P. putida

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 inoculated with the parental strain P . putida PP0301 or the inducible 2,4-D degrader P . putida PP0301(pR0101) . Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded . P . putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence . In unamended soil, survival of the plasmid-free parental strain P . putida PP0301 was similar to the survival of the GEM strain P . putida PP0301(pR0103) . However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.

Proteins, 1992 Aug, 13(4), 336 - 51
The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution; Mattevi A et al.; The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution . The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit . A crystallographic 2-fold axis generates the dimer which is observed in solution . The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2 . The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region . The C-terminus has been proven to be important for activity by site-directed mutagenesis . However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated . In this crystal form LipDH Val contains one NAD+ molecule per subunit . Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase . However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution . Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit . Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes . For instance, the two subunits in the dimer are shifted by 6 A with respect to each other . So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH . This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.

Appl Environ Microbiol, 1992 Aug, 58(8), 2531 - 5
Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli; Dong FM et al.; Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation . They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively . The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.

Biochem J, 1992 Jul 15, 285 ( Pt 2), 661 - 6
Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein; Silvestrini MC et al.; Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida . The purified recombinant enzyme contains haem c but no haem d1 . Nonetheless, like the holoenzyme from Ps . aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1) . Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps . aeruginosa (namely optical and c.d . spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).

J Bacteriol, 1992 Jul, 174(14), 4638 - 46
Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of promoters for the edd-eda operon; Egan SE et al.; The nucleotide sequence of the entire Escherichia coli edd-eda region that encodes the enzymes of the Entner-Doudoroff pathway was determined . The edd structural gene begins 236 bases downstream of zwf . The eda structural gene begins 34 bases downstream of edd . The edd reading frame is 1,809 bases long and encodes the 602-amino-acid, 64,446-Da protein 6-phosphogluconate dehydratase . The deduced primary amino acid sequences of the E . coli and Zymomonas mobilis dehydratase enzymes are highly conserved . The eda reading frame is 642 bases long and encodes the 213-amino-acid, 22,283-Da protein 2-keto-3-deoxy-6-phosphogluconate aldolase . This enzyme had been previously purified and sequenced by others on the basis of its related enzyme activity, 2-keto-4-hydroxyglutarate aldolase . The data presented here provide proof that the two enzymes are identical . The primary amino acid sequences of the E . coli, Z . mobilis, and Pseudomonas putida aldolase enzymes are highly conserved . When E . coli is grown on gluconate, the edd and eda genes are cotranscribed . Four putative promoters within the edd-eda region were identified by transcript mapping and computer analysis . P1, located upstream of edd, appears to be the primary gluconate-responsive promoter of the edd-eda operon, responsible for induction of the Entner-Doudoroff pathway, as mediated by the gntR product . High basal expression of eda is explained by constitutive transcription from P2, P3, and/or P4 but not P1.

Appl Environ Microbiol, 1992 Jul, 58(7), 2237 - 44
Biodegradation of mixtures of substituted benzenes by Pseudomonas sp . strain JS150; Haigler BE et al.; Pseudomonas sp . strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp . strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds . Pseudomonas sp . strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes . We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously . Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150 . Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida . The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols . Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture . Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol . These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.

J Bacteriol, 1992 Jul, 174(14), 4657 - 66
Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida; Parales RE et al.; beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway . This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme . The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter . DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon . A comparison of the deduced amino acid sequence of the P . putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution . Conserved functional groups important to the catalytic activity of CoA transferases were also identified.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1317 - 23
Lack of homology between two haloacetate dehalogenase genes encoded on a plasmid from Moraxella sp . strain B; Kawasaki H et al.; Two genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxella sp . strain B . H-1 predominantly acts on fluoroacetate, but H-2 does not . To elucidate the molecular relationship between the two enzymes, we compared their structural genes . Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined . The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass . The genes for H-1 and H-2, designated dehH1 and dehH2, respectively, had different sizes (885 bp and 675 bp) and G+C contents (58.3% and 53.4%) . Sequence analysis revealed no homology between the two genes . We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship . The deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalogenase of Xanthobacter autotrophicus . The dehH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of dehH2 from the plasmid.

Mikrobiol Zh, 1992 Jul-Aug, 54(4), 43 - 8
{The effect of physicochemical factors on the growth of Pseudomonas putida BS-2 on a medium with diethylene glycol}; Sedina SA; The physicochemical factors of medium have been studied for their effect on the physiological indices of growth of Pseudomonas putida BS-2 culture utilizing diethylenglycol as the only source of carbon . Action of the supraoptimal temperature on the growth process of P . putida BS-2 is accompanied by a decrease (more than twice) in economic coefficient of substrate and specific growth rate as compared with their maximal values . Dependences of specific growth rate of P . putida BS-2 in the medium with diethylenglycol on the presence of NaCl in it within the range of its concentrations from 0 to 4% and methanol in the concentration range of 0-20 g/l follow the noncompetitive inhibition equation . When NaCl concentration in the medium is more than 4%, complete separation of constructive and energy metabolism processes is observed.

