystal structure of Pseudomonas putida cytochrome P-450cam in the substrate-free form has been refined at 2.20-A resolution and compared to the substrate-bound form of the enzyme . In the absence of the substrate camphor, the P-450cam heme iron atom is hexacoordinate with the sulfur atom of Cys-357 providing one axial heme ligand and a water molecule or hydroxide ion providing the other axial ligand . A network of hydrogen-bonded solvent molecules occupies the substrate pocket in addition to the iron-linked aqua ligand . When a camphor molecule binds, the active site waters including the aqua ligand are displaced, resulting in a pentacoordinate high-spin heme iron atom . Analysis of the Fno camphor - F camphor difference Fourier and a quantitative comparison of the two refined structures reveal that no detectable conformational change results from camphor binding other than a small repositioning of a phenylalanine side chain that contacts the camphor molecule . However, large decreases in the mean temperature factors of three separate segments of the protein centered on Tyr-96, Thr-185, and Asp-251 result from camphor binding . This indicates that camphor binding decreases the flexibility in these three regions of the P-450cam molecule without altering the mean position of the atoms involved.

J Biol Chem, 1986 Sep 5, 261(25), 11832 - 9
Purification and characterization of a novel enzyme, N-carbamoylsarcosine amidohydrolase, from Pseudomonas putida 77; Kim JM et al.; N-Carbamoylsarcosine amidohydrolase, a novel enzyme involved in the microbial degradation of creatinine in Pseudomonas putida 77, was purified 27-fold to homogeneity with a 63% overall recovery through simple purification procedures including successive ammonium sulfate fractionation, DEAE-cellulose chromatography, and crystallization . The relative molecular mass of the native enzyme estimated by the ultracentrifugal equilibrium method is 102,000 +/- 5000, and the subunit Mr is 27,000 . The Km and Vm values for N-carbamoylsarcosine are 3.2 mM and 1.75 units/mg protein, respectively . Ammonia, carbon dioxide, and sarcosine were formed stoichiometrically from N-carbamoylsarcosine through the action of the purified enzyme preparation . N-Carbamoyl amino acids with a methyl group or hydrogen atom on the amino-N atom and possessing glycine, D-alanine, or one of their derivatives as an amino acid moiety served well as substrates for N-carbamoylsarcosine amidohydrolase . N-Carbamoylsarcosine, N-methyl-N-carbamoyl-D-alanine, N-carbamoylglycine, and N-carbamoyl-D-alanine were hydrolyzed at relative rates of 100, 12.8, 9.8, and 7.3, respectively, by the enzyme . N-Carbamoyl derivatives of D-tryptophan, D-phenylalanine, and those of some other amino acids including D-phenylglycine and p-hydroxy-D-phenylglycine were also hydrolyzed by the enzyme . For the L-isomers of all N-carbamoyl amino acids tested there was no production of ammonia, carbon dioxide, or the corresponding amino acids due to the action of the enzyme . Cupric, mercuric, and silver ions inhibited the enzyme strongly, and some thiol reagents were also found to be inhibitory.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2209 - 18
Adaptation of Pseudomonas putida mt-2 to growth on aromatic amines; McClure NC et al.; Pseudomonas putida mt-2 (ATCC 33015) carrying the TOL plasmid pWW0 could adapt to growth on the aromatic amines aniline and m- and p-toluidine . In strain UCC2, a derivative adapted to rapid growth on these compounds, they were oxidatively deaminated to catechol or 4-methylcatechol, which in turn were dissimilated by a meta-cleavage pathway . The aniline/toluidine oxygenase and the meta-cleavage pathway enzymes were inducible by aromatic amines . Evidence is presented that in strain UCC2, plasmid pWW0 has undergone deletion of its catabolic genes, and that it is a novel plasmid, pTDN1, which is involved in the catabolism of aniline and m- and p-toluidine . The meta-cleavage pathway genes which are carried by pTDN1 were shown not to have originated in pWW0.

Appl Environ Microbiol, 1986 Aug, 52(2), 334 - 9
Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2; Zeyer J et al.; Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen . ONP was converted by a nitrophenol oxygenase to nitrite and catechol . Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway . ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2 . Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized . Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions . It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP . In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase . Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols . 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria . Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.

J Bacteriol, 1986 Aug, 167(2), 447 - 54
Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria; Mermod N et al.; A pKT231-based broad-host-range plasmid vector was constructed which enabled regulation of expression of cloned genes in a wide range of gram-negative bacteria . This vector, pNM185, contained upstream of its EcoRI, SstI, and SstII cloning sites the positively activated pm twin promoters of the TOL plasmid and xylS, the gene of the positive regulator of these promoters . Expression of cloned genes was induced with micromolar quantities of benzoate or m-toluate, the inexpensive coinducers of the pm promoters . Expression of a test gene, xylE, which specifies catechol 2,3-dioxygenase, cloned in this vector was tested in representative strains of a variety of gram-negative bacteria . Regulated expression of xylE was observed in most strains examined, and induced levels of enzyme representing up to 5% of total cellular protein and ratios of induced:noninduced levels of enzyme up to a factor of 600 were observed . The level of xylE gene expression in different bacteria tended to be correlated with their phylogenetic distance from Pseudomonas putida.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2195 - 204
A comparative study of acquired amidase activity in Pseudomonas species; Wyndham RC et al.; Pseudomonas putida PP3 carrying dehalogenases I and II and Pseudomonas aeruginosa PAU3 carrying dehalogenase I coded for by plasmid pUU2 were able to grow on 2-monochloropropionic acid (2MCPA) . Neither strain utilized 2-chloropropionamide (2CPA) as a carbon or nitrogen source for growth . Mutations in both strains to 2Cpa+ phenotypes (designated P . putida PPW3 and P . aeruginosa PAU5, respectively) involved the expression of an acquired 2CPA-amidase activity . The amidase followed by dehalogenase reactions in these strains constituted a novel metabolic pathway for growth on 2CPA . P . putida PPW3 synthesized a constitutive amidase of molecular mass 59 kDa consisting of two identical subunits of 29 kDa . For those amides tested this acquired enzyme was most active against chlorinated aliphatic amides, although substrate affinities (Km) and maximum rates of activity (Vmax) were poor . P . aeruginosa PAU5 acquired a 2Cpa+ phenotype by overproducing the A-amidase normally used by this species to hydrolyse aliphatic amides . The A-amidase had only slight activity towards 2CPA . However, with constitutive synthesis the mutant grew on the chlorinated substrates . Chloroacetamide (CAA) was a toxic substrate analogue for these Pseudomonas strains . A strain resistant to CAA was isolated from P . aeruginosa PAU5 when exposed to 1-10 mM-CAA . This mutant, P . aeruginosa PAU6, synthesized an inducible A-amidase . CAA-resistance depended upon the simultaneous expression of CAA-inducible amidase and dehalogenase activities.

Arch Biochem Biophys, 1986 Jul, 248(1), 130 - 7
Reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol dioxygenases; Pascal RA Jr et al.; The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined . Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme . Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates . 3-Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl-cis,cis-muconic acid . However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM) . The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported.

Biochem J, 1986 Jun 1, 236(2), 401 - 8
Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B . Purification and properties of the P1 enzyme; Bateman TJ et al.; The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose . The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1 . Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14) . Increasing the chain length from C6 to C12 led to diminishing Km . Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions . Vmax./Km values increased with increasing chain length . Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding . The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol . The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously . Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.

