Gene, 1993 Aug 16, 130(1), 41 - 6 Engineering of alkyl- and haloaromatic-responsive gene expression with mini-transposons containing regulated promoters of biodegradative pathways of Pseudomonas; de Lorenzo V et al.; Four recombinant mini-Tn5 transposons are described which contain outward-facing Pm, Pu or Psal promoters from the catabolic plasmids TOL and NAH of Pseudomonas putida, along with their cognate wild-type regulatory genes (xylS, xylR, nahR) or mutant varieties (xylS2) . Transcription from such promoters is activated when the host bacteria encounters certain aromatic compounds, such as alkyl- and halobenzoates (XylS, XylS2), alkyl- and halotoluenes (XylR) or salicylates (NahR) . These transposons enable the generation of conditional phenotypes dependent on the presence of specific effectors, as well as the engineering of strains expressing heterologous genes that are regulated by aromatic inducers . A mini-Tn5 xylS/Pm::luxAB, was used to construct Pseudomonas strains emitting light upon exposure to concentrations of m-toluate as low as 5-10 microM . The broad-host-range transposition system of Tn5 and the stability of the inserted genes due to the loss of the transposase-encoding gene during delivery of the mobile element make these transposons particularly well suited for the construction of stable strains exhibiting halo/alkyl aromatic-regulated conditional phenotypes in the absence of antibiotic selection, as is required for some uncontained bioremediation and biomonitoring applications. Gene, 1993 Aug 16, 130(1), 33 - 9 The Pseudomonas putida ML2 plasmid-encoded genes for benzene dioxygenase are unusual in codon usage and low in G+C content; Tan HM et al.; Benzene dioxygenase, catalyzing the oxidation of benzene to cis-1,2-dihydroxy-cyclohexa-3,5-diene, comprises four polypeptides that are encoded by plasmid pHMT112 of Pseudomonas putida ML2 . In this study, the nucleotide (nt) sequences of four genes encoding this enzyme (bedC1C2BA) were determined, and the amino acid (aa) sequences were deduced . The sequence showed significant homology with the chromosomally encoded benzene dioxygenase and toluene dioxygenase genes (73-77% for nt and 83-99% for aa), but not the plasmid-encoded naphthalene dioxygenase genes (20-26% for nt and 32-36% for aa) . A conserved motif (Cys-Xaa-His-15-to-17 aa-Cys-Xaa2-His, where Xaa is any aa), proposed to bind the Rieske-type {2Fe-2S} cluster, was identified in the deduced aa sequence of the iron-sulfur proteins . Three regions were also identified in the flavoprotein which are likely to be involved in FAD and NAD+ binding . The gene order of bedC1C2BA is consistent with most ring-hydroxylating dioxygenases isolated from Pseudomonas . However, the G+C content of 47% is in contrast to the high G+C content of the Pseudomonas chromosome (63%) and other Pseudomonas plasmids (57%), and with its unique codon usage preference this suggests that bedC1C2BA originated from a host derived from a different genus. J Bacteriol, 1993 Aug, 175(16), 5224 - 32 Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon); Furukawa K et al.; bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P . putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G . J . Zylstra and D . T . Gibson, J . Biol . Chem . 264:14940-14946, 1989; K . Taira, J . Hirose, S . Hayashida, and K . Furukawa, J . Biol . Chem . 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism . The gene components responsible for substrate specificity between the bph and tod operons were investigated . The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results . (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P . pseudoalcaligenes KF707 and P . putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway . Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P . putida F1 permitted growth on biphenyl . (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another . (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4. EMBO J, 1993 Aug, 12(8), 3339 - 47 In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways; Polissi A et al.; The meta-cleavage operon of the TOL plasmid pWW0 of Pseudomonas putida contains 13 genes responsible for the oxidation of benzoate and toluates to Krebs cycle intermediates via estradiol (meta) cleavage of (methyl)catechol . The functions of all the genes are known with the exception of xylT . We constructed pWW0 mutants defective in the xylT gene, and found that these mutants were not able to grow on p-toluate while they were still capable of growing on benzoate and m-toluate . In the xylT mutants, all the meta-cleavage enzymes were induced by p-toluate with the exception of catechol 2,3-dioxygenase whose activity was 1% of the p-toluate-induced activity in wild-type cells . Addition of 4-methylcatechol to m-toluate-grown wild-type and xylT cells resulted in the inactivation of catechol 2,3-dioxygenase in these cells . In the wild-type strain but not in the xylT mutant, the catechol 2,3-dioxygenase activity was regenerated in a short time . The regeneration of the catechol 2,3-dioxygenase activity was also observed in H2O2-treated wild-type cells, but not in H2O2-treated xylT cells . We concluded that the xylT product is required for the regeneration of catechol 2,3-dioxygenase. J Bacteriol, 1993 Aug, 175(15), 4597 - 604 Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa; Poole K et al.; Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyroverdine . By using a phagemid-based in vivo cloning system, attempts were made to clone the receptor gene by complementing this growth defect . Several recombinant phagemids carrying P . aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyroverdine receptor in strain K437 were isolated . These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the receptor in K437 . Nucleotide sequencing of the SphI fragment revealed a single large open reading frame, designated fpvA (ferripyoverdine uptake), of 2439 bp . The predicted translation product of fpvA has a molecular mass of 89,395 Da . N-terminal amino acid sequence analysis of the purified ferripyoverdine receptor confirmed fpvA as the receptor gene . Moreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein . The deduced FpvA polypeptide exhibited homology to regions shown to be conserved in TonB-dependent receptor proteins . FpvA also shared strong homology (41.3% identity) with the PupA protein of Pseudomonas putida WCS358 . This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very similar to pyoverdine. Can J Microbiol, 1993 Aug, 39(8), 787 - 94 Characterization of cell surface properties in agglutinable and nonagglutinable mutants of Pseudomonas putida; Buell CR et al.; Cells of an aggressive, root-colonizing isolate of Pseudomonas putida are agglutinated by a root surface glycoprotein . The agglutination phenotype in P . putida isolate Corvallis is lacking in mutants (Agg-) derived by Tn5 insertion and chemical mutagenesis . Specific mutation in the aggA locus by Tn5 insertion results in loss of agglutinability that is complemented in trans by a wild-type copy of the P . putida aggA locus . We examined the biochemical bases of agglutination in P . putida by comparing cell surface features in Agg+, Agg- mutants, and a genetically restored aggA mutant . No changes in gross cell surface features involving hydrophobic or hydrophilic binding or net negative charge were observed . Three macromolecular features, pili, flagella, and lipopolysaccharide size, did not differ between Agg+ and Agg- mutants . Protein profiles of cell envelope, periplasmic, and outer membrane preparations revealed pleiotropic effects of mutation in agglutination phenotype including alterations of an outer membrane protein of 47,000 molecular weight and periplasmic proteins of 56,000 and 60,000 molecular weight . The protein alterations seen in the aggA::Tn5 Agg- mutant 5123 reverted to wild-type patterns upon introduction of a wild-type copy of the aggA locus . These data suggest agglutinability may be conditioned by more than one proteinaceous component associated with the bacterial envelope layers. Biosci Biotechnol Biochem, 1993 Aug, 57(8), 1264 - 9 Survival and impact of genetically engineered Pseudomonas putida harboring mercury resistance gene in aquatic microcosms; Iwasaki K et al.; The survival of wild-type and genetically engineered Pseudomonas putida PpY101 that contained a recombinant plasmid pSR134 conferring mercury resistance were monitored in aquatic microcosms . We used lake, river, and spring water samples . The density of genetically engineered and wild-type P . putida decreased rapidly within 5 days (population change rate k -0.87 approximately -1.00 day-1), then moderately after 5 to 28 days (-0.10 approximately -0.14 day-1) . The population change rates of genetically engineered and wild-type P . putida were not significantly different . We studied the important factors affecting the survival of genetically engineered and wild-type P . putida introduced in aquatic microcosms . Visible light exerted an adverse effect on the survival of the two strains . The densities of genetically engineered and wild-type P . putida were almost constant until 7 days after inoculation in natural water filtered with a 0.45-micron membrane filter, or treated with cycloheximide to inhibit the growth of protozoa . These results suggested that protozoan predation was one of the most important factors for the survival of two strains . We examined the impact of the addition of genetically engineered and wild-type P . putida on indigenous bacteria and protozoa . Inoculation of genetically engineered or wild-type P . putida had no apparent effect on the density of indigenous bacteria . The density of protozoa increased in microcosms inoculated with genetically engineered or wild-type P . putida at 3 days after inoculation, but after 5 to 21 days, the density of protozoa decreased to the same level as the control microcosms. Biochim Biophys Acta, 1993 Jul 18, 1174(1), 91 - 4 Complete nucleotide sequence of the 5-exo-hydroxycamphor dehydrogenase gene on the CAM plasmid of Pseudomonas putida (ATCC 17453); Aramaki H et al.; We determined the complete nucleotide sequence of the first gene of Pseudomonas putida cytochrome P-450cam hydroxylase operon, camD, which encodes 5-exo-hydroxycamphor dehydrogenase . This dehydrogenase apparently consists of 361 amino acids and has a molecular mass of 38.4 kDa . Structural relationships to other zinc-containing alcohol dehydrogenases also became evident. Appl Environ Microbiol, 1993 Jul, 59(7), 2139 - 44 Hydroxylation and biodegradation of 6-methylquinoline by pseudomonads in aqueous and nonaqueous immobilized-cell bioreactors; Rothenburger S et al.; Selective culturing of pseudomonads that could degrade quinoline led to enrichment cultures and pure cultures with expanded substrate utilization and transformation capabilities for substituted quinolines in immobilized and batch cultures . Immobilized cells of the pseudomonad cultures rapidly transformed quinolines to hydroxyquinolines in bioreactors and were able to tolerate higher substrate concentrations compared with batch cultures . After prolonged incubation on a mixture of quinoline and 6-methylquinoline, a quinoline-degrading culture of Pseudomonas putida developed the ability to biodegrade 6-methylquinoline, which initially was resistant to microbial attack, as a sole source of carbon and energy . 