Z Naturforsch {C}, 1992 Jul-Aug, 47(7-8), 487 - 502
{Pyoverdins from Pseudomonas putida}; Gwose I et al.; The structures of two pyoverdins (Pp1 and Pp2) and one dihydropyoverdin (dihydro-Pp2) from a strain of Pseudomonas putida have been elucidated by spectroscopic methods and degradation studies . The pyoverdins Pp1 and Pp2 consist of a chromophore which was identified as (1S)-5-amino-2,3-dihydro-8,9-dihydroxy-1 H-pyrimido{1,2-a}quinoline-1- carboxylic acid substituted at the amino group with a 3-carboxypropanoyl or a succinamoyl residue and at the carboxy group with the N-terminus of L-Ser-L-Thr-D-Ser-L-Orn-L-threo-(OH)Asp-{D-Glu + L-Dab}*-L-Ser-D-allo-Thr- L-c(OH)Orn . Dihydro-Pp2 differs from Pp2 only in the chromophore, which is saturated at carbons 5 and 6 . All compounds contain a tetrahydropyrimidine moiety ({D-Gln + L-Dab}*) resulting from the condensation of 2,4-diaminobutyric acid and glutamine.

Mol Microbiol, 1992 Jul, 6(13), 1785 - 99
Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms; Antoine R et al.; A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87 . After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B . bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation . Conjugation was possible only when the IncP group transfer functions were provided in trans . As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups . DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization . Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria . In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids . We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms . Determination of the plasmid copy number in E . coli and B . pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.

Gene, 1992 Jun 15, 115(1-2), 19 - 25
Histidine ammonia-lyase from Streptomyces griseus; Wu PC et al.; Histidine ammonia-lyase (histidase; HutH) has been purified to homogeneity from Streptomyces griseus and the N-terminal amino acid (aa) sequence used to clone the histidase-encoding structural gene, hutH . The purified enzyme shows typical saturation kinetics and is inhibited competitively by D-histidine and histidinol phosphate . High concentrations of K.cyanide inactivate HutH unless the enzyme is protected by the substrate or histidinol phosphate . On the basis of the nucleotide sequence, the hutH structural gene would encode a protein of 53 kDa with an N terminus identical to that determined for the purified enzyme . Immediately upstream from hutH is a region that strongly resembles a class of Streptomyces promoters active during vegetative growth; however, there is no obvious ribosome-binding site adjacent to the hutH translation start codon . The deduced aa sequence of an upstream partial open reading frame shows no similarity with other proteins, including HutP of Bacillus subtilis and HutU of Pseudomonas putida . Promoter-probe analysis indicates that promoter activity maps within the DNA surrounding the hutH start codon . Pairwise comparisons of the primary structures of bacterial and mammalian histidases, together with the unique kinetic properties and gene organization, suggest that streptomycete histidase may represent a distinct family of histidases.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5567 - 71
Crystallization and preliminary x-ray diffraction analysis of P450terp and the hemoprotein domain of P450BM-3, enzymes belonging to two distinct classes of the cytochrome P450 superfamily; Boddupalli SS et al.; Cytochromes P450 are members of a superfamily of hemoproteins that are involved in the metabolism of various physiologic and xenobiotic organic compounds . This superfamily of proteins can be divided into two classes based on the electron donor proximal to the P450: an iron-sulfur protein for class I P450s or a flavoprotein for class II . The only known tertiary structure of any of the cytochromes P450 is that of P450cam, a class I soluble enzyme isolated from Pseudomonas putida (product of the CYP101 gene) . To understand the details of the structure-function relationships within and between the two classes, structural studies on additional cytochromes P450 are crucial . We report here characterization of the crystal forms of two soluble, bacterial enzymes: cytochrome P450terp {class I enzyme from a Pseudomonas species (product of CYP108 gene)} and the hemoprotein domain of cytochrome P450BM-3 {class II enzyme from Bacillus megaterium (product of the CYP102 gene)} . The crystals of cytochrome P450terp are hexagonal and belong to the space group P6(1)22 (or its enantiomorph, P6(5)22) with unit cell dimensions a = b = 68.9 A and c = 458.7 A . The crystals of the hemoprotein domain of cytochrome P450BM-3 are monoclinic and belong to the space group P2(1) with unit cell dimensions a = 59.4 A, b = 154.0 A, c = 62.2 A, and beta = 94.7 degrees . Diffraction data for the crystals of these two proteins were obtained to a resolution better than 2.2 A . Assuming the presence of two molecules in the asymmetric unit for the hemoprotein domain of P450BM-3 and one molecule for P450terp, the calculated values of Vm are 2.6 and 3.3 A3/Da, respectively.

J Biol Chem, 1992 Jun 5, 267(16), 11120 - 5
Cloning, sequencing, and overexpression of a {2Fe-2S} ferredoxin gene from Escherichia coli; Ta DT et al.; Escherichia coli contains a soluble, {2Fe-2S} ferredoxin of unknown function (Knoell, H.-E., and Knappe, J . (1974) Eur . J . Biochem . 50, 245-252) . Using antiserum to the purified protein to screen E . coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein . The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358) . The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam . The overall sequence identity is approximately 36% when compared with human mitochondrial and P . putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster . The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein . The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P . putida ferredoxin.

Appl Environ Microbiol, 1992 Jun, 58(6), 1874 - 7
Naphthalene degradation via salicylate and gentisate by Rhodococcus sp . strain B4; Grund E et al.; Rhodococcus sp . strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy . Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene . In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp . strain B4 . The key enzymes of naphthalene degradation in Rhodococcus sp . strain B4 have unusual cofactor requirements . The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.