J Bacteriol, 1986 Jun, 166(3), 1028 - 39
Identification of cis-diols as intermediates in the oxidation of aromatic acids by a strain of Pseudomonas putida that contains a TOL plasmid; Whited GM et al.; Pseudomonas putida BG1 was isolated from soil by enrichment with p-toluate and selection for growth with p-xylene . Other hydrocarbons that served as growth substrates were toluene, m-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene . The enzymes responsible for growth on these substrates are encoded by a large plasmid with properties similar to those of TOL plasmids isolated from other strains of Pseudomonas . Treatment of P . putida BG1 with nitrosoguanidine led to the isolation of a mutant strain which, when grown with fructose, oxidized both p-xylene and p-toluate to (-)-cis-1,2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylic acid (cis-p-toluate diol) . The structure of the diol was determined by conventional chemical techniques including identification of the products formed by acid-catalyzed dehydration and characterization of a methyl ester derivative . The cis-relative stereochemistry of the hydroxyl groups was determined by the isolation and characterization of an isopropylidene derivative . p-Xylene-grown cells contained an inducible NAD+-dependent dehydrogenase which formed catechols from cis-p-toluate diol and the analogous acid diols formed from the other hydrocarbon substrates listed above . The catechols were converted to meta ring fission products by an inducible catechol-2,3-dioxygenase which was partially purified from p-xylene-grown cells of P . putida BG1.

J Bacteriol, 1986 Jun, 166(3), 739 - 45
Nucleotide sequence of a DNA segment promoting transcription in Pseudomonas putida; Inouye S et al.; A DNA segment that promotes gene expression in Pseudomonas putida was identified in pTN8, a mutant plasmid of an RP4-TOL recombinant . A promoter on the segment was cloned with a promoter-probe vector containing the xylE gene of the TOL plasmid . The xylE gene was expressed under the control of the promoter, and the gene product catechol 2,3-dioxygenase was constitutively synthesized . As analyzed by an S1 nuclease protection assay, the amount of mRNA produced in P . putida was more than that in Escherichia coli . Fine S1 nuclease mapping and reverse transcriptase mapping revealed three tandem transcription start sites in both P . putida and E . coli . The nucleotide sequence preceding the transcription start sites was determined; a part of this sequence contained a sequence homologous to E . coli promoter sequences . A tentative consensus sequence for P . putida constitutive promoters is proposed.

J Bacteriol, 1986 Jun, 166(3), 1089 - 95
camR, a negative regulator locus of the cytochrome P-450cam hydroxylase operon; Koga H et al.; A 4.27-kilobase insert from a HindIII DNA library of Pseudomonas putida carrying the CAM plasmid allowed coordinate expression of genes camD and camC under control of camR, an upstream regulator . The camC gene specifies cytochrome P-450cam, and camD specifies the 5-exo-alcohol dehydrogenase . A 1.38-kilobase deletion from the insert results in the constitutive expression of genes camC and camD; transformation in trans restores the substrate control, indicating that camR is a negative regulator.

J Biol Chem, 1986 May 15, 261(14), 6454 - 60
The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B; Linden KG et al.; We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B . The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536 . The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background . The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds . The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation . The full sequence was deduced by the overlapping of the various peptides . A search for homologous proteins was performed . Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar . Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P . testosteroni isomerase . The P . putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P . testosteroni isomerase lacks these amino acids . The two proteins are not highly conserved.

J Bacteriol, 1986 Apr, 166(1), 155 - 61
Naphthalene association and uptake in Pseudomonas putida; Bateman JN et al.; Two methods for bacterial membrane transport, filtration and flow dialysis, were used to study cellular association of Pseudomonas putida with naphthalene . It is not technically possible to determine the exact cellular or vesicular location of the naphthalene, and because it is hydrophobic, it could be at the membrane(s) rather than inside the cells . As an index of naphthalene having crossed the inner membrane we used the intracellular formation of its first catabolite . An energized membrane or ATP was not essential for association or movement into the cell . Evidence for a nonspecific association and a movement into cells by simple diffusion are the lack of saturation of association, an absence of inhibition of association by protein inhibitors and structural analogs, and the passage of naphthalene through cell membranes in the presence of iodoacetamide . Specific naphthalene metabolism gene expression was not required for association.

Mol Gen Genet, 1986 Apr, 203(1), 129 - 36
The xylABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli; Dixon R; The xylABC promoter (OP1), located on the TOL plasmid of Pseudomonas putida contains sequences homologous to the conserved regions found in nitrogen fixation (nif) promoters and in other promoters subject to nitrogen control . XylA-lac fusions were constructed in order to monitor expression from the OP1 promoter in Escherichia coli . Transcription was activated in the presence of the heterologous regulatory genes ntrC or nifA from Klebsiella pneumoniae as well as by the homologous P . putida regulatory gene xylR . In all cases activation was also dependent on the ntrA gene, whose product has been implicated as a specific sigma factor for ntr activatable operons . The 5' ends of xylA mRNA, detected by S1 nuclease mapping of in vivo transcripts, were identical in strains containing xylR, ntrC or nifA as transcriptional activators . However, activation of the K . pneumoniae nifL or nifH promoters by xylR was not detected.

Eur J Biochem, 1986 Apr 1, 156(1), 59 - 64
Formaldehyde dismutase, a novel NAD-binding oxidoreductase from Pseudomonas putida F61; Kato N et al.; A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F61 . The enzyme catalyzes the dismutation of aldehydes and alcohol:aldehyde oxidoreduction in the absence of an exogenous electron acceptor . The enzyme is composed of four identical subunits with a Mr of 44 000 . Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol . The ratio of NAD+ and NADH in a crystalline preparation of the enzyme was about 7:3 . The enzyme-bound coenzyme was completely reduced and oxidized on the addition of a large amount of an alcohol and an aldehyde respectively . Both the oxidized and reduced enzymes catalyzed the dismutation reaction to the same extent . Steady-state kinetics of the enzyme were investigated using an oxidoreduction reaction between an alcohol and p-nitroso-N, N-dimethylaniline . The enzyme obeys a ping-pong mechanism and is competitively inhibited by an alcoholic substrate analogue, pyrazole, but not coenzyme analogues, such as AMP, N-methylnicotinamide . These results indicate that NAD(H) binds firmly (but not covalently) at each active site . The enzyme-bound NAD(H) was reduced and oxidized only by the added second substrates, alcohol and aldehyde respectively, and not by exogenous electron acceptors {including NAD(H)}.

Biochemistry, 1986 Mar 25, 25(6), 1390 - 4
Thermodynamic properties of oxidation-reduction reactions of bacterial, microsomal, and mitochondrial cytochromes P-450: an entropy-enthalpy compensation effect; Huang YY et al.; An optically transparent thin-layer electrode cell with a very small volume was used for determination of the formal reduction potentials of bacterial, microsomal, and mitochondrial cytochromes P-450 . At an extrapolated zero concentration of dye, the bacterial cytochrome from Pseudomonas putida catalyzing the hydroxylation of camphor and the adrenal mitochondrial cytochrome catalyzing the cholesterol side-chain cleavage reaction had formal reduction potentials of -168 and -285 mV (pH 7.5 and 25 degrees C), respectively . The oxidation-reduction potentials for the rabbit liver microsomal cytochrome P-450 induced by 3-methylcholanthrene and the mitochondrial cytochrome for steroid 11 beta-hydroxylation were found as -360 and -286 mV, respectively . Potential measurements at different temperatures allowed documentation of the standard thermodynamic parameters for cytochrome P-450 reduction for the first time . All cytochromes tested were found to have a relatively large negative entropy change upon reduction . The extent of these changes is comparable to that observed for the ferric-ferrous couple of cytochrome c . An entropy-enthalpy compensation effect was observed among the four cytochromes P-450 examined although the correlation is weaker than that observed with cytochrome c isolated from various sources.