6-Methylquinoline was also degraded in a nonaqueous solution by this strain of P . putida when a solution of 6-methylquinoline in decane was flowed through an immobilized-cell fixed-bed bioreactor. J Bacteriol, 1993 Jul, 175(13), 4213 - 7 A new amino acid racemase with threonine alpha-epimerase activity from Pseudomonas putida: purification and characterization; Lim YH et al.; We have found that Pseudomonas putida ATCC 17642 cells grown in a medium containing D-threonine as the sole nitrogen source produce an enzyme that catalyzes epimerization of threonine . Proton nuclear magnetic resonance analysis of the enzyme reaction in deuterium oxide clearly showed epimerization from L- to D-allo-threonine and also from D- to L-allo-threonine . This is the first example of an enzyme that was clearly shown to epimerize threonine . The enzyme has been purified to homogeneity, which was shown by polyacrylamide gel electrophoresis . The enzyme has a molecular weight of about 82,000 and consists of two subunits identical in molecular weight (about 41,000) . The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of subunit as a cofactor, and its absorption spectrum exhibits absorption maxima at 280 and 420 nm . The enzyme catalyzes not only epimerization of threonine by stereoconversion at the alpha position but also racemization of various amino acids, except acidic and aromatic amino acids . The enzyme is similar to amino acid racemase with low substrate specificity (EC 5.1.1.10) in enzymological properties but is distinct from it in the action on threonine. J Bacteriol, 1993 Jul, 175(13), 3934 - 40 The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli; Madhusudhan KT et al.; Branched-chain keto acid dehydrogenase is a multienzyme complex which is required for the metabolism of the branched-chain amino acids in Pseudomonas putida . The structural genes encoding all four proteins of the bkd operon have been cloned, and their nucleotide sequences have been determined (G . Burns, K . T . Madhusudhan, K . Hatter, and J . R . Sokatch, p . 177-184 in S . Silver, A . M . Chakrabarty, B . Iglewski, and S . Kaplan {ed.}, Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology, American Society for Microbiology, Washington D.C., 1990) . An open reading frame which encoded a protein with 36.5% amino acid identity to the leucine-responsive regulatory protein (Lrp) of Escherichia coli was found immediately upstream of the bkd operon . Chromosomal mutations affecting this gene, named bkdR, resulted in a loss of ability to use branched-chain amino acids as carbon and energy sources and failure to produce branched-chain keto acid dehydrogenase . These mutations were complemented in trans by plasmids which contained intact bkdR . Mutations affecting bkdR did not have any effect on transport of branched-chain amino acids or transamination . Therefore, the bkdR gene product must affect expression of the bkd operon and regulation must be positive . Mutations affecting bkdR could also be complemented by plasmids containing lrp of E . coli . This is the first instance of a Lrp-like protein demonstrated to regulate expression of an operon outside of E . coli. Biol Chem Hoppe Seyler, 1993 Jul, 374(7), 479 - 88 Microbial metabolism of quinoline and related compounds . XIX . Degradation of 4-methylquinoline and quinoline by Pseudomonas putida K1; Ruger A et al.; A bacterial strain, designated K1, which utilizes 4-methylquinoline and quinoline as sole source of carbon, nitrogen and energy was isolated from soil . Based on its morphological and physiological characteristics, it was classified as Pseudomonas putida biovar B . Four metabolites of 4-methylquinoline degradation were isolated from the culture supernatant and identified as 4-methyl-2-oxo-1,2-dihydroquinoline, 8-hydroxy-4-methyl-2-oxo-1,2-dihydroquinoline, 7,8-dihydroxy-4-methyl-2-oxo-1,2-dihydroquinoline and 6-hydroxy-5-(2-carboxyethenyl)-4-methyl-1H-2-pyridone . Formation of the latter compound is suggested to proceed by decarbonylation of a putative meta-cleavage product of the 7,8-dihydroxy derivative . During growth on quinoline four compounds were released into the culture fluid, too . Upon isolation they were identified as 2-oxo-1,2-dihydroquinoline, 6-hydroxy-2-oxo-1,2-dihydroquinoline, 5-hydroxy-6-(3-carboxy-3-oxopropenyl)-1H-2-pyridone and 2H-pyran-2-on-{3,2b}-5H-6-pyridone . Thus it is proved, that Pseudomonas putida possesses two different catabolic pathways for various quinoline derivatives, which are induced selectively depending on the growth substrate. Biol Chem Hoppe Seyler, 1993 Jul, 374(7), 427 - 34 Intrinsic stability and extrinsic stabilization of creatinase from Pseudomonas putida; Schumann J et al.; Creatinase (creatine amidinohydrolase, EC 3.5.3.3), a homodimer of 45 kDa subunit molecular mass, shows only limited functional stability, and is inaccessible to reconstitution after preceding deactivation, denaturation and dissociation . The enzyme has been characterized regarding its native and denatured states . Studying its unfolding characteristics in the presence of "extrinsic factors", such as DTE, BSA and glycerol, it was possible to define solvent conditions where the stability of the enzyme is significantly improved . Apart from protecting essential thiol groups and charge screening effects, the stabilization is caused mainly by preferential solvation . In the presence of 20% (w/v) glycerol, the kinetic analysis of the time course of denaturation indicates that a partially active folding intermediate, rather than the whole molecule, is involved in the stabilization . The mixed solvent improves the thermal stability, as well as the stability toward GdmCl and urea. Appl Environ Microbiol, 1993 Jul, 59(7), 2166 - 70 Biological synthesis of the analgesic hydromorphone, an intermediate in the metabolism of morphine, by Pseudomonas putida M10; Hailes AM et al.; The morphine alkaloid hydromorphone (dihydromorphinone) was identified as an intermediary metabolite in the degradation of morphine by Pseudomonas putida M10 . A constitutive NADH-dependent morphinone reductase capable of catalyzing the reduction of the 7,8-unsaturated bond of morphinone and codeinone, yielding hydromorphone and hydrocodone, respectively, was shown to be present in cell extracts . The structures have been identified by 1H nuclear magnetic resonance and mass spectrometry . Morphinone reductase has been partially purified by anion-exchange and gel filtration chromatography . This enzyme has potential applications as a biocatalyst for the synthesis of the highly potent analgesic hydromorphone and the antitussive hydrocodone. FEMS Microbiol Lett, 1993 Jun 1, 110(1), 65 - 70 Analysis of the DNA damage-mediated induction of Pseudomonas putida and Pseudomonas aeruginosa lexA genes; Calero S et al.; A fusion between the lexA gene of Pseudomonas aeruginosa and Pseudomonas putida and the lacZ gene was constructed in vitro and cloned in a mini-Tn5 transposon derivative to obtain chromosomal insertions which enable to quantitatively examine their transcriptional regulation in both Pseudomonas and E . coli . Analysis of DNA damage-mediated induction of these lexA-lacZ fusions showed that expression of P . putida and P . aeruginosa lexA genes was always higher and earlier than the expression of the lexA gene of E . coli . Furthermore, and in contrast to the lexA gene fusion of E . coli, the rates and extent of the induction of lexA gene fusion of P . putida and P . aeruginosa were largely independent of the UV doses applied . The behaviour of the lexA-lacZ fusions of two Pseudomonas species was the same regardless of whether they were inserted into their own chromosome or into E . coli. J Biol Chem, 1993 May 25, 268(15), 10842 - 50 Kinetic studies on benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO . A member of the zinc-containing long chain alcohol dehydrogenase family; Shaw JP et al.; The nucleotide sequence of the structural gene for benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO of Pseudomonas putida has been determined . Benzyl alcohol dehydrogenase is a member of the long-chain zinc alcohol dehydrogenase family and, like other alcohol dehydrogenases of this family, contains two zinc atoms per subunit . Benzyl alcohol dehydrogenase, while sharing 31% identical residues with horse liver alcohol dehydrogenase, contains several amino acid substitutions near the active site, some of which may be responsible for the substrate specificity of benzyl alcohol dehydrogenase, which oxidizes exclusively aromatic substrates . Benzyl alcohol dehydrogenase also notably lacks the His51 residue of horse liver alcohol dehydrogenase . Contrary to the results obtained with a mutant human liver alcohol dehydrogenase lacking this residue, the concentration and pKa of solvent proton acceptors had no effect on the catalytic efficiency of benzyl alcohol dehydrogenase . The electronic nature of substituents on the aromatic ring of the substrate influenced the kcat of the enzyme in low concentrations of external proton acceptor, but not in high concentrations . Product inhibition studies demonstrated that benzyl alcohol dehydrogenase followed a general Ordered Bi Bi kinetic mechanism in low proton acceptor conditions, while following a Theorell-Chance kinetic mechanism at high proton acceptor conditions. J Biol Chem, 1993 May 25, 268(15), 11217 - 21 Human glyoxalase I . cDNA cloning, expression, and sequence similarity to glyoxalase I from Pseudomonas putida; Kim NS et al.; Glyoxalase I (EC 4.4.1.5) catalyzes the transformation of methylglyoxal and glutathione to S-lactoylglutathione . We have isolated human cDNA clones encoding glyoxalase I from a phorbol myristate acetate-treated U937 cDNA library . This cDNA encodes a protein of 184 amino acids with a calculated M(r) of 20,719 . The amino acid composition calculated from the deduced amino acid sequence agreed with that reported for glyoxalase I purified from human erythrocytes . The Escherichia coli cells carrying the expression vector of this cDNA acquired methylglyoxal resistance and the cell lysate showed the high activity of glyoxalase I . The amino acid sequence of human glyoxalase I exhibited 57% identity with Pseudomonas putida glyoxalase I at the C-terminal two-thirds, suggesting that the two enzymes may have originated from a common ancestor. Gene, 1993 May 15, 127(1), 31 - 7 Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4; Simon MJ et al.; The multicomponent enzyme, naphthalene dioxygenase, initiates the metabolism of naphthalene by Pseudomonas putida strains G7 (PpG7) and NCIB 9816-4 (Pp9816-4) . The genes involved (nahAaAbAcAd) are encoded by the NAH7 and pDTG1 plasmids, respectively, and form part of the nah operon . The locations of the structural genes were determined on previously cloned fragments of DNA . The nucleotide (nt) sequences were determined for nahAaAb from Pp9816-4 and for nahAaAbAcAd from PpG7 . The appropriate open reading frames were identified using N-terminal amino acid sequences determined from the purified proteins . The two nt sequences showed 93% homology, with the least homology seen upstream from the promoter region. Gene, 1993 May 15, 127(1), 23 - 9 In-vivo-generated fusion promoters in Pseudomonas putida; Nurk A et al.; Plasmid pEST1463 carrying the promoterless pheBA operon was cloned into Pseudomonas putida PaW85, and phenol-utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source . In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequencing of pheA (encoding phenol monooxygenase) . Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P . putida cells . These promoter sequences show similarity to the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence . In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn4652 into DNA containing potential -10 sequences and point mutations in these sequences . These mutations made the -10 sequences more like the sigma 70 promoter consensus sequences. Mol Gen Genet, 1993 May, 239(1-2), 281 - 8 Physical organization of the upper pathway operon promoter of the Pseudomonas TOL plasmid . Sequence and positional requirements for XylR-dependent activation of transcription; Abril MA et al.; The upper pathway operon of the Pseudomonas putida TOL plasmid belongs to the -12/-24 class of promoters . These promoters exhibit three regions critical for regulated transcription, namely, the -12/-24 site for RNA polymerase/sigma 54 binding, the -55/-67 region for IHF protein binding, and the -130(UAS2)/-170(UAS1) region, where two sites for XylR binding are located . The XylR-protected G residues located at -131, -139, -160 and -169 were replaced with As, and the activity of the mutant promoters was assayed after fusion to a promoterless lacZ gene . The mutation (G(-169)-->A) resulted in a 50% decrease in expression from the promoter (Pu), whereas the other three changes had no significant effect . The XylR recognition sequence UAS2 has a perfect inverted repeat (5'-ATTTN4-AAAT-3') while UAS1 shows two mismatches (5'-CCTTN4AAAT-3') . The two Cs (located at -172 and -173), which interrupt the inverted repeat, were changed as follows: C(-172)-->T; C(-173)-->A, CC(-172, -173)-->AT . Transcription activation from the mutant promoters was measured as beta-galactosidase activity after fusion to lacZ; the better the palindromic sequence, the higher the rate of transcription from Pu, with increases in activity of up to 50% . The introduction of one or two full helix turns between the IHF and the XylR binding sites did not significantly affect transcription from Pu; however, the insertion of three helix turns resulted in a drop of 90% in the activity . The non-permissive effect of insertion of three full helix turns between the IHF and XylR binding sites was not evident in an IHF- background.