Appl Environ Microbiol, 1992 Jun, 58(6), 1847 - 52
Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity; Heipieper HJ et al.; A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8 . The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, and infrared absorption spectrometry . Growing cells of P . putida P8 reacted to the presence of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids, thereby increasing the degree of saturation . At phenol concentrations which completely inhibited the growth of P . putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid . This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate . The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system . The cis-to-trans modification of the fatty acids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols, thereby compensating for the elevation of membrane permeability induced by these toxic substances.

Mol Gen Genet, 1992 Jun, 233(3), 419 - 26
Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid of Pseudomonas putida; Gomada M et al.; Transcription from the promoter of a positive regulatory gene, xylS, on the TOL plasmid of Pseudomonas putida is activated by another positive regulator, XylR, in the presence of m-xylene and is dependent on RNA polymerase containing the NtrA protein (sigma 54) . Deletion analysis of the upstream region of the xylS gene revealed an upstream regulatory sequence (URS), located between 145 and 188 bp upstream from the transcription start site . The URS is active in either orientation and can be placed 3.9 kb further upstream without loss of activity . Dependence of activation on helical periodicity was observed in the region between the URS and the promoter of the xylS gene, suggesting DNA loop formation between these two sites, which are located about 100 bp apart . The expression of xylR was autogenously repressed by XylR protein . This autogenous repression is decreased in an NtrA- background, irrespective of the presence of the xylS promoter in cis, indicating that NtrA protein, or NtrA-containing RNA polymerase that is not bound to the xylS promoter, is involved in the binding of XylR protein to the URS.

Biol Chem Hoppe Seyler, 1992 Jun, 373(6), 343 - 9
Microbial metabolism of quinoline and related compounds . XIV . Purification and properties of 1H-3-hydroxy-4-oxoquinoline oxygenase, a new extradiol cleavage enzyme from Pseudomonas putida strain 33/1; Block DW et al.; 1H-3-Hydroxy-4-oxoquinoline oxygenase was purified to apparent homogeneity from Pseudomonas putida strain 33/1 which can use 1H-4-oxoquinoline as sole source of carbon . The molecular mass of the enzyme was determined to 26,000 Da by gel chromatography and by SDS polyacrylamide gel electrophoresis . The enzyme is labile at temperatures above 30 degrees C and has a pH optimum of 8.0 . It requires oxygen for the reaction and is significantly inhibited by metal ions like Cu2+, Zn2+, Hg2+ and by 4-chloromercuribenzoate . The enzyme is specific only for 1H-3-Hydroxy-4-oxoquinoline; the apparent Km value for this substrate is 24 microM.

Can J Microbiol, 1992 Jun, 38(6), 520 - 5
Slow rehydration improves the recovery of dried bacterial populations; Kosanke JW et al.; Slow rehydration of bacteria from dried inoculant formulations provided higher viable counts than did rapid rehydration . Estimates were higher when clay and peat powder formulations of Rhizobium meliloti, Rhizobium leguminosarum biovar trifolii, and Pseudomonas putida, with water activities between 0.280 and 0.650, were slowly rehydrated to water activities of approximately 0.992 before continuing the dilution plating sequence . Rhizobium meliloti populations averaged 6.8 x 10(8) cfu/g and 1328 cfu/alfalfa seed greater when slowly rehydrated from bulk powder and preinoculated seeds, respectively . Bulk powder samples were slowly rehydrated to 0.992 water activity by the gradual addition of diluent, followed by a 10-min period for moisture equilibration . Preinoculated seed samples were placed in an environmental chamber at 24 degrees C with relative humidity greater than 80% for 1 h to allow moisture absorption . "Upshock," osmotic cellular stresses that occur during rehydration, was reduced when dried microbial formulations were slowly rehydrated and equilibrated before becoming fully hydrated in the dilution plating sequence . These procedures may also be applicable when estimating total viable bacterial populations from dried soil or other dry formulations.

Microb Releases, 1992 Jun, 1(1), 35 - 9
Bioluminescence-based detection of genetically engineered microorganisms in nonsterile river water; Heller S et al.; The luminescence genes of the marine bacterium Vibrio fischeri were cloned into a lac expression vector and introduced into Escherichia coli and Pseudomonas putida . Survival of the cells in river water samples was monitored by light measurements . Whereas E . coli survived in sterilized river water for more than 29 days, it died off in nonsterile river water after 9 to 13 days . The engineered P . putida cells survived in nonsterile river water for more than 137 days . The detection limit for E . coli was 11 cells/ml.

Biochem J, 1992 May 15, 284 ( Pt 1), 87 - 93
Molecular biology of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4; Murdiyatmo U et al.; The structural gene (hdl IVa) for the Pseudomonas cepacia MBA4 2-haloacid halidohydrolase IVa (Hdl IVa) was isolated on a 1.6 kb fragment of Ps . cepacia MBA4 chromosomal DNA . The recombinant halidohydrolase was expressed in Escherichia coli and Pseudomonas putida and the structural gene was subcloned on to the tac expression vector pBTac1 . High-level expression from the tac promoter was seen to be temperature-dependent, a consequence of the nucleotide sequence adjacent to the fragment encoding the halidohydrolase . The nucleotide sequence of the fragment encoding the Hdl IVa was determined and analysed . Three ATG codons were identified in one of the open reading frames and the one corresponding to the start of the hdl IVa structural gene was determined by comparison of the predicted amino acid sequences with the experimentally determined N-terminal sequences of halidohydrolase IVa . The hdl IVa gene encoded a 231-amino acid-residue protein of M(r) 25,900 . The sequence and predicted structural data are discussed and comparison is made with sequence data for other halidohydrolases.