Environ Health Perspect, 1986 Mar, 65, 5 - 11
Cadmium-binding proteins in Pseudomonas putida: pseudothioneins; Higham DP et al.; Pseudomonas putida adapted to growth in 3 mM cadmium . The resistance mechanism involved complexation of cadmium in polyphosphate granules, changes in the structure of the cell membrane and induction of three cysteine-rich, low molecular weight proteins (3500-7000) containing 4 to 7 g-atoms per mole of cadmium, zinc, and copper . Each protein was produced during a different phase of growth, and the smallest protein (3500) was released into the environment when the cells lysed at the end of the exponential phase . The metal binding sites of the major protein were further characterized using a range of physical methods, including 113Cd NMR . The properties of the bacterial pseudothioneins are compared to those of metallothioneins.

Plasmid, 1986 Mar, 15(2), 132 - 46
Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis; Cross MA et al.; A range of Tn1723 transposon mutants of the oriV region of broad-host-range plasmid RK2 have been isolated, and the internal EcoRI fragment of the transposon has been deleted from each to reduce the insertion size from 9.6 kb (Tn1723) to 35 bp (delta Tn1723) . Sequencing from the delta Tn1723-derived EcoRI site has allowed the precise mapping of these insertions to various points dispersed through the origin region . Using these mutants we have determined which regions of oriV RK2 are of functional importance to plasmid establishment following transformation of the host species Escherichia coli, Pseudomonas putida, and P . aeruginosa . Insertions into an A/T-rich region, and a region containing five direct repeat sequences prevented successful transformation of each host species tested, but the continuity of sequences adjacent to the five repeats were essential only in E . coli and P . putida . The establishment and maintenance in E . coli of a mini-RK2 replicon was found to be inhibited by transcription from an inducible promoter positioned to read into oriV RK2 against the direction of replication . Assays of transcription emerging from Tn1723 demonstrated significant levels from one end of the transposon only . Four mutants with insertions downstream of oriV RK2 were unable to become established in E . coli, and contained Tn1723 in the orientation which would supply transcription toward the oriV RK2 region . These results demonstrate both that the sequence requirements for oriV RK2 function differ between host bacterial species, and that origin function may be further influenced by the genetic environment in which it lies.

J Bacteriol, 1986 Feb, 165(2), 489 - 97
Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453; Taylor DG et al.; The oxygenating component of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453 was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatography on DEAE-cellulose and polyanion SI-17 columns . It had an Mr of 78,000, bound one molecule of nonautooxidizable flavin mononucleotide (FMN), consisted of two subunits of equal molecular weight, and existed in two electrophoretically distinguishable active forms . The oxygenating complex was constructed from equimolecular amounts of an NADH oxidase, which could be purified separately (Mr, 36,000), and the oxygenating component . Most of the NADH oxidase dissociated from the oxygenating component during purification, although traces remained, to give the final preparation of the oxygenating component significant oxygenase activity . FMN did not dissociate significantly from the oxygenating component during purification, but it was not covalently bound and could be removed under a variety of conditions . Binding between the two proteins that made up the active complex was fairly weak and freely reversible . It probably occurred through the FMN which was strongly bound to the oxygenating component and for which the NADH had a weak binding site . Iron was not present at a significant level in the oxygenating component, and in common with other characterized Baeyer Villiger monooxygenases, 2,5-diketocamphane 1,2-monooxygenase was found to be a simple flavoprotein.

Mol Gen Genet, 1986 Feb, 202(2), 226 - 34
Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida; Harayama S et al.; Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida . A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon . In parallel, a series of N-methyl-N'-nitro-N-nitro-soguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon . Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined . Four cistrons for toluate 1,2-dioxygenase were thus identified . DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate . This analysis also identified four cistrons . Examination of the products of these cistrons, by means of E . coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xylY, encodes a 20 kilodalton protein.

J Bacteriol, 1986 Feb, 165(2), 650 - 3
Characterization of the OCT plasmid encoding alkane oxidation and mercury resistance in Pseudomonas putida; Harder PA et al.; Transformation of Pseudomonas putida and analysis for plasmid DNA revealed that both n-alkane oxidation and mercury resistance are encoded on a single 220-megadalton OCT plasmid molecule . Derivatives of OCT having lost the mercury resistance function could be readily isolated and contained a smaller plasmid estimated to be 170 megadaltons . The results show that segregation of the mercury resistance property occurs not by loss of a separate MER plasmid as previously thought but by a deletion in the OCT plasmid.

J Biol Chem, 1986 Jan 25, 261(3), 1158 - 63
Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli; Unger BP et al.; Cytochrome P-450cam catalyzes the stereospecific methylene hydroxylation of camphor to form 5-exohydroxycamphor and is encoded by the camC gene on the CAM plasmid of Pseudomonas putida, ATCC 17453 . The cytochrome P-450cam structural gene has been cloned by mutant complementation in P . putida (Koga, H., Rauchfuss, B., and Gunsalus, I . C . (1985) Biochem . Biophys . Res . Commun . 130, 412-417) . We report the complete nucleotide sequence of the camC gene along with 155 base pairs of 5' and 175 base pairs of 3' flanking sequence . Upon comparison of the amino acid sequence derived from the gene sequence to the one obtained from the purified protein (Haniu, M., Armes, L . G., Yasunobu, K . T., Shastry, B . A., and Gunsalus, I . C . (1982) J . Biol . Chem . 257, 12664-12671), five differences were found . The most significant was the addition of a Trp and a Thr residue between Val-54 and Arg-55, thereby increasing the amino acid numbering scheme by 2 after Val-54, bringing the total number of amino acids to 414 . Other differences were: Gln-274----Glu-276, Ser-359----His-361, and Asn-405----Asp-407 . N-terminal amino acid sequence analysis of the cloned cytochrome P-450cam enzyme expressed in Escherichia coli under the lac promoter showed a faithful translation of the hemo-protein, with the N-terminal Met removed by processing as found in P . putida . Purification to homogeneity of the cloned protein was accomplished by the method used for the CAM plasmid-encoded enzyme of P . putida . The G + C content of the camC gene was found to be 59.0%, caused by a preferred usage of G and C terminated codons . The gene encoding putidaredoxin reductase, camA, was located 22 nucleotides downstream from the cytochrome P-450cam gene . The camA gene initiated with a novel GUG codon, the first such initiator documented in Pseudomonas.

J Bacteriol, 1986 Jan, 165(1), 334 - 5
Plasmid-determined cadmium resistance in Pseudomonas putida GAM-1 isolated from soil; Horitsu H et al.; Cadmium-resistant Pseudomonas putida GAM-1, which was able to grow in concentrations of CdCl2 as high as 7 mM, was isolated from soil in a rice paddy . This bacterium harbored a DNA plasmid of about 52 kilobases . The plasmid (pGU100) transformed Escherichia coli C600 to cadmium resistance . A cadmium-resistant transformant of E . coli C600 contained a plasmid corresponding to that seen in P . putida GAM-1 . The transformant did not take up cadmium as well as P . putida GAM-1 did.