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Biochem, 1993 May 1, 213(3), 1225 - 33 Creatinase in its collapsed A state shows properties of a molten globule with dimeric quaternary structure; Schumann J et al.; In the past, the molten globule state at acidic pH (A state) has mainly been observed for small single-domain proteins . For more complex proteins such as immunoglobulin, alternatively folded states, with certain characteristics of the molten globule but different thermodynamic properties, were observed . In the present work, the acid-induced structural characteristics of a homodimer, creatinase from Pseudomonas putida, are described . The 91-kDa protein at pH 2 shows molten globule behavior in that there is (a) a high content of native-like secondary structure (monitored by far-ultraviolet circular dichroism), (b) changes in the solvent accessibility of intrinsic fluorophores (acrylamide quenching of protein fluorescence), (c) increased hydrophobic surface area (indicated by anomalous dye binding) and (d) a slight expansion of the hydrodynamic volume (calculated from S20,solv, obtained from analytical ultracentrifugation) . The enzyme at pH 2 shows reversible cooperative transitions in guanidinium chloride or urea (at elevated ionic strength) . Its quaternary structure remains unaltered, indicating that native-like subunit interactions are involved in the stabilization of the A state of the enzyme . Anions stabilize the compact conformation due to reduced intramolecular charge repulsion; for the same reason, the enzyme in its A state shows a strong tendency to aggregate at > 0.3 M NaCl. Mol Microbiol, 1993 May, 8(3), 591 - 601 Identification and characterization of the pupB gene encoding an inducible ferric-pseudobactin receptor of Pseudomonas putida WCS358; Koster M et al.; Pseudomonas putida WCS358 can transport iron complexed to a wide variety of pseudobactins produced by other Pseudomonas strains . The pupB gene encoding an outer membrane ferric-pseudobactin receptor was isolated from a genomic library of P . putida WCS358 . The PupB receptor facilitated iron transport via two distinct heterologous siderophores, i.e . pseudobactin BN8 and pseudobactin BN7 . The amino acid sequence deduced from the nucleotide sequence consisted of 804 amino acids (molecular weight 88,369) of which the N-terminal part was very similar to a prokaryotic leader peptide . The mature protein shared significant homology with the receptor for ferric-pseudobactin 358 (PupA) and contained three regions common to TonB-dependent receptor proteins of Escherichia coli . Interestingly, PupB expression was only observed in cells cultured in iron-deficient medium containing pseudobactin BN8 or pseudobactin BN7 . This expression required a transcriptional unit, pupR, identified upstream of the structural pupB gene . Transposon Tn5 insertion mutants defective in PupB production still exhibited uptake of iron via pseudobactin BN8, although with reduced efficiency . Apparently, an additional transport system for this ferric-siderophore complex operates in this strain . In addition to pseudobactin BN8 also other heterologous siderophores were capable of inducing synthesis of specific high-molecular-weight outer membrane proteins in strain WCS358, which suggests the existence of multiple siderophore-inducible iron transport systems in this strain. Mol Plant Microbe Interact, 1993 May-Jun, 6(3), 331 - 40 Expression of the aggA locus of Pseudomonas putida in vitro and in planta as detected by the reporter gene, xylE; Buell CR et al.; In vitro agglutinability by Pseudomonas putida, isolate Corvallis, with a plant root surface agglutinin is correlated with rapid adhesion of cells of the fluorescent pseudomonad to bean (Phaseolus vulgaris) root surfaces . Agglutinability in P . putida cells is regulated by nutrient status as well as growth phase . Cells grown in three different nutrient complex media are agglutinable at early and mid-late logarithmic phase but become nonagglutinable at stationary phase . Cells grown in a minimal medium are weakly agglutinable, but the addition of lysine, aspartic acid, or histidine increases agglutinability . Cells in the same minimal medium supplemented with bean root surface components grow in a highly agglutinated state . Previous data indicate both agglutination and rapid adhesion to roots by P . putida Corvallis involves the aggA locus, which contains two putative open reading frames (ORF), ORF-AGG1 and ORFAGG2, on complementary strands . Sequence and deletion analyses suggest ORFAGG1 is the most probable ORF responsible for agglutination and adhesion . Chimeric fusion of an Escherichia coli lac promoter with ORFAGG1, but not with ORFAGG2, complemented agglutinability of an aggA::Tn5 P . putida Agg mutant, providing further evidence that ORFAGG1, not ORFAGG2, is responsible for agglutination . Heterologous expression of ORFAGG1 yields a 50-kDa precursor and a 48-kDa mature periplasmic protein . Fusions of ORFAGG1 and ORFAGG2 to the reporter gene, xylE, and detection of the reporter enzyme, catechol-2,3-oxygenase reveal an active promoter in the 5' noncoding region of ORFAGG1 . The ORFAGG1 promoter is active during growth of the cells in liquid culture and is regulated by growth medium . Greatest activity of the catechol-2,3-oxygenase is observed in stationary phase when the cells are nonagglutinable . Expression of the ORFAGG1 promoter is detected in P . putida cells extracted from the root surface of bean at 48 and 72 hr after inoculation. J Mol Biol, 1993 Apr 5, 230(3), 699 - 703 Identification of a cis-acting sequence within the Pm promoter of the TOL plasmid which confers XylS-mediated responsiveness to substituted benzoates; Kessler B et al.; The DNA sequences within the Pm promoter/operator region of the meta operon of the TOL plasmid of Pseudomonas putida, which confer XylS-mediated responsiveness to substituted benzoates, have been identified through deletion analysis and mutagenesis of the region . Integrity and proper phasing of two homologous tandem sequences 5'-TGCAAPuAAPu-PyGGNTA-3', separated by six base-pairs and overlapping with the -35 region of the Pm promoter, was essential for m-toluate activation of a Pm-lacZ fusion in xylS+ strains . The spacing between equivalent bases in each of the half-sites is 21 base-pairs, i.e . two turns of DNA helix . The activity of Pm varieties containing identical half-sites suggested that the XylS-responsive element is arranged as a direct repeat, the distal sequence being the one which provided the most stringent regulation when duplicated . The role of the repeats as the target for XylS binding to Pm is discussed. J Gen Microbiol, 1993 Apr, 139 ( Pt 4), 797 - 805 Diketocamphane enantiomer-specific 'Baeyer-Villiger' monooxygenases from camphor-grown Pseudomonas putida ATCC 17453; Jones KH et al.; Pseudomonas putida ATCC 17453 grew with either (+)- or (-)-camphor as sole carbon source . Enantiomer-specific 'biological Baeyer-Villiger' monooxygenases were synthesized irrespective of the camphor isomer used for growth . The two enzymes are probably the products of separate genes but showed many similarities . Each consisted of two electrophoretically identical subunits, bound flavin mononucleotide (FMN) non-covalently and accepted electrons from an induced NADH dehydrogenase which interacted with the FMN bound to the oxygenating component . They showed minor differences in M(r) with 3,6-diketocamphane 1,6-monooxygenase being the smaller enzyme . Isoelectric focussing showed the two enzymes to have different acidic pI values . Polyclonal antibodies raised against 3,6-diketocamphane 1,6-monooxygenase also cross-reacted with 2,5-diketocamphane 1,2-monooxygenase and its subunits. Can J Microbiol, 1993 Apr, 39(4), 420 - 9 Superoxide dismutase activity in root-colonizing pseudomonads; Katsuwon J et al.; Several saprophytic fluorescent pseudomonads that are aggressive root colonizers express similar specific activities of superoxide dismutase during growth in liquid culture . The pseudomonads have the potential to produce hydrogen peroxide sensitive and hydrogen peroxide insensitive isoforms of superoxide dismutase with distinct mobilities in nondenaturing polyacrylamide gel electrophoresis . Synthesis of the hydrogen peroxide insensitive form is enhanced by limited iron availability, by exposure to Mn2+, and to a lesser extent by external sources of superoxide anion . Unlike Pseudomonas aeruginosa, a root-colonizing strain of Pseudomonas putida did not show regulation of isoform pattern by phosphate availability . A plasmid potentially encoding the pseudomonad hydrogen peroxide sensitive form complemented the superoxide dismutase deficiency in a mutant of Escherichia coli lacking expression of both Fe and Mn genes . Contact between the plant root and pseudomonad or E . coli cells that lack or express superoxide dismutase did not influence superoxide anion production from root surface enzymes . The pseudomonad and the superoxide dismutase deficient and producing E . coli strains survived exposure to the root equally well . Only the hydrogen peroxide sensitive isoform of superoxide dismutase was detected in P . putida cells associated with bean root surfaces. Can J Microbiol, 1993 Apr, 39(4), 357 - 62 Molecular analysis of the plasmid-borne bed gene cluster from Pseudomonas putida ML2 and cloning of the cis-benzene dihydrodiol dehydrogenase gene; Tan HM et al.; Pseudomonas putida ML2 contains a large catabolic plasmid, pHMT112, carrying genes that encode the dioxygenase and dehydrogenase involved in the catabolism of benzene via the ortho or beta-ketoadipate pathway . pHMT112 was derived from a larger and less stable plasmid in P . putida ML2 following growth on succinate as carbon and energy source but was, however, stably maintained in P . putida even in the absence of selection for growth on benzene . Cleavage sites for the restriction endonucleases DraI, XbaI, and BamHI were mapped on the plasmid . A region of the plasmid, downstream of the benzene dioxygenase genes (bedC1C2BA), was found to encode the cis-benzene dihydrodiol dehydrogenase gene (bedD) . Recombinant Escherichia coli containing bedC1C2BAD genes was found to express benzene dioxygenase and dehydrogenase activity, indicated by the production of catechol when incubated in the presence of benzene . Hybridization using benzene dioxygenase genes as probes was used to construct a restriction map of the 35.5-kb XhoI-DraI fragment on which the bed operon was located. Appl Environ Microbiol, 1993 Apr, 59(4), 1194 - 200 Enhanced mineralization of polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading bacteria; Hickey WJ et al.; An Altamont soil containing no measurable population of chlorobenzoate utilizers was examined for the potential to enhance polychlorinated biphenyl (PCB) mineralization by inoculation with chlorobenzoate utilizers, a biphenyl utilizer, combinations of the two physiological types, and chlorobiphenyl-mineralizing transconjugants . Biphenyl was added to all soils, and biodegradation of 14C-Aroclor 1242 was assessed by disappearance of that substance and by production of 14CO2 . Mineralization of PCBs was consistently greatest (up to 25.5%) in soils inoculated with chlorobenzoate degraders alone . Mineralization was significantly lower in soils receiving all other treatments: PCB cometabolizer (10.7%); chlorobiphenyl mineralizers (8.7 and 14.9%); and mixed inocula of PCB cometabolizers and chlorobenzoate utilizers (11.4 and 18.0%) . However, all inoculated soils had higher mineralization than did the uninoculated control (3.1%) . PCB disappearance followed trends similar to that observed with the mineralization data, with the greatest degradation occurring in soils inoculated with the chlorobenzoate-degrading strains Pseudomonas aeruginosa JB2 and Pseudomonas putida P111 alone . While the mechanism by which the introduction of chlorobenzoate degraders alone enhanced biodegradation of PCBs could not be elucidated, the possibility that chlorobenzoate inoculants acquired the ability to metabolize biphenyl and possibly PCBs was explored . When strain JB2, which does not utilize biphenyl, was inoculated into soil containing biphenyl and Aroclor 1242, the frequency of isolates able to utilize biphenyl and 2,5-dichlorobenzoate increased progressively with time from 3.3 to 44.4% between 15 and 48 days, respectively . Since this soil contained no measurable level of chlorobenzoate utilizers yet did contain a population of biphenyl utilizers, the possibility of genetic transfer between the latter group and strain JB2 cannot be excluded. Appl Environ Microbiol, 1993 Apr, 59(4), 1149 - 54 Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates; Fernandez-Valverde M et al.; Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase {ACoAS}) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source . The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol . Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively . Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids) . The broad substrate specificity of ACoAS from P . putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins. J Bacteriol, 1993 Apr, 175(8), 2278 - 83 Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene; Duque E et al.; A bacterium, Pseudomonas sp . strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated . The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite . Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp . clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium . Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene . Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp . clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source . All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives . Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found. Eur J Biochem, 1993 Mar 15, 212(3), 819 - 26 Nicotinoprotein {NAD(P)-containing} alcohol/aldehyde oxidoreductases . Purification and characterization of a novel type from Amycolatopsis methanolica; Van Ophem PW et al.; Extracts of Gram-positive bacteria like Rhodococcus rhodochrous, Rhodococcus erythropolis and Amycolatopsis methanolica, but not those of several Gram-negative ones, showed dehydrogenase activity for ethanol as well as for methanol when 4-nitroso-N,N-dimethylaniline (NDMA) was used as electron acceptor . Chromatography of extracts of the first two organisms revealed one activity for both substrates, that of A . methanolica two activities, one of which is able to oxidize methanol and has been purified (Bystrykh, L.V., Govorukhina, N.I., van Ophem, P.W., Hektor, H.J., Dijkhuizen, L . and Duine, J.A., unpublished results) . The other, indicated as NDMA-dependent alcohol dehydrogenase (NDMA-ADH), was purified to homogeneity . It is a trimeric enzyme consisting of subunits of 39 kDa and one firmly bound NAD as cofactor . Although NDMA-ADH shows structural similarity with the long-chain, zinc-containing, NAD(P)-dependent alcohol dehydrogenases with respect to the N-terminal sequence up to residue 41 (56% identity with horse liver alcohol dehydrogenase), the enzymes are catalytically different since NDMA-ADH is unable to use NAD(P)(H) as a coenzyme and NAD(P)-dependent alcohol dehydrogenases are inactive with NDMA (in the absence of NAD) . Comparison of the NDMA-ADH properties with those of the methanol-oxidizing enzyme of A . methanolica, Mycobacterium gastri and Bacillus methanolica C1, and formaldehyde dismutase of Pseudomonas putida F61 revealed large differences in structural as well as catalytic properties, in spite of the fact that all are nicotinoproteins {enzymes which have bound NAD(P) as a cofactor} . It is concluded, therefore, that NDMA-ADH is a novel type of nicotinoprotein alcohol dehydrogenase. Biochem Mol Biol Int, 1993 Mar, 29(4), 785 - 91 Oxidation of aniline to nitrobenzene by nonheme bromoperoxidase; Itoh N et al.; Nonheme bromoperoxidase found in Pseudomonas putida catalyzed the bromination of aniline with hydrogen peroxide and bromide ions to give o- and p-bromoanilines . However, in the absence of bromide ions, it oxidized aniline via azobenzene and azoxybenzene finally into nitrobenzene . This is the first report of the biological oxidation of an arylamine to the corresponding nitrocompound at the enzyme level . In addition, nitrobenzene was not formed by a nonheme bromoperoxidase from Corallina pilulifera (marine alga), implying that the alga enzyme has a different reaction mechanism. Biochem J, 1993 Mar 1, 290 ( Pt 2), 539 - 44 Nucleotide sequence and over-expression of morphine dehydrogenase, a plasmid-encoded gene from Pseudomonas putida M10; Willey DL et al.; Pseudomonas putida M10 was originally isolated from factory waste liquors by selection for growth on morphine . The NADP(+)-dependent morphine dehydrogenase that initiates morphine catabolism is encoded by a large plasmid of 165 kb . Treatment of P . putida M10 with ethidium bromide led to the isolation of a putative plasmid-free strain that was incapable of growth on morphine . The structural gene for morphine dehydrogenase, morA, has been located on the plasmid by oligonucleotide hybridization, by coupled transcription-translation of cloned restriction fragments and by nucleotide sequence analysis and is contained within a 1.7 kb SphI fragment that has been cloned into Escherichia coli . The cloned dehydrogenase enzyme is expressed at high levels in E . coli resulting in a 65-fold increase in morphine dehydrogenase activity in cell-free extracts compared with P . putida M10 . Morphine dehydrogenase was rapidly purified to homogeneity, as judged by SDS/PAGE, by a one-step affinity chromatography procedure on Mimetic Orange 3 A6XL . The properties of the purified enzyme were identical with those previously reported for P . putida M10 morphine dehydrogenase . The morA gene was sequenced and the deduced amino acid sequence confirmed by N-terminal amino acid sequencing of the over-expressed protein . The predicted amino acid sequence of morA, deduced from the nucleotide sequence, indicated that morphine dehydrogenase did not belong to the non-metal-requiring short-chain class of dehydrogenases, but was more closely related to the aldo-ketoreductases. FEMS Microbiol Lett, 1993 Feb 15, 107(1), 107 - 10 Energy conservation by pyrroloquinoline quinol-linked xylose oxidation in Pseudomonas putida NCTC 10936 during carbon-limited growth in chemostat culture; Hardy GP et al.; When grown in carbon source-limited chemostat cultures with lactate or glucose as the carbon and energy source and xylose as an additional source of reducing equivalents . Pseudomonas putida NCTC 10936 oxidized xylose to xylonolactone and xylonate . No other products were formed from this pentose, nor was it incorporated into biomass . The presence of xylose in these cultures resulted in higher Yglucose and Ylactate values as compared to cultures without xylose indicating that biologically useful energy was conserved during the periplasmic oxidation of xylose . As the Y0 values for growth on glucose or on lactate alone were equal to the Y0 values for growth with xylose as co-substrate, it is concluded that for glucose- or lactate-limited growth energy conservation by PQQH2 oxidation is as efficient as by NADH2 oxidation. J Bacteriol, 1993 Feb, 175(4), 1187 - 90 Proteins induced by sulfate limitation in Escherichia coli, Pseudomonas putida, or Staphylococcus aureus; Kertesz MA et al.; Two-dimensional gel electrophoresis of proteins from Escherichia coli, Pseudomonas putida, and Staphylococcus aureus, grown with methionine or one of a variety of organosulfates and organosulfonates as the sole source of sulfur, showed expression of specific sets of 7 to 14 proteins which were not observed during growth with sulfate or cysteine for all three species or with thiocyanate for P . putida and S . aureus . Under the same conditions, arylsulfatase activity in P . putida and S . aureus was seen to increase by up to 140-fold, suggesting that the proteins induced under these conditions may be involved in sulfur metabolism . We propose that these proteins are members of a sulfate starvation-induced stimulon. J Bacteriol, 1993 Feb, 175(4), 1095 - 102 Cloning, sequencing, and genetic characterization of regulatory genes, rinA and rinB, required for the activation of staphylococcal phage phi 11 int expression; Ye ZH et al.; The int gene of staphylococcal bacteriophage phi 11 is the only viral gene responsible for the integrative recombination of phi 11 . To study the regulation of int gene expression, we determined the 5' end of the transcript by S1 mapping . The presumed promoter is located just 22 nucleotides upstream of the int open reading frame in a region which is conserved between phi 11 and a closely related staphylococcal phage, L54a . To clone the possible regulatory gene, a vector which contained the reporter gene, xylE, of Pseudomonas putida under the control of the phi 11 int promoter was constructed . Subsequently, a 2-kb DNA fragment from the phi 11 genome, which mapped distal to the int gene, was shown to increase the XylE activity from the int promoter . Sequencing and subsequent deletion analysis of the 2-kb fragment revealed that two phi 11 regulatory genes, rinA and rinB, were both required to activate expression of the int gene . Northern (RNA) analysis suggested that the activation was, at least partly, at the transcriptional level . In addition, one of these regulatory genes, rinA, was capable of activating L54a int gene transcription. FEMS Microbiol Lett, 1993 Jan 15, 106(2), 211 - 6 Increased expression of the plasmid-determined 2,3-dihydroxybiphenyl dioxygenase gene in strains of Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa; Andreyeva AL et al.; A 6.5-kb EcoRI fragment containing the gene encoding 2,3-dihydroxybiphenyl dioxygenase from the plasmid pBS312 was cloned into broad host range plasmid RSF1010 and expressed in Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa strains . The increased expression of the gene was orientation-dependent and probably due to the transcription read through from the streptomycin promoter of the vector . Subcloning experiments of the PstI fragments of pBS312 plasmid using vector pBR322 revealed that the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase is localized on the 2.1-kb fragment . In Escherichia coli JM109, transformed by the plasmid pBS314 carrying the 2.1-kb insert in orientation which allowed expression of the bphC gene from the ampicillin promoter of pBR322, the enzyme activity of 2,3-dihydroxybiphenyl dioxygenase was ten times higher than that in parental strain Pseudomonas putida SU83 . The results presented show the first case of the increased expression of Pseudomonas degradative gene in Escherichia coli. J Biol Chem, 1993 Jan 5, 268(1), 315 - 9 Heteronuclear NMR analysis of unsaturated fatty acids in poly(3-hydroxyalkanoates) . Study of beta-oxidation in Pseudomonas putida; de Waard P et al.; Poly(3-hydroxyalkanoates) (PHAs) were isolated from Pseudomonas putida KT2442 cultivated on petroselenic acid, oleic acid, and linoleic acid to study beta-oxidation of unsaturated fatty acids . Both saturated and unsaturated medium chain length 3-hydroxy fatty acids were found to be constituents of these polymers . With the aid of proton-detected multiple quantum coherence and proton-detected multiple bond coherence NMR spectra the structures of the unsaturated monomers were identified as 3-hydroxy-5-cis-tetradecanoate for PHA produced on oleic acid, and 3-hydroxy-6-cis-dodecanoate and 3-hydroxy-5-cis-8-cis-tetradecadienoate for PHA produced on linoleic acid . The identified structures, which are derived from fatty acid degradation intermediates, indicate a degradation of oleic acid via the enoyl-CoA isomerase-dependent route and a degradation of linoleic acid via the dienoyl-CoA reductase-dependent route. Mol Microbiol, 1993 Jan, 7(1), 117 - 30 Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric-pseudobactin transport; Bitter W et al.; Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway . Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics . These mutants failed to grow under iron-limiting conditions, and could not utilize different ferric-siderophores . The mutants fall in three complementation groups . The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively . The deduced amino acid sequence of P . putida ExbB showed 58.6% homology with its E . coli homologue, but, unlike the E . coli protein, it has a N-terminal extension of 91 amino acids . The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology . The P . putida exbB gene could complement an E . coli exbB mutation, but the TonB proteins were not interchangeable between the species . It is concluded that P . putida WCS358 contains an energy-coupling system between the membranes, for active transport across the outer membrane, which is comprised of a TonB-like energy-transducing protein and two accessory proteins . This system is similar to, but not completely compatible with, the E . coli system. J Bacteriol, 1993 Jan, 175(2), 417 - 27 Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida; Coco WM et al.; The 3-chlorocatechol operon clcABD is central to the biodegradative pathway of 3-chlorobenzoate . The clcR regulatory gene, which activates the clcABD operon, was cloned from the region immediately upstream of the operon and was shown to complement an insertion mutation for growth on 3-chlorobenzoate . ClcR activated the clcA promoter, which controls expression of the clcABD operon, in trans by 14-fold in an in vivo promoter probe assay in Pseudomonas putida when cells were incubated with 15 mM 3-chlorobenzoic acid . Specific binding of ClcR to the clcR-clcA intergenic promoter region was observed in a gel shift assay . Nucleotide sequence analysis of the clcR gene predicts a polypeptide of 32.5 kDa, which was confirmed by using specific in vivo 35S labeling of the protein from a T7 promoter-controlled ATG fusion construct . ClcR shares high sequence identity with the LysR family of bacterial regulator proteins and has especially high homology to a subgroup of the family consisting of TcbR (57% amino acid sequence identity), TfdS, CatR, and CatM . ClcR was shown to autoregulate its own production in trans to 35% of unrepressed levels but partially relieved this autorepression under conditions that induced transcription at the clcA promoter . Several considerations indicate that the clcR-clcABD locus is most similar to the tcbR-tcbCDEF regulon. Biometals, 1993 Summer, 6(2), 119 - 23 Indirect utilization of the phytosiderophore mugineic acid as an iron source to rhizosphere fluorescent Pseudomonas; Jurkevitch E et al.; The phytosiderophore mugineic acid (MA) was studied as a source of iron for rhizosphere fluorescent pseudomonads . 55Fe supplied as Fe-MA was taken up by Pseudomonas putida WCS358, B10 and St3 grown under iron deficient conditions . The uptake decreased when the bacteria were grown in the presence of iron . However, no differences in uptake were observed when a siderophore deficient mutant was tested . Since ligand exchange between pseudobactin and MA was shown to occur rapidly with a half-life of 2 h, MA mediated iron uptake probably proceeds through this indirect mechanism . The ecological implications of these findings are discussed. Appl Biochem Biotechnol, 1993 Spring, 39-40, 655 - 66 Degradative capability of Pseudomonas putida on acetonitrile; Chapatwala KD et al.; Pseudomonas putida, capable of utilizing acetonitrile as a sole source of carbon and nitrogen, was isolated from contaminated soil and water samples collected from industrial sites . The P . putida cells were immobilized in calcium alginate beads . The degradation of acetonitrile by the immobilized cells of P . putida was investigated . The immobilized cells degraded different concentrations of acetonitrile into ammonia and carbon dioxide . The effect of aeration on the degradation rate was also studied . Oxygen limitation was suggested in the alginate-immobilized system . The rate of degradation of acetonitrile increased with increase in the rate of aeration. Ann Biol Clin (Paris), 1993, 51(9), 815 - 9 Engineering enzymes for clinical diagnosis; Schumacher G et al.; The enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3.) is a major limiting factor in the enzymatic creatinine determination because of its comparatively poor catalytic activity and stability in the native form . The gene from Pseudomonas putida coding for creatinase was cloned and used for overexpression of the protein in E coli and Pseudomonas . In addition, it was possible by means of 'random' mutagenesis in vivo and subsequent screening using an activity plate assay to isolate creatinase derivatives that are more stable towards detergents and elevated temperature under test kit conditions . This example shows that enzymes can be optimized for use in given assay conditions by mutagenesis of cloned DNA and suitable screening methods. Biodegradation, 1993, 4(1), 59 - 69 Aerobic, phenol-induced TCE degradation in completely mixed, continuous-culture reactors; Coyle CG et al.; Both Pseudomonas putida F1 and a mixed culture were used to study TCE degradation in continuous culture under aerobic, non-methanotrophic conditions . TCE mass balance studies were performed with continuous culture reactors to determine the total percent removed in the reactors, and to quantify the percent removed by air stripping and biodegradation . Adsorption of TCE to biomass was assumed to be negligible . This research demonstrated the feasibility of treating TCE-contaminated water under aerobic, non-methanotrophic conditions with a mixed-culture, continuous-flow system . Initially glucose and acetate were fed as primary substrates . Pnenol, which has been shown to induce TCE-degrading enzymes, was fed at a much lower concentration (20 mg/L) . Little degradation of TCE was observed when acetate and glucose were the primary substrates . After omitting glucose and acetate from the feed and increasing the phenol concentration to 50 mg/L, TCE biotransformation was observed at a significant level (46%) . When the phenol concentration in the feed was increased to 420 mg/L, 85% of the incoming TCE was estimated to have been biodegraded . Under the same conditions, phenol utilization by the mixed culture was greater than that of P . putida F1, and TCE degradation by the mixed culture (85%) exceeded that of P . putida F1 (55%) . The estimated percent-of-TCE biodegraded by the mixed culture was consistently greater than 80% when phenol was fed at 420 mg/L . Biodegradation of TCE was also observed in mixed-culture, batch experiments. Biodegradation, 1993, 4(1), 39 - 50 Cosubstrate effects in reductive dehalogenation by Pseudomonas putida G786 expressing cytochrome P-450CAM; Logan MS et al.; Cytochrome P-450CAM was shown to be the primary catalyst mediating reductive dehalogenation of polychlorinated ethanes by Pseudomonas putida G786 . Under anaerobic conditions, the enzyme catalyzed reductive elimination reactions in vivo with the substrates hexachloroethane, pentachloroethane, and 1,1,1,2-tetrachlorethane; the products were tetrachloroethylene, trichloroethylene, and 1,1-dichloroethylene, respectively . In vivo reaction rates were determined . No reaction was observed with 1,1,2,2-tetrachloroethane or 1,1,1-trichloroethane . Purified cytochrome P-450CAM was used to measure dissociation constants of polychlorinated ethanes for the enzyme active site . Observed rates and dissociation constants were used to predict the course of a reaction with the three substrates simultaneously . Data obtained from experiments with P . putida G786 generally followed the simulated reaction curves . Oxygen suppressed the reductive dechlorination reactions and, in the case of 1,1,1,2-tetrachlorethane, 2,2,2-trichloroacetaldehyde was formed . Significant rates of reductive dechlorination were observed at 5% oxygen suggesting that these reactions could occur under partially aerobic conditions . These studies highlight the potential to use an aerobic bacterium, P . putida G786, under a range of oxygen tensions to reductively dehalogenate mixed wastes which are only degraded at very low rates by obligately anaerobic bacteria. J Biol Chem, 1992 Dec 25, 267(36), 25848 - 55 p-Hydroxyphenylacetate-3-hydroxylase . A two-protein component enzyme; Arunachalam U et al.; p-Hydroxyphenylacetate-3-hydroxylase, an inducible enzyme isolated from the soil bacterium Pseudomonas putida, catalyzes the conversion of p-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate . The enzyme requires two protein components: a flavoprotein and a colorless protein referred to as the coupling protein . The flavoprotein alone in the presence of p-hydroxyphenylacetate and substrate analogs catalyzes the wasteful oxidation of NADH with the stoichiometric generation of H2O2 . A 1:1 complex of the flavoprotein and coupling protein is required for stoichiometric product formation . Such complex formation also eliminates the nonproductive NADH oxidase activity of the flavoprotein . A new assay measuring the product formation activity of the enzyme was developed using homoprotocatechuate-2,3-dioxygenase, as monitoring the oxidation of NADH was not sufficient to demonstrate enzyme activity . The coupling protein does not seem to have any redox center in it . Thus, this 2-component flavin hydroxylase resembles the other aromatic hydroxylases in that the only redox chromophore present is FAD. J Bacteriol, 1992 Dec, 174(23), 7798 - 806 Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida; Parsek MR et al.; In Pseudomonas putida, the catBC operon encodes enzymes involved in benzoate degradation . Previous studies have determined that these enzymes are induced when P . putida is grown in the presence of benzoate . Induction of the enzymes of the catBC operon requires an intermediate of benzoate degradation, cis,cis-muconate, and a regulatory protein, CatR . It has been determined that CatR binds to a 27-bp region of the catBC promoter in the presence or absence of inducer . We have called this the repression binding site . In this study, we used a gel shift assay to demonstrate that the inducer, cis,cis-muconate, increases the affinity of CatR for the catBC promoter region by 20-fold . Furthermore, in the absence of cis,cis-muconate, CatR forms two complexes in the gel shift assay . The inducer cis,cis-muconate confers specificity primarily for the formation of complex 2 . DNase I footprinting showed that an additional 27 bp of the catBC promoter region is protected by CatR in the presence of cis,cis-muconate . We have named this second binding site the activation binding site . Methylation interference footprinting determined that in the presence or absence of inducer, five G nucleotides of the catBC promoter region were necessary for CatR interaction with the repression binding site, while a single G residue was important for CatR interaction with the activation binding site in the presence of cis,cis-muconate . Using polymerase chain reaction-generated constructs, we found that the binding of CatR to the repression binding site is independent of the activation binding site . However, binding of CatR to the activation binding site required an intact repression binding site. Microb Releases, 1992 Dec, 1(3), 155 - 9 The effect of microcosm design on the survival of recombinant Pseudomonas putida in lake water; Morgan JA et al.; The survival of Pseudomonas putida marked with the xylE gene was monitored in lake-water microcosms . Various designs of microcosms were compared . These ranged from 250-ml conical flasks containing 100 ml surface lake water to 12-1 glass containers with lake water overlying sediment, continuous aeration and a supply of fresh surface lake water . The presence of a low flow-through rate was shown to have little effect on the survival of P . putida . An increase in the size of microcosm, presence of sediment and aeration had a significant effect on survival in lake water and increased the rate of decline of released cells . The implication of these results in predicting the survival of P . putida in lake water using microcosms is discussed. Biokhimiia, 1992 Dec, 57(12), 1883 - 91 {Purification and properties of pyrocatechase II from Pseudomonas putida strain 87}; Solianikova IP et al.; Induction of modified ortho-pathway enzymes (catechol 1.2-dioxygenase II, muconate cycloisomerase II, dienelactone hydrolase, and maleylacetate reductase) was found in Pseudomonas putida 87, when 3-chlorobenzoic acid was used as a sole carbon and energy source . Catechol 1.2-dioxygenase II, the key chlorocatechol cleaving enzyme, was purified and characterized . The enzyme molecular mass as determined by gel filtration was 65,000 Da; the minimum molecular mass upon SDS electrophoresis was 33,000 Da . The pH and temperature optima for the enzyme were 7.2-7.8 and 35 degrees C, respectively . The highest stability of catechol 1.2-dioxygenase II upon storage was observed in 50 mM Tris-HCl buffer pH 7.8 at 4 degrees C . The relative values of Vmax for catechol 1.2-dioxygenase II with 3-chloro-, 4-chloro-, and 3.5-dichlorocatechols were 28%, 50%, and 41% of those for catechol . The enzyme affinity for chlorocatechols was 3-9 times higher than for methylcatechols and 10-20 times higher than for unsubstituted catechol. Biochemistry, 1992 Nov 24, 31(46), 11383 - 9 Genetic variants in the putidaredoxin-cytochrome P-450cam electron-transfer complex: identification of the residue responsible for redox-state-dependent conformers; Davies MD et al.; Camphor is hydroxylated in Pseudomonas putida by a three-component system comprised of an oxidase, cytochrome P-450cam, and a two-protein electron-transfer chain, putidaredoxin and putidaredoxin reductase {Tyson et al . (1972) J . Biol . Chem . 