Biochemistry, 1992 May 12, 31(18), 4384 - 93
Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420; Wells AV et al.; High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported . The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate . In ferric, camphor-bound, P450 (mos), the E . coli-expressed P450 is found to be spectroscopically indistinguishable from the native material . Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state . A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states . In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron . Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands . The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb . Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state . The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding . Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis . Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.

Biol Chem Hoppe Seyler, 1992 May, 373(5), 249 - 54
Microbial metabolism of quinoline and related compounds . XIII . Purification and properties of 1H-4-oxoquinoline monooxygenase from Pseudomonas putida strain 33/1; Block DW et al.; 1H-4-Oxoquinoline monooxygenase was purified to homogeneity from Pseudomonas putida strain 33/1 which can use 1H-4-oxoquinoline as sole source of carbon and energy . The apparent M(r) of the native enzyme was determined to be 126,000 by gel chromatography . SDS polyacrylamide gel electrophoresis of the enzyme revealed one protein band corresponding to M(r) 42,000 . The enzyme consists of three probably identical subunits with a relative molecular mass of about 42,000 . The enzyme requires oxygen and NADH for the reaction and is significantly inhibited by metal ions like Cu2+, Zn2+, Hg2+ . The enzyme is specific only for 1H-4-oxoquinoline and the Km values of the enzyme for NADH and 1H-4-oxoquinoline were determined to be 87 microM and 25 microM, respectively.

Appl Environ Microbiol, 1992 May, 58(5), 1699 - 704
Acyloin formation by benzoylformate decarboxylase from Pseudomonas putida; Wilcocks R et al.; Whole cells and cell extracts of Pseudomonas putida grown in a medium containing ammonium mandelate have the capacity to produce the acyloin compound 2-hydroxypropiophenone when incubated with benzoylformate and acetaldehyde . Benzaldehyde and benzyl alcohol were formed as reaction by-products . The enantiomeric excess of the 2-hydroxypropiophenone product was found to be 91 to 92% . The absolute configuration of the enzymatically prepared product at the carbinol carbon was found to be S . The thiamine PPi-linked enzyme benzoylformate decarboxylase, purified to give a single protein band on polyacrylamide gel electrophoresis, was shown to be responsible for the catalysis of this novel condensation reaction.

Biochem J, 1992 May 1, 283 ( Pt 3), 789 - 94
Substrate-specificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by TOL plasmid pWW0 . Metabolic and mechanistic implications; Shaw JP et al.; The substrate-specificities of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, encoded by TOL plasmid pWW0 of Pseudomonas putida mt-2, were determined . The rates of benzyl alcohol dehydrogenase-catalysed oxidation of substituted benzyl alcohols and reduction of substituted benzaldehydes were independent of the electronic nature of the substituents at positions 3 and 4 . Substitutions at position 2 of benzyl alcohol affected the reactivity of benzyl alcohol dehydrogenase: the velocity of the benzyl alcohol dehydrogenase-catalysed oxidation was lower for compounds possessing electron-withdrawing substitutions . In the reverse reaction of benzyl alcohol dehydrogenase, none of the substitutions tested influenced the apparent kcat . values . The rates of benzaldehyde dehydrogenase-catalysed oxidation of substituted benzaldehydes were influenced by the electronic nature of the substitutions: electron-withdrawing groups at positions 3 and 4 favoured the oxidation of benzaldehydes . Substitution at position 2 of benzaldehyde greatly diminished the benzaldehyde dehydrogenase-catalysed oxidation . Substitution at position 2 with electron-donating groups essentially abolished reactivity, and only substitutions that were strongly electron-withdrawing, such as nitro and fluoro groups, permitted enzyme-catalysed oxidation . The influence of the electronic nature and the position of substitutions on the aromatic ring of the substrate on the velocity of the catalysed reactions provided some indications concerning the transition state during the oxidation of the substrates, and on the rate-limiting steps of the enzymes . Pseudomonas putida mt-2 containing TOL plasmid pWW0 cannot grow on toluene derivatives substituted at position 2, nor can it grow on 2-substituted benzyl alcohols or aldehydes . One of the reasons for this may be the substrate-specificities of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase.

J Bacteriol, 1992 May, 174(9), 2903 - 12
Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400; Erickson BD et al.; The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced . Six open reading frames were identified, four of which are homologous to the components of toluene dioxygenase from Pseudomonas putida F1 and have been named bphA, bphE, bphF, and bphG . From this comparison, biphenyl dioxygenase was found to be a multicomponent enzyme containing a two-subunit iron-sulfur protein, a ferredoxin, and a reductase . Comparison of the large subunit of the iron-sulfur protein and the ferredoxin with other multicomponent dioxygenases identified amino acid sequences similar to Rieske iron-sulfur proteins for binding a {2Fe-2S} cluster . Sequences have also been identified in the reductase component that match the consensus sequence for FAD or NAD binding . Transcription of the biphenyl dioxygenase region was examined, and three transcription initiation sites were identified . Transcription initiating at the site furthest upstream is greatly increased when the LB400 cells are grown on biphenyl as the sole carbon source.