Mikrobiologiia, 1986 Jan-Feb, 55(1), 100 - 4
{Selection of Escherichia coli and Pseudomonas putida cultures with an enhanced resistance to immobilization on polyacrylamide gel}; Starostina NG et al.; Clones of Escherichia coli (A4, A70, G60) and Pseudomonas putida (A70, G30) with an elevated resistance to the process of immobilization in polyacrylamide gel and to the action of monomeric acrylamide were selected from the parent E . coli IBPM B115 and P . putida . The isolated cultures remained resistant to the above actions for a long time . The frequency at which cells with the elevated resistance appeared was comparable with the frequency of bacterial mutations . The plasmid analysis did not reveal the presence of plasmid DNA in the cells of the isolated cultures . The decrease in the viability index of bacterial populations caused by their immobilization in polyacrylamide gel and by the action of monomeric acrylamide did depend on the growth phase . The cells were more resistant to these actions in the stationary phase . The isolated cultures were more resistant as compared to the parent cultures irrespective of the growth phase.

Microbios, 1986, 47(192-193), 149 - 57
Utilization of pyrimidines and pyrimidine analogues by fluorescent pseudomonads; West TP et al.; The fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens and Pseudomonas putida were examined for their ability to utilize pyrimidines and pyrimidine analogues . Both P . aeruginosa and P . aureofaciens grew upon dihydrouracil, dihydrothymine, uridine and cytidine as either a sole nitrogen or carbon source while uracil, thymine and cytosine served only as sole nitrogen sources for both pseudomonads . The only difference between the observed growth of P . aeruginosa and P . aureofaciens on pyrimidines was that deoxycytidine sustained the growth of P . aeruginosa as a nitrogen source . With respect to P . putida, uracil, cytosine and cytidine were found to be nitrogen sources while dihydrothymine, uridine and cytidine served as carbon sources . The fluorescent pseudomonads investigated had fourteen nutritional characteristics which were determined in common for the pyrimidines screened . All the fluorescent pseudomonads were sensitive to a low concentration of 5-fluorouracil . Inhibition by 5-fluorouridine of P . aureofaciens growth was observed at a low concentration while a high concentration of this analogue was required to halt P . putida growth . Neither P . aureofaciens nor P . putida could grow in the presence of a high concentration of 5-fluorocytosine . Only P . aureofaciens growth was noted to be inhibited by a high concentration of 6-azauracil.

Gene, 1986, 44(2-3), 235 - 42
Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product; Inouye S et al.; The xylS gene is a regulatory gene which positively controls expression of the genes on the TOL plasmid for degradation enzymes of benzoate or m-toluate in Pseudomonas putida . Cloning of the gene in Escherichia coli and determination of the nucleotide sequence revealed an open reading frame of 963 bp which corresponds to a protein with an Mr of 36,502 . The xylS gene was recloned onto a tac-promoter vector, and the product was identified by the maxicell procedure as a protein with an approximate Mr of 37,000 . The predicted amino acid sequence of XylS protein showed a basic character and contained a region similar to those in other DNA-binding proteins.

Proc Natl Acad Sci U S A, 1986 Jan, 83(2), 369 - 73
Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida; Schell MA; The in vivo transcription start sites of the nah and sal operons of the NAH7 plasmid were determined by S1 nuclease mapping and the nucleotide sequence surrounding these transcription start sites was determined . Since expression of both of these operons is coordinately controlled by the product of the transcriptional activator gene nahR, the sequences were compared to locate potential sites involved in common regulation . In the 100-base-pair region preceding transcription start sites of both operons, three regions of extensive homology were found and may be involved in nahR-mediated transcriptional control: between -80 and -60 with 81% homology; between -40 and -28 with 75% homology; between -1 and +15 with 70% homology . Comparison of the promoter sequences of nah and sal with the analogous sequences of the xylABC and xylDEFG operons of the TOL plasmid showed little homology between the 5' regions of these two sets of positively regulated hydrocarbon degradation operons . In addition, the transcription start site of the nahR regulatory gene was located and its promoter sequence was determined . The nahR promoter overlapped at the -35 position of the sal promoter; however, the nahR gene is transcribed in the opposite direction . Sequences similar to the consensus sequences of Escherichia coli promoters (at -35 and -10) were found in nah, sal, and nahR at the appropriate positions.

J Biol Chem, 1985 Dec 25, 260(30), 16122 - 30
The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450; Poulos TL et al.; The crystal structure of Pseudomonas putida cytochrome P-450cam in the ferric, camphor bound form has been determined and partially refined to R = 0.23 at 2.6 A . The single 414 amino acid polypeptide chain (Mr = 45,000) approximates a triangular prism with a maximum dimension of approximately 60 A and a minimum of approximately 30 A . Twelve helical segments (A through L) account for approximately 40% of the structure while antiparallel beta pairs account for only approximately 10% . The unexposed iron protoporphyrin IX is sandwiched between two parallel helices designated the proximal and distal helices . The heme iron atom is pentacoordinate with the axial sulfur ligand provided by Cys 357 which extends from the N-terminal end of the proximal (L) helix . A substrate molecule, 2-bornanone (camphor), is buried in an internal pocket just above the heme distal surface adjacent to the oxygen binding site . The substrate molecule is held in place by a hydrogen bond between the side chain hydroxyl group of Tyr 96 and the camphor carbonyl oxygen atom in addition to complementary hydrophobic contacts between the camphor molecule and neighboring aliphatic and aromatic residues . The camphor is oriented such that the exo-surface of C5 would contact an iron bound, "activated" oxygen atom for stereoselective hydroxylation.

Eur J Biochem, 1985 Dec 2, 153(2), 407 - 12
Protein-protein interactions and antigenic relationships between the components of 4-methoxybenzoate monooxygenase and of benzene 1,2-dioxygenase from Pseudomonas putida; Eich F et al.; The investigations presented in this paper were performed on two enzyme systems from Pseudomonas putida: (a) 4-methoxybenzoate monooxygenase, consisting of a NADH: putidamonooxin oxidoreductase and putidamonooxin, the oxygen-activating component, and (b) benzene 1,2-dioxygenase, a three-component enzyme system with an NADH: ferredoxin oxidoreductase, functioning together with a plant-type ferredoxin as electron-transport chain, and an oxygen-activating component similar to putidamonooxin in its active sites . The influence of temperature, ionic strength, and pH on the activities of 4-methoxybenzoate monooxygenase and of NADH: putidamonooxin oxidoreductase were investigated . The studies revealed that the activity of 4-methoxybenzoate monooxygenase is determined by the behaviour of the reductase . Spectroscopic measurements showed that the interaction between the two components of 4-methoxybenzoate monooxygenase influences the optical-absorption behaviour of one or both components . As a criterion for the affinity between the two components of 4-methoxybenzoate monooxygenase, the Km value of the reductase for putidamonooxin was determined and found to be 31 +/- 11 microM . Antibodies against both components of 4-methoxybenzoate monooxygenase were obtained from rabbits . The antibodies against putidamonooxin inhibited the O-demethylation reaction (up to 80%) and also the reduction of putidamonooxin by the reductase (up to 40%) . The antibodies against putidamonooxin did not interact with the oxygen-activating component of benzene 1,2-dioxygenase . The electron-transport chains of 4-methoxybenzoate monooxygenase and benzene 1,2-dioxygenase could not be replaced by one another without a complete loss of enzyme activity.