274, 5777-5784} . The enzymatic removal of putidaredoxin's C-terminal tryptophan is known to cause a much reduced rate of enzymatic activity in the reconstituted camphor hydroxylase system {Sligar et al . (1974) Proc . Natl . Acad . Sci . U.S.A . 71, 3906-3910} . To further study the role of tryptophan in the association and/or electron-transfer reactions of putidaredoxin, the gene coding for the iron-sulfur protein was altered so that the tryptophan codon was either deleted or replaced by Phe, Tyr, Asp, Leu, Val, or Lys . Although the initial evaluation of these variant proteins {Davies et al . (1990) J . Am . Chem . Soc . 112, 7396-7398} showed much reduced velocities of electron transfer between P-450cam and the nonaromatic C-terminal proteins, the relative contributions of the binding specificity and intracomplex electron-transfer rates were not addressed . We report here a complete kinetic characterization of these proteins where the dependence of the rate constant on the putidaredoxin concentration was used to determine the intracomplex electron-transfer rate constants and the association energies for all the putidaredoxins in both oxidation states . The sum of forward and reverse intracomplex electron-transfer rate constants varies from 4.90 s-1 for the Lys C-terminal variant to 172 s-1 for the native protein.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1992 Nov 5, 267(31), 22587 - 94 Functional domains of aromatase cytochrome P450 inferred from comparative analyses of amino acid sequences and substantiated by site-directed mutagenesis experiments; Chen S et al.; Several functional domains, especially the active site regions, in aromatase cytochrome P450 were inferred by alignment of amino acid sequences of the enzyme from five species, human, rat, mouse, chicken, and trout, and that of Pseudomonas putida cytochrome P450cam, whose x-ray structure has been determined (Poulos, T.L., Finzel, B.C., and Howard, A.J . (1987) J . Mol . Biol . 195, 687-700) . The predicted functions of these domains have been evaluated by site-directed mutagenesis . Eighteen mutants, including seven new mutants, have been generated in this laboratory . The seven newly prepared mutants are Q123E, Q123H, T310S, T310C, R365K, R365A, and N delta 20 (a mutant without the first 20 amino acids) . The preparation and characterization of these new mutants are described . The structural model described in this paper should be very useful for future structure-function studies of aromatase by site-directed mutagenesis. Mol Biol (Mosk), 1992 Nov-Dec, 26(6), 1251 - 62 {Electron-conformational interactions at the active site of reduced bacterial cytochrome P450cam induced by a substrate and analysis of the electron structure of heme}; Sharonov IuA; Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K . The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme . In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state . In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts . The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region . The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam . The following conclusions were obtained from a theoretical analysis of the temperature profiles . In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively) . In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron . More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate . Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state. Appl Environ Microbiol, 1992 Nov, 58(11), 3630 - 7 Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss; Sobecky PA et al.; When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions . In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs . Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations . Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media . Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs . Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth) . However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water . Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1992 Nov, 235(2-3), 406 - 12 XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida; Michan C et al.; The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida . This protein is activated by a variety of benzoate analogues . To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to beta-galactosidase were constructed but all are inactive . In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions . The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein . The intraallelic dominance of the Ile229 (Ser229-->Ile) and Val274 (Asp274-->Val) substitutions over the N-terminal His41 (Arg41-->His) substitution, and the intraallelic dominance of Thr45 (Arg45-->Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally . Combination of the Leu88 (Trp88-->Leu) and Arg256 (Pro256-->Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates . In contrast, the Leu88 semiconstitutive phenotype was suppressed by Val288 (Asp288-->Val), and the double mutant was susceptible to activation by benzoates.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1992 Nov, 6(21), 3121 - 36 DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans; van Beilen JB et al.; The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes . In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides . The alkJ gene encodes a protein of 59 kilodaltons . The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases . AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes . The properties of AlkJ suggest that it is linked to the electron transfer chain . AlkJ is necessary for growth on alkanes only in P . putida alcohol dehydrogenase (AlcA) mutants . AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate . This list includes the acetate, coumarate and long-chain fatty acid CoA ligases . The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase . AlkK is a 60 kilodalton protein located in the cytoplasm . AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E . coli . AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins . The alkL gene product was found in the outer membrane of E . coli W3110 containing the alk-genes . The alkL gene can be deleted without a clear effect on growth rate . Its function remains unknown . The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P . putida chromosome. J Bacteriol, 1992 Nov, 174(22), 7253 - 61 Identification of a new gene, tmoF, in the Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase; Yen KM et al.; Five genes, tmoABCDE, encoding toluene-4-monooxygenase (T4MO) were previously mapped to a 3.6-kb region of a 10.2-kb SacI DNA fragment isolated from Pseudomonas mendocina KR1 (K.-M . Yen, M . R . Karl, L . M . Blatt, M . J . Simon, R . B . Winter, P . R . Fausset, H . S . Lu, A . A . Harcourt, and K . K . Chen, J . Bacteriol . 173:5315-5327, 1991) . In this report, we describe the identification and characterization of a DNA region in the SacI fragment whose expression enhances the T4MO activity determined by the tmoABCDE gene cluster . This region was mapped immediately downstream of the putative transcription termination sequence previously located at the end of the tmoABCDE gene cluster (Yen et al., J . Bacteriol., 1991) and was found to stimulate T4MO activity two- to threefold when expressed in Escherichia coli or Pseudomonas putida . Determination of the nucleotide sequence of this region revealed an open reading frame (ORF) of 978 bp . Expression of the ORF resulted in the synthesis of an approximately 37-kDa polypeptide whose N-terminal amino acid sequence completely matched that of the product predicted from the ORF . The ORF thus defines a gene, which has now been designated tmoF . The TmoF protein shares amino acid sequence homology with the reductases of several mono- and dioxygenase systems . In addition, the reductase component of the naphthalene dioxygenase system, encoded by the nahAa gene of plasmid NAH7 from P . putida G7, could largely replace the TmoF protein in stimulating T4MO activity, and TmoF could partially replace the NahAa protein in forming active naphthalene dioxygenase . The overall properties of tmoF suggest that it is a member of the T4mo gene cluster and encodes the NADH:ferredoxin oxidoreductase of the T4MO system. Mol Gen Mikrobiol Virusol, 1992 Nov-Dec, (11-12), 23 - 5 {Cloning and expression of the Arthrobacter globiformis fcb genes in Bacillus subtilis}; Mikhailiuk NV et al.; The fcb genes of Arthrobacter globiformis KZT1 coding for the dehalogenase (4-chlorobenzoate-4-hydroxylase) activity have been cloned . The characteristics of fcb genes expression have been studied . The recombinant strains of Bacillus subtilis 6JM15 (pCBS 311) and 6JM15 (pCBS1) have shown the decreased level of substrate dehalogenation as compared with the one in the parent strain KZT1 and the recombinant strains of Escherichia coli and Pseudomonas putida. Mikrobiologiia, 1992 Nov-Dec, 61(6), 1098 - 100 {Preservation of Pseudomonas putida bacteria at low temperatures}; Vysekantsev IP et al.; We have found that the mode of cooling, composition of cryopreservation medium, original concentration of cells and storage temperature affect viability of Pseudomonas putida bacteria during low-temperature preservation . We have elaborated the conditions of preservation, providing for a high survival of bacteria, namely: one-stage cooling with the rates of 30, 40 degrees C/min or immersion into liquid nitrogen in the culturing medium with addition of sucrose, glycerol or dimexide in the concentration of 0.5 M; storage temperature is -80 degrees C divided by -196 degrees C. FEBS Lett, 1992 Oct 26, 311(3), 206 - 8 Isolation, sequencing and expression in E . coli of the urocanase gene from white clover (Trifolium repens); Koberstaedt A et al.; The urocanase gene was detected in a clone obtained from a genomic library of white clover . The entire gene has been sequenced and expressed in the pT7-7/E . coli BL 21 (DE 3) system . The deduced sequence of the plant urocanase is 72% homologous with that of the well-characterized urocanase from Pseudomonas putida . The purification procedure, as well as kinetic and electrophoretic behaviour, of the new enzyme are described. Biochemistry, 1992 Oct 13, 31(40), 9768 - 76 3-Carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida is homologous to the class II fumarase family: a new reaction in the evolution of a mechanistic motif; Williams SE et al.; The gene (pcaB) for 3-carboxymuconate lactonizing enzyme (CMLE; 3-carboxymuconate cycloisomerase; EC 5.5.1.2) from Pseudomonas putida has been cloned into pMG27NS, a temperature-sensitive expression vector, and expressed in Escherichia coli N4830 . The specific activity and kinetic parameters of the recombinant CMLE were comparable to those previously reported . A comparison of the deduced amino acid sequence of CMLE with sequences available in the PIR and Genbank databases revealed that CMLE has highly significant sequence homology to the class II fumarase family, particularly to adenylosuccinate lyase from Bacillus subtilis . CMLE has no significant homology to muconate lactonizing enzyme (MLE) from P . putida, its sister enzyme in the beta-ketoadipate pathway . These findings fully corroborate a prediction made by us on the basis of mechanistic and stereochemical analyses of CMLE and MLE {Chari, R . V . J., Whitman, C . P., Kozarich, J . W., Ngai, K.