J Bacteriol, 1992 May, 174(9), 2978 - 85
Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440; Ramos-Gonzalez MI et al.; Assessment of potential risks involved in the release of genetically engineered microorganisms is facilitated by the availability of monoclonal antibodies (MAbs), a tool potentially able to monitor specific organisms . We raised a bank of MAbs against the soil bacterium Pseudomonas putida 2440, which is a host for modified TOL plasmids and other recombinant plasmids . Three MAbs, 7.3B, 7.4D, and 7.5D, were highly specific and recognized only P . putida bacteria . Furthermore, we developed a semiquantitative dot blot assay that allowed us to detect as few as 100 cells per spot . A 40-kDa cell surface protein was the target for MAbs 7.4D and 7.5D . Detection of the cell antigen depended on the bacterial growth phase and culture medium . The O antigen of lipopolysaccharide seems to be the target for MAb 7.3B, and its in vivo detection was independent of the bacterial growth phase and culture medium . MAb 7.3B was used successfully to track P . putida (pWW0) released in unsterile lake mesocosms.

Appl Microbiol Biotechnol, 1992 May, 37(2), 252 - 9
Degradation of phenol and phenolic compounds by Pseudomonas putida EKII; Hinteregger C et al.; The phenol-degrading strain Pseudomonas putida EKII was isolated from a soil enrichment culture and utilized phenol up to 10.6 mM (1.0 g.l-1) as the sole source of carbon and energy . Furthermore, cresols, chlorophenols, 3,4-dimethylphenol, and 4-chloro-m-cresol were metabolized as sole substrates by phenol-grown resting cells of strain EKII . Under conditions of cell growth, degradation of these xenobiotics was achieved only in co-metabolism with phenol . Phenol hydroxylase activity was detectable in whole cells but not in cell-free extracts . The specificity of the hydroxylating enzyme was found during transformation of cresols and chlorophenols: ortho- and meta-substituted phenols were degraded via 3-substituted catechols, while degradation of para-substituted phenols proceeded via 4-substituted catechols . In cell-free extracts of phenol-grown cells a high level of catechol 2,3-dioxygenase as well as smaller amounts of 2-hydroxymuconic semialdehyde hydrolyase and catechol 1,2-dioxygenase were detected . The ring-cleaving enzymes were characterized after partial purification by DEAE-cellulose chromatography.

J Inorg Biochem, 1992 May 1, 46(2), 129 - 42
Effects of gold(I) antiarthritic drugs and related compounds on Pseudomonas putida; Rhodes MD et al.; The effects of the antiarthritic drugs aurothiomalate (AuTm), aurothioglucose (AuTg), auranofin, its metabolite triethylphosphinegold(I)thioglucose (Et3PAuTg), and several related complexes on the growth of Pseudomonas putida were studied . Two strains were used, one of which (BK135) was more sensitive to Et3PAuTg (tolerant up to 4 microM) than the other (BK403; tolerant to at least 500 microM) . Gold thiolate complexes and thiolate ligands alone had little effect on growth . Gold phosphine complexes increased the length of the lag phase of growth and reduced oxygen uptake . Marked changes in cellular morphology were determined by electron microscopy . Copper(II) compounds and aurothiomalate were synergistic in their growth inhibitory effects towards these bacteria . Experiments with 195Au suggested that a mechanism does not exist for the short term (minutes) uptake of gold by sensitive or resistant bacteria, but the resistant strain appeared to limit gold uptake over a longer term (hours).

J Biol Chem, 1992 Apr 25, 267(12), 8073 - 80
NMR studies of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins . Direct assignment of beta-cysteinyl carbon NMR resonances and further proton NMR assignments of cysteinyl and aromatic resonances; Cheng H et al.; Ferredoxins are proteins which contain iron and inorganic sulfide and are capable of electron transport . They are found in a wide range of organisms, from anaerobic bacteria, to plants and mammals . Although NMR spectroscopy has been used to study ferredoxins since the 1970s, little important structural or biochemical information has resulted from these investigations . The major difficulty has been the effect of the paramagnetic iron-sulfur clusters on the peptide resonances, hindering nuclear Overhauser effect (NOE) studies and causing broad line widths . These effects are most pronounced on resonances arising from the nuclei closest to the iron-sulfur center . Unfortunately, these are likely to be the most interesting nuclei, as they report the events and geometry in the vicinity of the active sites . In this paper, the first direct assignment of beta-cysteinyl 13C resonances for any iron-sulfur protein is reported for the spectrum of Pseudomonas putida ferredoxin . These resonances are of special significance, as they arise from the atoms on the protein closest to the iron centers, with the exception of the directly bound cysteinyl sulfur atoms . In addition, cysteinyl and ring system 1H NMR resonance assignments are made for the spectra of P . putida ferredoxin and Azotobacter vinelandii ferredoxin I.

Appl Environ Microbiol, 1992 Apr, 58(4), 1292 - 300
Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10; Hill KE et al.; This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance . Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom) . Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C . The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance . Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals . Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings . Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C . Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria . In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies . Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.