J Bacteriol, 1985 Dec, 164(3), 1171 - 81
Scanning electron microscope study of Pseudomonas putida colonies; Shapiro JA; Pseudomonas putida colonies were examined by scanning electron microscope . A variety of cell morphologies, multicellular arrangements, and extracellular materials were observed in the fixed material . Different regions of a single colony showed characteristic organizations of these architectural elements . In some cases, the detailed microstructure of the fixed colony surfaces observed by scanning electron microscopy could be correlated with macroscopic patterns visualized by histochemical staining and surface relief photography of live colonies . Extracellular materials were seen to extend onto the agar surface beyond the boundaries of the cell mass, and the final structures of these materials, after fixation and desiccation, were colony specific . The significance of these features of colony microstructure for formulating hypotheses about the control of colony morphogenesis is discussed.

Appl Environ Microbiol, 1985 Dec, 50(6), 1409 - 13
Use of cloned genes of Pseudomonas TOL plasmid to effect biotransformation of benzoates to cis-dihydrodiols and catechols by Escherichia coli cells; Zeyer J et al.; DNA fragments containing the xylD and xylL genes, which specify the broad-specificity enzymes toluate-1,2-dioxygenase and 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid dehydrogenase, respectively, of TOL plasmid pWW0-161 of Pseudomonas putida have previously been cloned in the pBR322 vector plasmid (P.R . Lehrbach, J . Zeyer, W . Reinecke, H.-J . Knackmuss, and K . N . Timmis, J . Bacteriol . 158:1025-1032, 1984) . In this study, Escherichia coli cells containing hybrid plasmids carrying the cloned xylD or xylDL genes quantitatively transformed 14C-ring- and 14C-carboxy-labeled benzoate to the pathway intermediates 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (cis-dihydrodiol) and catechol, respectively . Like P . putida cells, E . coli cells containing the xylD gene transformed a variety of chloro- and hydrocarbon-substituted benzoates . The toluate-1,2-dioxygenase produced in E . coli thus exhibited the broad-substrate-specificity properties of the enzyme in P . putida . Turnover rates by the enzymes in these two bacteria are compared.

Genetika, 1985 Dec, 21(12), 1937 - 44
{Expression of Pseudomonas aeruginosa phage transposons in Pseudomonas putida PpG1 cells . II . Zygotic induction--a necessary condition for the formation of defective lysogens}; Gorbunova SA et al.; The transfer of hybrid plasmid RP4::PT (where PT is the genome of a transposable phage specific for Pseudomonas aeruginosa) into recipient cells of P . putida strain PpG1 occurs with the same frequency as into P . aeruginosa, the homologous host for PT . Approximately 1/3 of all PpG1 exconjugants carrying RP4 markers lost the capability to produce viable PT phage . In contrast, in a cross with homologous recipient P . aeruginosa all exconjugant clones contained nondefective prophages in the hybrid plasmids . Zygotic induction is an obligatory condition for detection of PpG1 exconjugants with defective phages . The defective prophages in RP4::PT hybrid plasmids have deletions of different size; the other carry mutations indistinguishable from point mutations in an essential phage gene . Some of deletions also cover plasmid genes . At least some of the defective prophages, including deleted ones, have arisen in the recipient cells of P . putida after transfer of the hybrid plasmid.

J Biol Chem, 1985 Nov 15, 260(26), 14035 - 44
{17O}Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase . Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases; Arciero DM et al.; Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates . The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2 . Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes . NO is not displaced by either substrates or inhibitors . The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water . The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands . Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex . Nitric oxide binding is found to be substrate-dependent for each enzyme . Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites . Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM . One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively . The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.

Biochemistry, 1985 Nov 5, 24(23), 6696 - 701
Tyrosine motions in relation to the ferric spin equilibrium of cytochrome P-450cam; Fisher MT et al.; Second derivative spectroscopy was used to determine the percentage of tyrosine residues that are exposed to solvent in cytochrome P-450cam isolated from Pseudomonas putida . The ratio between two peak to trough second derivative absorbance differences has been shown to be dependent on the polarity of the microenvironment surrounding tyrosine residues {Ragone, R., Colonana, G., Balestrieri, C., Servillo, L., & Irace, G . (1984) Biochemistry 23, 1871} . With a number of camphor analogues that independently vary the spin equilibrium of the ferric cytochrome P-450 cam, experiments have demonstrated that the percentage of tyrosine residues exposed to solvent is linearly dependent on the percentage of ferric high-spin species present . This is not simply a function of the extent of substrate binding since in all cases the substrate concentration was sufficient to ensure saturation of the cytochrome . The local microenvironment of approximately one tyrosine residue appears to be linearly correlated with the percentage of ferric high-spin cytochrome . Structural studies of cytochrome P-450cam using small-angle X-ray scattering {Lewis, B . A., & Sligar, S . G . (1983) J . Biol . Chem . 258, 3599} and high-pressure difference spectroscopy {Fisher, M . T., Scarlata, S . F., & Sligar, S . G . (1985) Arch . Biochem . Biophys . 240, 456} imply that global conformational changes linked to the spin equilibria are small . Together with the data reported herein, these results suggest that one tyrosine residue is involved in a conformational change that is directly linked with the spin equilibrium.

Mikrobiologiia, 1985 Nov-Dec, 54(6), 914 - 8
{Catabolism of biphenyl by Pseudomonas putida BS 893 strain containing the biodegradation plasmid pBS241}; Starovoitov II et al.; The isolation and identification of biphenyl catabolism products in Pseudomonas putida BS 893 (pBS241) showed the presence of benzoic, m-hydroxybenzoic and cinnamic acids . The two latter compounds were not found in biphenyl degradation by other bacterial strains . P . putida BS 893 (pBS241) differed from other biphenyl-positive Pseudomonas strains in the enzyme activity . These differences may stem from peculiarities in the pathway of biphenyl catabolism controlled by plasmid pBS241.

Mol Gen Mikrobiol Virusol, 1985 Nov, (11), 11 - 6
{Hybrid plasmid pBS251 containing genes for n-alkane degradation}; Andreeva AL et al.; The strain of Pseudomonas aeruginosa BS316 utilizing H-alkanes of the C6-C12 series (Alk+) harbours the 96 Md plasmid pBS250 . The use of plasmid RP4 to mobilize Alk+ markers in conjugal transfer to Pseudomonas aeruginosa and Pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid RP4) and capable of growth on H-alkanes . A transconjugant from this series harbours plasmid pBS251, a derivative of plasmid RP4 containing the genes for octane and octanol catabolism . A fragment of DNA inserted into RP4 has a mol mass 3.8 Md, possesses two restriction sites for EcoRI, one site for PstI, is not restricted by SmaI and BamHI restriction endonucleases, and is localized in the region 4.5-5.7 Md on the physical map of plasmid RP4.