-L., & Ornston, L . N . (1987) J . Am . Chem . Soc . 109, 5514-5519} and suggest that CMLE and MLE were recruited into this specialized pathway from two different enzyme families. Mol Microbiol, 1992 Oct, 6(20), 3065 - 76 Evidence that the hrpB gene encodes a positive regulator of pathogenicity genes from Pseudomonas solanacearum; Genin S et al.; The hrp gene cluster of Pseudomonas solanacearum GMI1000 strain encodes functions that are essential for pathogenicity on tomato and for the elicitation of the hypersensitive response on tobacco . In this study, we present the nucleotide sequence of one of the hrp genes (hrpB) located at the left-hand end of the cluster and we show that hrpB encodes a positive regulator controlling the expression of hrp genes . hrpB has a coding capacity for a 477-amino-acid polypeptide, which shows significant similarity to several prokaryotic transcriptional activators including the AraC protein of Escherichia coli, the XylS protein of Pseudomonas putida and the VirF protein of Yersinia enterocolitica . The predicted hrpB gene product belongs to a family of bacterial regulators different from the previously described HrpS protein of the hrp gene cluster of Pseudomonas syringae pv . phaseolicola . Genetic evidence demonstrates that the hrpB gene product acts as a positive regulator of the expression in minimal medium of all but one of the putative transcription units of the hrp gene cluster and also controls the expression of genes located outside this cluster . We also show in this paper that the transcription of hrpB is induced in minimal medium and is partly autoregulated. Can J Microbiol, 1992 Oct, 38(10), 1074 - 83 Expression of the 4-chlorobenzoate dehalogenase genes from Pseudomonas sp.-CBS3 in Escherichia coli and identification of the gene translation products; Savard P et al.; The genes encoding the 4-chlorobenzoate dehalogenase of Pseudomonas sp . strain CBS3 were, in an earlier study, cloned in Escherichia coli DH1 with the cosmid vector pPSA843 and then mobilized to the 4-chlorobenzoate dehalogenase minus strain Pseudomonas putida KT2440 . In this paper we report on the expression of 4-chlorobenzoate dehalogenase in these clones and on the polypeptide composition of the active enzyme . The dehalogenase activity in whole cells suspended in 3.2 mM 4-chlorobenzoate (30 degrees C) was determined to be approximately 27 units (micromoles 4-hydroxybenzoate produced per minute) per 100 g of E . coli-pPSA843 cells and approximately 28 units per 100 g of P . putida-pPSA843 cells . Dehalogenase activity in fresh cellular extracts (pH 7.4, 30 degrees C) prepared from the E . coli and P . putida clones was unstable and at least 20-fold lower than that observed with the whole cells . The polypeptide components of the dehalogenase were identified by selective expression of the cloned dehalogenase genes and analysis of the gene translation products . Analysis of dehalogenase activity in omega insertion mutants and deletion mutants circumscribed the dehalogenase genes to a 4.8-kilobase (4.8 kb) stretch of the 9.5-kb DNA fragment . Selective expression of the dehalogenase genes from a cloned 4.8-kb DNA fragment in a maxicell system revealed a 30-kDa polypeptide as one of the components of the dehalogenase system . Selective expression of the dehalogenase genes using the T7 polymerase promoter system revealed the 30-kDa polypeptide and 57- and 16-kDa polypeptide products . Determination of which of the three polypeptides were translated in deletion mutants provided the relative positions of the encoding genes on a single DNA strand and the direction in which they are transcribed. Appl Environ Microbiol, 1992 Oct, 58(10), 3407 - 9 Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1; Brand JM et al.; Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol . Under similar conditions, P . putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol . The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P . putida F39/D that preferentially oxidizes (+)-(1S)-indanol. J Toxicol Environ Health, 1992 Oct, 37(2), 247 - 64 Performance of an aquatic multispecies system in evaluating the effects of a model microbial pest control agent on nontarget organisms; Williams RR et al.; A recirculating multispecies test system was developed in conjunction with a study of the fate and persistence of a model microbial pest control agent on non-target marine and freshwater organisms . The basic unit of the system was a 113-I glass aquarium with vertical biological filters in the center of the aquarium, such that two compartments were formed . This allowed the sequestration of predator and prey species within the same system . Organisms from six phyletic groups were subjected to a genetically altered strain of Pseudomonas putida for 15-29 d in either artificial seawater or fresh water . The system was able to maintain the animals for these periods with a minimum of maintenance . Additionally, the system design lent itself to disinfection, dismantling, and rebuilding between experiments with a minimum of labor, and has potential for longer-term studies. Eur J Biochem, 1992 Oct 1, 209(1), 375 - 82 Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2; Shimizu S et al.; A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2 . The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE . The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits . The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 mumol.min-1.mg-1 at 30 degrees C . It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters . The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine . The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenylethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively . The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+. Mol Ecol, 1992 Oct, 1(3), 137 - 43 Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain; Genthner FJ et al.; Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples . This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA . The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate . This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces . The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization . Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected . Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater . In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms . These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples. Eur J Biochem, 1992 Oct 1, 209(1), 51 - 61 Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2; Shaw JP et al.; The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes . Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively . In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity . Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity . The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration . The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one {2Fe-2S} cluster/protein molecule . The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm . These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite . The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5 . The reductase was found to have a Km value for NADH of 22 microM . The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme) . The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the {2Fe-2S} center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD . (semiquinone form), with a calculated midpoint redox potential of -244 mV . The reduction of FAD . to FAD. . (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV . The {2Fe-2S} center could be removed from the protein by treatment with an excess of mersalyl acid . The {2Fe-2S}-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> {2Fe-2S} in this reductase . The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate. Biosci Biotechnol Biochem, 1992 Oct, 56(10), 1644 - 8 In vitro interactions of Pseudomonas RNA polymerases with tac and RNA I promoters; Fujita M et al.; The activities of RNA polymerases from Pseudomonas putida and Pseudomonas aeruginosa were compared with that of Escherichia coli RNA polymerase in an in vitro transcription system . All three enzymes initiated transcription at the tac promoter and the RNA I promoter of E . coli . We measured the rate of open complex formation between the RNA polymerases and the promoters, and the saturation level of open complex formation at equilibrium in single-round transcription . The relative rates of open complex formation were P . putida > E . coli > P . aeruginosa and the relative saturation levels of open complex formation at equilibrium were E . coli > P . putida > P . aeruginosa for the tac and RNA I promoters . The interaction of the RNA polymerases with the promoters was also studied by DNase I footprinting . The patterns of protection of the Pseudomonas RNA polymerases on the tac promoter were similar to that of E . coli RNA polymerase . However, the protection patterns of the Pseudomonas RNA polymerases on the RNA I promoter were slightly different from that of E . coli RNA polymerase. Appl Environ Microbiol, 1992 Oct, 58(10), 3387 - 94 Bacterioplankton community structure and dynamics after large-scale release of nonindigenous bacteria as revealed by low-molecular-weight-RNA analysis; Hofle MG; A set of freshwater mesocosms (1.7 m3 each) was inoculated with large amounts of Escherichia coli, Pseudomonas putida, and their culture medium to substantially disturb the natural microbial community . To monitor microbial community dynamics, low-molecular-weight RNA (5S rRNA and tRNA) obtained directly from bacterioplankton was analyzed by using high-resolution electrophoresis . The introduced bacteria showed no significant effect on the community structure of the natural bacterial assemblage and its dynamics for 16 days . In contrast, the addition of culture medium resulted within 2 days in a reduction of community diversity due to dominance of a single 5S rRNA band from an indigenous bacterium . Partial sequencing of several 5S rRNAs demonstrated the molecular homogeneity of most of the abundant bands and enabled the identification of corresponding bacterial isolates and/or species . The dominating bacterium (around 54% of the total 5S rRNA) in the nutrient-amended mesocosms could be identified by partial sequencing as a member of the Aeromonas hydrophila complex . Another bloom of heterotrophic bacteria belonging to the Cytophaga johnsonae complex was detected in the nutrient-amended mesocosms after 13 days . The dominance of this C . johnsonae-like bacterium could even be seen in the environmental tRNAs of the bacterioplankton, where its specific tRNAs prevailed from day 13 onward . This event was also independent of the introduced nonindigenous bacteria because it occurred at the same time in all nutrient-amended mesocosms . By contrast, in the unamended experiments, a different small 5S rRNA could by observed from day 10 onward with less pronounced dominance.