Appl Environ Microbiol, 1992 Apr, 58(4), 1142 - 52
Differential bioavailability of soil-sorbed naphthalene to two bacterial species; Guerin WF et al.; Prediction of the fate of hydrophobic organic contaminants in soils is complicated by the competing processes of sorption and biodegradation . To test the hypothesis that sorbed naphthalene is unavailable to degradative microorganisms, we developed a simple kinetic method to examine the rates and extents of naphthalene degradation in soil-free and soil-containing systems in a comparison of two bacterial species . The method is predicated on the first-order dependence of the initial mineralization rate on the naphthalene concentration when the latter is below the Michaelis-Menten half-saturation constant (Km) for naphthalene for the organism under study . Rates and extents of mineralization were estimated by nonlinear regression analysis of data by using both a simple first-order model and a three-parameter, coupled degradation-desorption model described for the first time here . Bioavailability assays with two bacterial species (Pseudomonas putida ATCC 17484 and a gram-negative soil isolate, designated NP-Alk) gave dramatically different results . For NP-Alk, sorption limited both the rate and extent of naphthalene mineralization, in accordance with values predicted on the basis of the equilibrium aqueous-phase naphthalene concentrations . For strain 17484, both the rates and extents of naphthalene mineralization exceeded the predicted values and resulted in enhanced rates of naphthalene desorption from the soils . We conclude that there are important organism-specific properties which make generalizations regarding the bioavailability of sorbed substrates inappropriate.

Mol Gen Genet, 1992 Apr, 232(3), 431 - 9
Expression and regulation of a dnaA homologue isolated from Pseudomonas putida; Ingmer H et al.; A gene homologous to the Escherichia coli dnaA gene was isolated from Pseudomonas putida and its transcription was investigated in E . coli as well as in P . putida . In both species the P . putida dnaA gene is transcribed from two promoters, one of which shows strong homology to promoters recognized by the sigma 54 factor found in both bacteria . In E . coli transcription of the P . putida dnaA gene can be repressed by overproduction of E . coli DnaA protein, presumably due to the presence of several DnaA-box-like sequences found in the promoter region . Likewise the P . putida DnaA protein is able to regulate expression of the E . coli dnaA gene but we failed to demonstrate autoregulation of the P . putida dnaA gene . A point mutation was introduced into the P . putida dnaA gene, equivalent to the ATP binding site mutation present in E . coli dnaA5 and dnaA46 mutants, and this alteration abolished the ability of the protein to repress the expression of the E . coli dnaA gene . These results indicate that DnaA proteins from other species than E . coli have maintained the ability to recognize the DnaA box sequence and that the conservation between the DnaA proteins reflects functionally similar domains.

J Gen Microbiol, 1992 Apr, 138 ( Pt 4), 675 - 83
Nucleotide sequence of the structural gene encoding a 2-haloalkanoic acid dehalogenase of Pseudomonas putida strain AJ1 and purification of the encoded protein; Jones DH et al.; The nucleotide sequence of a gene encoding an L-2-haloalkanoic acid halidohydrolase from Pseudomonas putida strain AJ1 was determined . The ORF (hadL) codes for a polypeptide of 227 amino acids (Mr 25,687) which has significant homology to two other L-2-haloalkanoic acid halidohydrolases of Pseudomonas sp., DehcI and DehcII; these show 38% and 51% amino acid identity respectively to HadL . All three enzymes produce products of an opposite optical configuration to that of the substrates . Comparison of the three sequences shows several highly conserved motifs which indicate the possible position of the enzyme active site . The enzyme was purified to homogeneity and appears to exist as a tetramer.

J Bacteriol, 1992 Apr, 174(8), 2612 - 9
Cloning and partial sequencing of an operon encoding two Pseudomonas putida haloalkanoate dehalogenases of opposite stereospecificity; Barth PT et al.; We have cloned fragments of DNA (up to 13 kb), from Pseudomonas putida AJ1, that code for two stereospecific haloalkanoate dehalogenases . These enzymes are highly specific for D and L substrates . The two genes, designated hadD and hadL, have been isolated and independently expressed in Escherichia coli and P . putida hosts by using broad-host-range vectors . They are closely adjacent and inducible in what appears to be an operon with an upstream open reading frame of unknown function . Nucleotide sequence determination of hadD predicts a mature, cytoplasmic protein of 300 amino acid residues (molecular weight of 33,601) . This has no significant homology with the L-specific haloalkanoate dehalogenases from Pseudomonas sp . strain CBS3 (B . Schneider, R . Muller, R . Frank, and F . Lingens, J . Bacteriol . 173:1530-1535, 1991) nor with any other known DNA or protein sequences.

J Bacteriol, 1992 Apr, 174(7), 2087 - 94
Identification of a cocaine esterase in a strain of Pseudomonas maltophilia; Britt AJ et al.; A strain of Pseudomonas maltophilia (termed MB11L) which was capable of using cocaine as its sole carbon and energy source was isolated by selective enrichment . An inducible esterase catalyzing the hydrolysis of cocaine to ecgonine methyl ester and benzoic acid was identified and purified 22-fold . In the presence of the solubilizing agent cholate, cocaine esterase had a native Mr of 110,000 and was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a monomer . In the absence of cholate, cocaine esterase had a native Mr of 410,000 and probably existed as a tetramer . The pH optimum of the enzyme was 8.0, and the Km values for cocaine, ethyl benzoate, and ethyl 2-hydroxybenzoate were 0.36, 1.89, and 1.75 mM, respectively . Inhibition studies indicated that the enzyme was a serine esterase, possibly possessing a cation-binding site similar to those of mammalian acetylcholinesterase and the atropine esterase of Pseudomonas putida PMBL-1 . The cocaine esterase of P . maltophilia MB11L showed no activity with atropine, despite the structural similarity of cocaine and atropine.