J Bacteriol, 1985 Nov, 164(2), 887 - 95
Evolutionary conservation of genes coding for meta pathway enzymes within TOL plasmids pWW0 and pWW53; Keil H et al.; Pseudomonas putida MT53 contains a TOL plasmid, pWW53, that encodes toluene-xylene catabolism . pWW53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal TOL plasmid pWW0 . An RP4::pWW53 cointegrate plasmid, pWW53-4, containing about 35 kbp of pWW53 DNA, including the entire catabolic pathway genes, was formed, and a restriction map for KpnI, HindIII, and BamHI was derived . The entire regulated meta pathway genes for the catabolism of m-toluate were cloned into pKT230 from pWW53 on a 17.5-kbp HindIII fragment . The recombinant plasmid supported growth on m-toluate when mobilized into plasmid-free P . putida PaW130 . A restriction map of the insert for 10 restriction enzymes was derived, and the locations of xylD, xylL, xylE, xylG, and xylF were determined by subcloning and assaying for their gene products in both Escherichia coli and P . putida hosts . Good induction of the enzymes by m-toluate and m-methylbenzyl alcohol but not by m-xylene was measured in P . putida, but little or no regulation was found in E . coli . The restriction map and the gene order showed strong similarities with published maps of the DNA encoding both the entire meta pathway operon (xylDLEGFJIH) and the regulatory genes xylS and xylR on the archetype TOL plasmid pWW0, suggesting a high degree of conservation in DNA structure for the catabolic operon on the two different plasmids.

J Bacteriol, 1985 Nov, 164(2), 563 - 70
Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358; Marugg JD et al.; The plant-growth-stimulating Pseudomonas putida WCS358 was mutagenized with transposon Tn5 . The resulting mutant colony bank was screened for mutants defective in the biosynthesis of the fluorescent siderophore . A total of 28 mutants, divided into six different classes, were isolated that were nonfluorescent or defective in iron acquisition or both . These different types of mutants together with the probable overall structure of the siderophore, i.e., a small peptide chain attached to a fluorescing group, suggest a biosynthetic pathway in which the synthesis of the fluorescing group is preceded by the synthesis of the peptide part . A gene colony bank of P . putida WCS358 was constructed with the broad-host-range cosmid vector pLAFR1 . This genomic library, established in Escherichia coli, was mobilized into the 28 individual mutants, screening for transconjugants restored in fluorescence or growth under iron-limiting conditions or both . A total of 13 cosmids were found to complement 13 distinct mutants . The complementation analysis revealed that at least five gene clusters, with a minimum of seven genes, are needed for siderophore biosynthesis . Some of these genes seem to be arranged in an operon-like structure.

Mol Biol Evol, 1985 Nov, 2(6), 557 - 67
Dehalogenase genes of Pseudomonas putida PP3 on chromosomally located transposable elements; Slater JH et al.; Pseudomonas putida PP3 utilizes halogenated alkanoic acids (HAA) such as 2,2-DCPA as its sole carbon and energy sources . Spontaneous HHA- mutants, isolated by selection for resistance to the toxic analogs monochloroacetic acid and dichloroacetic acid, arose at frequencies several orders of magnitude higher than expected for spontaneous mutations . Analysis of the five classes of mutants isolated suggested that the dehalogenase and HAA permease genes were on chromosomally located transposable elements and that the spontaneous mutations involved excision of these elements . This suggestion was confirmed by the observation that one of the elements can transpose to a target DNA molecule . The frequency of the excision event was strongly influenced by environmental conditions . Possible relationships between expression of cryptic genes and their location on transposable elements are discussed.

Biochem J, 1985 Oct 15, 231(2), 383 - 7
Formation and properties of flavoprotein-cytochrome hybrids by recombination of subunits from different species; Koerber SC et al.; p-Cresol methylhydroxylases from four different pseudomonads differ in their isoelectric points and, to a lesser extent, in Mr values and substrate specificity . The enzymes from three species were isolated in homogeneous form, then resolved into their flavoprotein and cytochrome subunits, and the subunits were recombined to yield the nine possible hybrids (i.e . three intraspecies and six interspecies) . The resulting flavocytochromes showed extensive similarities in steady-state kinetic parameters and in the dissociation constants of their subunits . Evidence is also presented that a fourth type of p-cresol methylhydroxylase, from Pseudomonas putida (N.C.I.B . 9869, form 'B'), the subunits of which cannot be isolated by the isoelectric focusing technique used to separate the subunits of the other flavocytochromes, nevertheless dissociates slowly at high dilution . The dissociation is reflected by a decline of catalytic activity with time . This process for the 'B' enzyme is prevented by the presence of substrate or an excess of a cytochrome subunit isolated from another enzyme species . Incubation of the dissociated subunits with p-cresol brings about extensive, albeit incomplete, re-association and regeneration of activity.

J Biol Chem, 1985 Oct 5, 260(22), 12190 - 3
Activation of urocanase from Pseudomonas putida by electronically excited triplet species; Venema RC et al.; Urocanase from Pseudomonas putida becomes inactive in growing and resting cells and, as shown previously, is activated by the direct absorption of ultraviolet light . In this study, we describe the activation of urocanase by energy transfer from triplet indole-3-aldehyde, generated in the peroxidase-catalyzed aerobic oxidation of indole-3-acetic acid . The activation was time-, temperature-, and pH-dependent . The involvement of reactive oxygen intermediates was excluded by the lack of effect of appropriate quenchers and traps . Triplet quenchers, in contrast, reduced the level of activation . Photoexcited rose bengal, a triplet species of a different nature and origin, was also effective in promoting activation . These results demonstrate a potential mechanism of urocanase regulation not dependent on an environmental source of light, but rather brought about by an enzymically generated excited species.

Biol Chem Hoppe Seyler, 1985 Oct, 366(10), 945 - 51
Degradation of lawsone by Pseudomonas putida L2; Wessendorf J et al.; From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon . This bacterium is capable of utilizing lawsone as sole source of carbon and energy . Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida . The organism is referred to as Pseudomonas putida L2 . The degradation of lawsone by Pseudomonas putida L2 was investigated . Salicylic acid and catechol were isolated and identified as metabolites . In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase . Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway . Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays . Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially . The enzymes of the beta-ketoadipate pathway are also inducible . Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells . This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.

Biochemistry, 1985 Sep 10, 24(19), 5276 - 80
Resolution of the flavocytochrome p-cresol methylhydroxylase into subunits and reconstitution of the enzyme; Koerber SC et al.; An improved procedure is described for the isolation of the flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida as well as methods for the separation of its subunits in native form and their recombination to reconstitute the original flavocytochrome . Under appropriate conditions, the reconstitution is stoichiometric and results in complete recovery of the catalytic activity of the flavocytochrome . The separated flavoprotein subunit shows only 2% of the catalytic activity of the original enzyme on p-cresol and is characterized by converging lines in bisubstrate kinetic analysis, while the intact and reconstituted enzymes show parallel line kinetics in steady-state experiments . van't Hoff plots of the dependence of the dissociation constant of the subunits of PCMH on temperature show a break near 15 degrees C . Above this temperature, KD is characterized by a positive delta H value of 12.6 kcal mol-1; below 15 degrees C, the dissociation is essentially temperature independent . The subunit dissociation is strongly dependent on ionic strength in the oxidized form of PCMH but not in the reduced form of the enzyme . Reduction also lowers the KD significantly, while substrates and nonoxidizable competitive inhibitors lower the dissociation constant even further, suggesting a conformation change . Combination of the subunits to form PCMH entails a small but measurable change in the absorption spectra of the component proteins.