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1992 Sep 5, 267(25), 17716 - 21 4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer; Chen LH et al.; The xylH gene encoding 4-oxalocrotonate tautomerase (4-OT) has been located on a subclone of the Pseudomonas putida mt-2 TOL plasmid pWW0 and inserted into an Escherichia coli expression vector . Several of the genes of the metafission pathway encoded by pWW0 have been cloned in E . coli, but the overexpression of their gene products has met with limited success . By utilizing the E . coli alkaline phosphatase promoter (phoA) coupled with the proper positioning of a ribosome-binding region, we are able to express functional 4-OT in yields of at least 10 mg of pure enzyme/liter of culture . 4-OT has been previously characterized and shown to be an extremely efficient catalyst (Whitman, C . P., Aird, B . A., Gillespie, W . R., and Stolowich, N . J . (1991) J . Am . Chem . Soc . 113, 3154-3162) . Kinetic and physical characterization of the E . coli-expressed protein show that it is identical with that of the 4-OT isolated from P . putida . The functional unit is apparently a pentamer of identical subunits, each consisting of only 62 amino acid residues . This is the smallest enzyme subunit reported to date . The amino acid sequence, determined in part from automated Edman degradation and also deduced from the primary sequence of xylH, did not show homology with any of the sequences in the current data bases nor with any of the sequences of enzymes that catalyze similar reactions . We propose that the active site of 4-OT may be established by an overlap of subunits and comprised of amino acid residues belonging to several, if not all, of the subunits. FEMS Microbiol Lett, 1992 Sep 1, 75(1), 81 - 7 Degradation of chlorotoluenes by in vivo constructed hybrid strains: problems of enzyme specificity, induction and prevention of meta-pathway; Brinkmann U et al.; The activities of the TOL plasmid-coded xylene oxygenase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase of Pseudomonas putida strain PaW1 were tested with substituted toluenes, benzylalcohols and benzaldehydes, respectively, as substrates . Several chlorinated toluenes were shown to induce enzymes of the xylene degradation sequence . Conjugative transfer of the TOL plasmid from Pseudomonas putida strain PaW1 to Pseudomonas sp . strain B13 and Pseudomonas cepacia strain JH230 allowed the isolation of hybrid strains capable of growing in the presence of 3-chloro-, 4-chloro- and 3,5-dichlorotoluene . Hybrid strains revealed new ways to prevent the dead-end meta-pathway for cholorocatechols. Mikrobiol Zh, 1992 Sep-Oct, 54(5), 53 - 9 {The kinetics of glycol destruction by a Pseudomonas putida BS-2 strain}; Sedina SA; Destruction of ethylene glycol and diethylene glycol by Pseudomonas putida BS-2 culture under conditions of its batch cultivation has been studied for its physiological regularities . The specific rate of the biomass growth in the region of limiting substrate concentrations depends on the diethylene glycol concentration in the medium and follows the Mono equation . A semisaturation constant for diethylene glycol is 209 +/- 17 mg/d . The specific rate of the culture growth is independent of the ethylene glycol concentration in the medium within a wide range from 0.08 to 10 g/l . Kinetics of the bacteria growth inhibition by excess of substrates is a complex character and obeys none of the known models of the substrate inhibition. Appl Environ Microbiol, 1992 Sep, 58(9), 2978 - 82 Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47; Lemanceau P et al.; Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture . This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone . The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own . P . putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F . oxysporum Fo47b10 . In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10 . Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358 . The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone. Appl Environ Microbiol, 1992 Sep, 58(9), 2723 - 9 Physiological properties of a Pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium; Cruden DL et al.; Pseudomonas putida Idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50% (vol/vol) . Growth also occurs on Luria-Bertani medium in the presence of a wide range of organic solvents . The ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethyl-phthalate (log P(OCT) = 2.3) being the most polar solvent tolerated . During growth with p-xylene (20% {vol/vol}), there was an initial lag period accompanied by cell death, which was followed by a period of exponential growth . The stationary phase of growth was characterized by a dramatic decrease in cell viability, although cell dry weight and turbidity measurements slowly increased . Electron micrographs revealed that during growth in the presence of p-xylene, the outer cell membrane becomes convoluted and membrane fragments are shed into the culture medium . At the same time, the cytoplasmic membrane invaginates, forming vesicles, and becomes disorganized . Electron-dense intracellular inclusions were observed in cells grown with p-xylene (20% {vol/vol}) and p-xylene vapors, which are not present in cells grown with succinate . Attempts to demonstrate the presence of plasmid DNA in P . putida Idaho were negative . However, polarographic studies indicated that the organism utilizes the same pathway for the degradation of toluene, m-xylene, and p-xylene as that used by P . putida mt-2 which contains the TOL plasmid pWWO.(ABSTRACT TRUNCATED AT 250 WORDS) Plasmid, 1992 Sep, 28(2), 101 - 14 Cloning and transposon vectors derived from satellite bacteriophage P4 for genetic manipulation of Pseudomonas and other gram-negative bacteria; Polissi A et al.; We developed transposon and cloning shuttle vectors for genetic manipulation of Pseudomonas and other gram-negative bacteria, exploiting the unique properties and the broad host range of the satellite bacteriophage P4 . P4::Tn5 AP-1 and P4::Tn5 AP-2 are suicide transposon vectors which have been used for efficient Tn5 mutagenesis in Pseudomonas putida . pKGB2 is a phasmid vector with a cloning capacity of about 7.5 kb; useful unique cloning sites are SacI and SacII in the streptomycin resistance determinant and PvuI and XhoI in the kanamycin resistance determinant . pKGB4 is a cosmid derived from pKGB2 and carries the additional cloning site SmaI in the kanamycin resistance determinant; its cloning capacity is about 18 kb . These vectors and their recombined derivatives were transferred from Escherichia coli to P . putida by transduction and may be used for other bacterial species susceptible to P4 infection. Mikrobiologiia, 1992 Sep-Oct, 61(5), 843 - 51 {Regulation of the expression of plasmid determination responsible for caprolactam degradation by bacteria of the genus Pseudomonas}; Esikova TZ et al.; On the basis of the study of some Tn5 induced mutants in Pseudomonas putida strain BS836 containing the plasmid pBS268 coding caprolactam degradation, growth on caprolactam and its intermediates, and the data on the induction of oxidative activities in plasmid containing P . putida strain BS831 it was shown that plasmid and chromosome genes regulated the expression of CAP-determinants . The regulation has some elements of the negative control mechanism . Caprolactam is the inducer of the synthesis of key enzymes cleaving it and its intermediates (aminocaproic and adipic acids) . At the same time each of its intermediates induced the synthesis of enzymes responsible for its cleavage. Mikrobiologiia, 1992 Sep-Oct, 61(5), 818 - 23 {Role of phenylalanine in the biosynthesis of fluorescent pigment in Pseudomonas putida bacteria}; Maksimova NL et al.; The contemporary data of the participation of phenylalanine in the biosynthesis of fluorescent pigment pyoverdine PM of Pseudomonas putida strain M are presented . Using aro1phu1 mutant of this strain, it has been shown that one of the precursors of the dihydroxyquinoline moiety of the pyoverdine PM is phenylalanine in the D- or L-form . These results were confirmed in experiments with 14C-phenylalanine incorporation . Pyoverdine PM that was synthesized by aro1phu1 mutant from exogenous phenylalanine is identical with the pigment from wild type cells. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1429 - 36 Microbial oxidation of adamantanone by Pseudomonas putida carrying the camphor catabolic plasmid; Selifonov SA; Intact cells of (+/-)camphor-grown Pseudomonas putida, ATCC17453(CAM), have been shown to oxidize readily the monoketone derivative of cage hydrocarbon adamantane, forming oxygenated products indicative of both biological Baeyer-Villiger and hydroxylation reactions . Formed products were identified as 4-oxahomoadamantan-5-one, 5-hydroxyadamantan-2-one and 1-hydroxy-4-oxahomoadamantan-5-one . Minor products formed as a result of secondary reactions were tentatively identified as syn- and anti-1,4-dihydroxyadamantanes and bicyclo{3.3.1}nonan-3-ol . Adamantanone initial concentrations determined whether 1-hydroxy-4-oxahomoadamantan-5-one was the sole product (below 120 mg/l) or 4-oxahomoadamantan-5-one was the principle (up to 92%) product (240-600 mg/l) . Formation of 1-hydroxy-4-oxahomoadamantan-5-one appears to occur by two routes determined by the sequence of lactonization and hydroxylation. Gene, 1992 Aug 1, 117(1), 157 - 8 Plasmids with easily excisable xylE cassettes; Stein DC; Two new vectors containing the xylE gene (encoding catechol-2,3-dioxygenase) of Pseudomonas putida were constructed that serve as the source of the xylE cassette . These vectors are based on the kanamycin-resistance-encoding plasmid, pKAN18 . The promoter-less xylE gene is flanked by several restriction enzyme sites that allow for easy excision of this gene in the form of a cassette containing a ribosome-binding site, 7 bp upstream from the start codon . These cassettes lack any transcriptional termination signals downstream from the stop codon. Appl Environ Microbiol, 1992 Aug, 58(8), 2643 - 8 Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction; Robertson JB et al.; Pseudomonas putida F1 and Pseudomonas sp . strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene . When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively . The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol . Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P . putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P . putida F1 and Pseudomonas sp . strain JS150 . These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent. J Ind Microbiol, 1992 Aug, 10(2), 79 - 85 Maintenance and killing efficiency of conditional lethal constructs in Pseudomonas putida; Bej AK et al.; Conditional lethal (suicidal) genetic constructs were designed and employed in strains of Pseudomonads as models for containment of genetically-engineered microbes that may be deliberately released into the environment . A strain of Pseudomonas putida was formed with a suicide vector designated pBAP24h that was constructed by cloning the host killing gene (hok) into the RSF1010 plasmid pVDtac24 and placing it under the control of the tac promoter . After hok induction in P . putida only 40% of surviving cells continued to bear the hok sequences within 4 h of induction; in contrast, 100% of the cells in uninduced controls bore hok . A few survivors that demonstrated resistance to hok-induced killing developed in P . putida, which may have been due to a mutation or physiological adaptation that rendered the membrane 'resistant' to hok . Conditional lethal strains of P . putida also were formed by inserting gef (a chromosomal homolog of hok) under the control of the tac promoter into the chromosome using a transposon . Constructs with chromosomal gef, as well as an RK2-derived plasmid construct containing gef, were only marginally more stable than the hok constructs; they were effective in killing P . putida when induced and within 2 h post-induction killing from either gef construct resulted in a 10(3)-10(5)-fold reduction in viable cell count compared to uninduced controls. Mol Ecol, 1992 Aug, 1(2), 89 - 94 Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2,4-dichlorophenoxyacetate; Short KA et al.; The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA) . P . putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA . PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period . No significant degradation of PAA occurred in soil in