Mol Plant Microbe Interact, 1992 Mar-Apr, 5(2), 154 - 62
Genetic analysis of the aggA locus involved in agglutination and adherence of Pseudomonas putida, a beneficial fluorescent pseudomonad; Buell CR et al.; An isolate of Pseudomonas putida, which rapidly adheres to plant roots, is agglutinated by a glycoprotein from root surfaces . Agglutination is prevented and adherence to the root surface is diminished by Tn5 insertion in mutant 5123 . Two cosmid clones from wild type P . putida and a 2.7-kbp EcoRI-HindIII subclone present in both cosmid clones restored agglutinable to wild type levels in transconjugants of the nonagglutinable (Agg-) Tn5 mutant 5123 . These three clones increased agglutinability in transconjugants of the parental Agg+ isolate . The 2.7-kbp EcoRI-HindIII subclone restored adherence to bean root surfaces of 5123 to wild type levels in a short-term binding assay . Deletion analysis of the 2.7-kbp fragment indicated only 1.45 kbp was necessary for complementation of agglutinability in 5123 . This sequence, termed the aggA locus, contains an open reading frame of 1,356 nucleotides encoding a predicted 50,509-Da protein . The distribution of the aggA locus in plant-associated bacteria, as detected through Southern hybridization, is limited to bacteria that express the agglutination phenotype.

Mol Microbiol, 1992 Mar, 6(5), 629 - 34
Genes and their organization in the replication origin region of the bacterial chromosome; Ogasawara N et al.; Genes and their organization are conserved in the replication origin region of the bacterial chromosome . To determine the extent of the conserved region in Gram-positive and Gram-negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida . Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B . subtilis and P . putida, respectively . Eight consecutive P . putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B . subtilis . Altogether, 12 genes and their organization are conserved in B . subtilis and P . putida in the origin region . We found that the conserved region terminated on one side after the orf290 in P . putida (orf282 in B . subtilis) . In the B . subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram-positive bacteria were diverged from the Gram-negative bacteria . One of the ORFs is a duplicate of the conserved gene . The third non-translatable region containing multiple repeats of DnaA-box (second in the case of P . putida) was found flanking gidA in both organisms . This result shows clearly that E . coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.

J Bacteriol, 1992 Mar, 174(6), 1941 - 7
Localization and functional analysis of structural and regulatory dehalogenase genes carried on DEH from Pseudomonas putida PP3; Thomas AW et al.; Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII . The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location . From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned . Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions . The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P . putida and Escherichia coli . The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI) . Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA) . Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters . Expression of dehI in P . putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent . The intact DEH element transferred to P . putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA . This indicated that the cloned DEH element contained functions associated with recombination.

J Bacteriol, 1992 Mar, 174(6), 1932 - 40
The dehalogenase gene dehI from Pseudomonas putida PP3 is carried on an unusual mobile genetic element designated DEH; Thomas AW et al.; As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy . The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain PP3 . DEH recombined with target plasmid DNAs at high frequencies (e.g . 3.8 x 10(-4) per RP4.5 plasmid transferred) . The regulated expression of dehI was detected in P . putida, Pseudomonas aeruginosa, and Escherichia coli strains containing derivative plasmids of RP4.5 and pWW0 recombined with DEH . Movement of DEH from the unstable RP4 derivatives pNJ5000 and pMR5 resulted in the insertion of DEH into the chromosome of RecA+ strains of P . putida but not in RecA+ nor RecA- strains of E . coli . Rescue of DEH from the chromosome of P . putida KT2441 onto plasmid RP4 involved recombination at a frequency (2.7 x 10(-4) per RP4 plasmid transferred) comparable to that observed in strain PP3 . The DEH element was not classified as a conventional transposon because it did not move as a discrete DNA fragment: dehI-containing inserts in plasmid DNA targets varied in size between 6 and 13 kb . In addition, DEH exhibited a marked preference for insertion into a specific site on the plasmid pWW0, but its transposition, independent of host recombinational systems, remains to be demonstrated . However, the transposonlike characteristics of DEH included the conservation of restriction endonuclease sites, high-frequency recombination with different target replicons (plasmid and chromosomal DNA), and promiscuous insertion into plasmid RP4-based replicons . Therefore, it is proposed that DEH is an unusual mobile genetic element.

Nucleic Acids Res, 1992 Feb 25, 20(4), 839 - 45
Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop; Sandler SJ et al.; We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels . To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al . (1) . We found that the RecF proteins encoded by these two genes contain respectively 92% and 38% amino acid identity with the E . coli RecF protein . Additionally, we have found that the S . typhimurium and P . putida recF genes will complement an E . coli recF mutant, but the recF gene from Bacillus subtilis {showing about 20% identity with E . coli (2)} will not . Amino acid sequence alignment of the four proteins identified four highly conserved regions . Two of these regions are part of a putative phosphate binding loop . In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon . We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E . coli chromosome for its effect on sensitivity to UV irradiation . The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain . The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain . We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity . Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S . typhimurium and E . coli recF genes . Both chimeras could complement E . coli chromosomal recF mutations.