FEBS Lett, 1985 Sep 2, 188(2), 295 - 301
Expression of plasmid encoded Escherichia coli 5S ribosomal ribonucleic acid in Pseudomonas putida; Hartmann RK et al.; The recombinant plasmid pNRK 36, which represents the plasmids RSF 1010, a small multicopy plasmid of the incompatibility group IncQ that confers resistance to streptomycin and sulfoamide to its host cells, and pKK 223-3, which contains the try-lac (tac) promoter followed by a polylinker and a DNA segment containing the 5S rRNA (rrn B) with the ribosomal RNA transcription terminators, was employed to transform Pseudomonas putida 2440 cells . The plasmid encoded 5 S rRNA from Escherichia coli was transcribed and processed properly in P . putida cells thus demonstrating for the first time the expression of a plasmid encoded ribosomal RNA in a heterologous system . The fact that the E . coli 5 S rRNA was not incorporated into assembled ribosomes suggests that the in vivo incorporation of 5 S rRNA into the 50 S ribosomal subunit is closely linked to 23 S rRNA and/or ribosomal protein synthesis.

Can J Microbiol, 1985 Sep, 31(9), 763 - 6
Effect of carbon dioxide on growth of Pseudomonas putida ATCC 11172 on asparagine, citrate, glucose, and lactate in batch and continuous culture; Molin G; The growth of Pseudomonas putida ATCC 11172 on L-asparagine, citrate, D-glucose, and L-lactate was followed in air and in 40% CO2 + air, using batch and carbon-limited continuous cultures . Batch cultures in air utilized a mixture of the carbon sources simultaneously . However, a change to 40% CO2 favoured the utilization of glucose . The maximum specific growth rate (mumax) in air was about 0.3 h-1 on glucose and 0.6 h-1 on the other carbon sources . In CO2, the mumax for glucose was reduced by 16% compared with almost 60-70% for the others . An order of preference for the different carbon sources in continuous cultures was determined by comparing the dilution rates at which the different carbon sources started to appear in the effluent . Glucose was the first compound to appear as the dilution rate increased (lowest preference when grown in air) . In 40% CO2, the mumax for glucose was slightly higher than the others and the recorded preference for glucose in continuous culture was equal to that for citrate but was somewhat lower than that of lactate and asparagine . D-Gluconate and glucono-delta-lactone were produced as a step in the utilization of glucose . The D-gluconate production was enhanced by CO2.

Genetika, 1985 Sep, 21(9), 1455 - 63
{Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells . I . The establishment of a lysogenic state and the effectiveness of lytic development}; Gorbunova SA et al.; Expression of transposable phages (TP) of Pseudomonas aeruginosa in the cells of P . putida was studied . The high efficiency of phage lytic development was shown both as a consequence of zygotic induction after transfer of the RP4::TPc+ plasmid into nonlysogenic recipients, and as a result of heat induction of lysogens PpG1 (D3112cts15) . The high phage yield (20-25 particles of D3112cts phage per one cell of P . putida) is an evidence for a high level of transposition in the cells of this bacterial species . Plasmids RP4::TP are transferred into cells of PpG1 and PAO1 with similar frequency . However, the efficiency of establishment of the lysogenic state is lower in PpG1 . Transposable phages of P . aeruginosa can integrate into the chromosome of PpG1 producing stable inducible lysogens . The presence of RP4 in the P . putida cells is not necessary for expression of transposable phages . The transposable phage D3112cts15 can be used in experiments of interspecies transduction of plasmids and chromosomal genes.

J Bacteriol, 1985 Sep, 163(3), 863 - 9
Determination of the transcription initiation site and identification of the protein product of the regulatory gene xylR for xyl operons on the TOL plasmid; Inouye S et al.; The xylR gene is a regulatory gene on the TOL plasmid, which acts in a positive manner on xyl operons for degradation of toluene and xylenes in Pseudomonas putida . A DNA fragment containing the xylR promoter region was cloned on promoter-probing vectors, and its nucleotide sequence was determined . The transcription initiation site of the xylR gene was determined in cells of P . putida and Escherichia coli by S1 nuclease and reverse transcriptase mapping . Two initiation sites were detected which were identical in both P . putida and E . coli . The amounts of mRNA synthesized in both bacterial cells were almost the same and independent of the inducers for xyl operons . The consensus sequences for E . coli promoters were found in the region preceding the respective transcription initiation sites . The product of the xylR gene was identified by the maxicell system as a protein with an approximate molecular weight of 67,000.

J Bacteriol, 1985 Aug, 163(2), 600 - 9
Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40; Woolfolk CA; The isolation of a xanthine dehydrogenase from Pseudomonas putida 40 which utilizes ferricyanide as an electron acceptor at high efficiency is presented . The new activity is separate from the NAD+ and oxygen-utilizing activities of the same organism but displays a broad pattern for reducing substrates typical of those of previously studied xanthine-oxidizing enzymes . Unlike the previously studied enzymes, the new enzyme appears to lack flavin but possess heme and is resistant to cyanide treatment . However, sensitivity of the purified enzyme to methanol and the selective elimination of the activity when tungstate is added to certain growth media suggest a role for molybdenum . The enzyme is subject to a selective proteolytic action during processing which is not accompanied by denaturation or loss of activity and which is minimized by the continuous exposure of the activity to EDTA and phenylmethylsulfonyl fluoride . Electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate suggests that the enzyme is constructed of subunits with a molecular weight of approximately 72,000 . Electrophoresis under native conditions of a purified enzyme previously exposed to magnesium ion reveals a series of major and minor activity bands which display some selectivity toward both electron donors and acceptors . An analysis of the effect of gel concentration on this pattern suggests that the enzyme forms a series of charge and size isomers with a pair of trimeric forms predominating . Comparison of the rate of sedimentation of the enzyme in sucrose gradients with its elution profile from standardized Sepharose 6B columns suggests a molecular weight of 255,000 for the major form of the native enzyme.

J Antimicrob Chemother, 1985 Aug, 16(2), 157 - 63
The kinetics of dihydrostreptomycin uptake in Pseudomonas putida membrane vesicles: absence of inhibition by cations; Thomson TB et al.; Dihydrostreptomycin was taken up in isolated cytoplasmic membrane vesicles of Pseudomonas putida by an active transport mechanism . Saturation kinetics were observed with an apparent Km and Vmax of 15 mM and 50 nmol/min/mg of protein respectively . The evidence suggested that the observed kinetics was that of the energy-dependent phase I component of dihydrostreptomycin uptake . Neither magnesium nor the polyamine, spermine, inhibited dihydrostreptomycin transport . Thus, the inhibition of aminoglycoside uptake in intact cells of Gram-negative bacteria and the increase in the minimal inhibitory concentration in the presence of multivalent cations and polyamines were interpreted to be effects that take place at the outer membrane level.

FEBS Lett, 1985 Jul 22, 187(1), 21 - 4
The site of cyclic AMP-dependent protein kinase catalyzed phosphorylation of cytochrome P-450 LM2; Muller R et al.; The phenobarbital-inducible form of cytochrome P-450 purified from rabbit liver microsomes is phosphorylated by cAMP-dependent protein kinase at a single site, the serine residue in position 128 of the amino acid sequence . The serine is located in a characteristic recognition sequence for cAMP-dependent protein kinase and is part of a primary structure which is conserved during evolution, present also in phenobarbital-inducible rat cytochrome and cytochrome P-450 CAM from Pseudomonas putida . The contribution of these findings to our understanding of the structure and membrane topology of cytochrome P-450 LM2 and its turnover regulated by phosphorylation is discussed.

J Mol Biol, 1985 Jul 20, 184(2), 311 - 7
Low resolution crystal structure of muconolactone isomerase . A decamer with a 5-fold symmetry axis; Katz BA et al.; Muconolactone isomerase from Pseudomonas putida crystallizes from sodium sulfate solution in space group P2(1) (a = 65.84 A, b = 105.70 A, c = 77.20 A, beta = 90.5 degrees) with ten 11,000 Mr subunits per asymmetric unit . The 7 A resolution crystal structure was solved by single isomorphous replacement followed by 10-fold symmetry averaging . The decameric enzyme has an uncommon non-crystallographic 5-fold symmetry axis and a large cavity in its center.

Biochim Biophys Acta, 1985 Jul 18, 830(1), 101 - 4
Evidence against a temperature-dependent conformational change in urocanase from Pseudomonas putida; Hug DH et al.; Enzymatic activity of urocanase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) has an unusual resistance to temperature changes, and a temperature-dependent conformational change has been suggested (Hug, D.H . and Hunter, J.K . (1974) Biochemistry 13, 1427-1431) . A conformational change or dissociation has been proposed in the range of 29-31 degrees C (Cohn, M.S., Lynch, M.C . and Phillips, A.T . (1975) Biochim . Biophys . Acta 377, 444-453) . In this work, no evidence was found for a temperature-dependent conformational change or dissociation . Arrhenius plots of Km and Vmax were linear; the sedimentation coefficient was independent of temperature; tryptophanyl fluorescence was a linear function of temperature; and heat capacity calorimetry showed no transitions below 60 degrees C.

Biochem Biophys Res Commun, 1985 Jul 16, 130(1), 412 - 7
P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli; Koga H et al.; The gene camC, which encodes the cytochrome P450 monoxygenase protein, was cloned into the shuttle vector pKT240 and recovered as the recombinant pKG201 with a 2.3 kb insert from the CAM plasmid in the PstI site . The gene product is expressed constitutively in P . putida and in E . coli whereas the inverted insert clone lacks expression, indicating absence of an insert promoter.

Mikrobiologiia, 1985 Jul-Aug, 54(4), 679 - 81
{Glucose consumption and dehydrogenase activity of the cells of the arsenite-oxidizing bacterium Pseudomonas putida}; Abdrashitova SA et al.; The rates of glucose assimilation and dehydrogenase activity were studied in Pseudomonas putida oxidizing arsenite . The rate of glucose utilization by the cells decreased in the presence of arsenites in the medium at the beginning because of the microbial adaptation to arsenite . The activity of dehydrogenase fell down when the cells were cultivated in the medium with arsenite . An inverse correlation existed between the rate of glucose assimilation and arsenite oxidation . Apparently, arsenites were oxidized under the action of metabolites produced by P . putida in the process of its heterotrophous growth.

Mikrobiologiia, 1985 Jul-Aug, 54(4), 610 - 5
{Stability of the NPL-1 and NPL-41 plasmids of naphthalene biodegradation in Pseudomonas putida populations in continuous culture}; Boronin AM et al.; The stability of biodegradation plasmids NPL-1 and NPL-41, which control the synthesis of enzymes for naphthalene oxidation to salicylate, was studied in Pseudomonas putida BSA under the conditions of its continuous cultivation with limitation in glucose or salicylate in the chemostat regime and without limitation in the pH-stat regime . Plasmid NPL-1, which controls the inducible synthesis of naphthalene oxygenase, is stable in the population of P . putida cells under the conditions of continuous cultivation on glucose, but is not stable in the course of cultivation on salicylate, an inductor of the naphthalene oxygenase synthesis . Plasmid NPL-41, which controls the constitutive synthesis of naphthalene oxygenase, is not stable in the population of P . putida cells under the conditions of continuous cultivation on glucose . The operation of genes, which control the oxidation of naphthalene to salicylate (nah), makes plasmids NPL-1 and NPL-41 unstable under the conditions of continuous cultivation in the absence of naphthalene from the medium, i.e . under the conditions when the expression of these genes is not necessary . In that case, cells containing plasmids with a deletion of nah-genes as well as cells without plasmids appear in the population of P . putida, which causes a decline in its futile energy and metabolic processes.

Biol Chem Hoppe Seyler, 1985 Jul, 366(7), 637 - 46
Purification and some properties of two isofunctional juglone hydroxylases from Pseudomonas putida J1; Rettenmaier H et al.; Juglone-induced cells of Pseudomonas putida J 1 were shown to contain two isofunctional juglone hydroxylases . Both enzymes were purified about 125-fold to homogeneity in polyacrylamide gel electrophoresis . The molecular masses of the native enzymes, as determined by Sephacryl S-200 gel filtration were 59 000 Da for enzyme 1 and 56 000 Da for enzyme 2 . The molecular masses of the subunits were determined by dodecyl sulfate polyacrylamide gel electrophoresis as 25 000 Da (enzyme 1) and 23 500 Da (enzyme 2) . Both enzymes hydroxylated juglone, naphthazarin, 1,4-naphthoquinone and 2-chloro-1,4-naphthoquinone, but they were completely inactive against naphtholes . The activity of both hydroxylases was not affected by chelating agents, thiol reagents, however, were found to be strong inhibitors . No external cofactors such as Fe2, NADH, NADPH, FAD, FMN were required for activity . Concomitant with the hydroxylation of juglone the consumption of oxygen in a molar ratio 2: 1 (juglone: oxygen) was observed but none of the enzymes incorporated 18O2 into the substrate juglone . By activity staining enzyme 1 was found to be present in induced and non-induced cells of P . putida J 1, enzyme 2, however, only in juglone-induced cells.

J Bacteriol, 1985 Jul, 163(1), 248 - 55
TOL plasmid pWW15 contains two nonhomologous, independently regulated catechol 2,3-oxygenase genes; Keil H et al.; Pseudomonas putida MT15 contains a 250-kilobase-pair (kbp) TOL plasmid pWW15, encoding toluene and xylene catabolism, which undergoes large spontaneous deletions to give two classes of mutants with altered catabolic phenotypes (H . Keil and P . A . Williams, J . Gen . Microbiol, 131:1023-1033, 1985) . Two structural genes for catechol 2,3-oxygenase (C23O) were cloned from pWW15 . The gene for C23OI was located on the 2.1-kbp XhoI fragment Xh, whereas that for C23OII was found on the 11.5-kbp BamHI fragment BJ . The two restriction fragments and the subcloned regions of them showed no similarity in the pattern of restriction digestion, nor did they hybridize with each other . The substrate specificities of the two enzymes were also substantially different . The two structural genes were separated on pWW15 by about 100 kbp . In plasmid pWW15-510 of a B5 mutant, the 90-kbp deletion in the plasmid removed most of the intervening DNA, but it also deleted 80% of the gene for C23OI from its 3' end . Thus, only C23OII was expressed in the host MT15-510 . Conversely, in RP4::pWW15 cointegrate plasmid pWW15-1003, only the C23OI gene was present . The expression of C23O activity from these two derivative plasmids and from the wild-type pWW15 showed that only C23OI was induced by growth in the presence of m-toluate, whereas both activities were induced in the only C23OI was induced by growth in the presence of m-toluate, whereas both activities were induced in the presence of m-xylene . These findings cast doubt on the earlier hypothesis that the deletions in B3 and B5 mutants remove a regulatory gene by which m-toluate induces the enzymes necessary for its own catabolism.

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