Appl Environ Microbiol, 1992 Feb, 58(2), 536 - 44
Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers; Huijberts GN et al.; The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied . PHAs isolated from P . putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition . In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate . The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance . The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis . Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature . These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis . Analysis of a P . putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.

J Chromatogr, 1992 Jan 31, 590(2), 247 - 53
Purification of pro- and eukaryotic superoxide dismutases by charge-controlled hydrophobic chromatography; Grunow M et al.; The process of purifying superoxide dismutases was simplified using charge-controlled hydrophobic chromatography on 10-carboxydecyl Sepharose . In only one chromatographic step following ammonium sulphate precipitation, Fe-containing superoxide dismutase from Pseudomonas putida and Cu,Zn-containing superoxide dismutase from bovine erythrocytes were purified with an overall yield of about 70% to electrophoretic homogeneity . The specific activities of the crystalline enzyme preparations were expressed in McCord and Fridovich units and were 3000 and 3200 U/mg, respectively.

FASEB J, 1992 Jan 6, 6(2), 674 - 9
Cytochrome P450cam: crystallography, oxygen activation, and electron transfer; Poulos TL et al.; Several crystal structures of various substrate and inhibited complexes of the camphor monoxygenase, cytochrome P450cam from Pseudomonas putida, are now available . These structures, together with mutagenesis, biochemical, and biophysical studies, have allowed for a detailed penetration into the problem of how P450s activate molecular oxygen, control stereoselectivity, and transfer electrons . This review will provide a summary of the crystallographic work in light of what these structures have taught us about P450 function.

J Biol Chem, 1992 Jan 5, 267(1), 83 - 90
Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences; Gotoh O; The substrate recognition regions in cytochrome P450 family 2 (CYP2) proteins were inferred by group-to-group alignment of CYP2 sequences and those of bacterial P450s, including Pseudomonas putida P450 101A (P450cam), whose substrate-binding residues have been definitely identified by x-ray crystallography of a substrate-bound form (Poulos T . L., Finzel, B . C., and Howard, A . J . (1987) J . Mol . Biol . 195, 687-700) . The six putative substrate recognition sites, SRSs, thus identified are dispersively located along the primary structure and constitute about 16% of the total residues . All the reported point mutations and chimeric fragments that significantly affect the substrate specificities of the parental CYP2 enzymes fell within or overlapped some of the six SRSs . Analysis of nucleotide substitution patterns in closely related members in four subfamilies, CYP2A, 2B, 2C, and 2D, consistently indicated that the SRSs have accumulated more nonsynonymous (amino acid-changing) substitutions than the rest of the sequence . This observation supports the idea that diversification of duplicate genes of drug-metabolizing P450s occurs primarily in substrate recognition regions to cope with an increasing number of foreign compounds.

J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 181 - 7
DNA homology between siderophore genes from fluorescent pseudomonads; Rombel IT et al.; Many species of pseudomonads produce fluorescent siderophores involved in iron uptake . We have investigated the DNA homology between the siderophore synthesis genes of an opportunist animal pathogen, Pseudomonas aeruginosa, and three plant-associated species Pseudomonas syringae, Pseudomonas putida and Pseudomonas sp . B10 . There is extensive homology between the DNA from the different species, consistent with the suggestion that the different siderophore synthesis genes have evolved from the same ancestral set of genes . The existence of DNA homology allowed us to clone some of the siderophore synthesis genes from P . aeruginosa, and genetic mapping indicates that the cloned DNA lies in a locus previously identified as being involved in siderophore production.

Folia Microbiol (Praha), 1992, 37(2), 128 - 32
Effect of glucose and ribose on microbial degradation of the herbicide bromoxynil continuously added to soil; Vokounova M et al.; Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was continuously added to chernozem (Haplic typic) soil inoculated with a suspension of Pseudomonas putida capable of cometabolic decomposition of the compound in a hetero-continuous-flow cultivation setup . In the steady state, when glucose or ribose were simultaneously added, 90 and 47% of the added herbicide was degraded per day, respectively . If the saccharides were absent, only 10-27% of the herbicide was decomposed . Addition and removal of glucose feeding resulted in an increase and decrease, respectively, of the degradation intensity, irrespective of the amount of the bacterial decomposers present . Two degradation products, 3,5-dibromo-4-hydroxy-benzamide and 3,5-dibromo-4-hydroxybenzoic acid, were formed during cultivation . The total amount of bromine-containing compounds was reduced only in the presence of glucose.

Folia Microbiol (Praha), 1992, 37(2), 122 - 7
Degradation of the herbicide bromoxynil in Pseudomonas putida; Vokounova M et al.; Biological conversion of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was studied in a batch culture of Pseudomonas putida by using HPLC . The process had a cometabolic character and proceeded only in the presence of another, simultaneously metabolizable, carbon and energy source . The intensity of degradation correlated with the growth rate, the degradation stopping when the cosubstrate becomes exhausted or the pH value of the medium falls below 6.5 . In a medium with glucose, no lag phase longer than one day was observed concerning growth, sugar and herbicide consumption and formation of metabolic herbicide derivatives (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid) . In a medium with ribose, the initial lag of the above processes took 2 d . No formation of other degradation products was detected . Growth inhibition was proportional to the concentration of bromoxynil.

Arch Microbiol, 1992, 158(3), 176 - 82
Environmentally directed mutations in the dehalogenase system of Pseudomonas putida strain PP3; Thomas AW et al.; Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids--HAAs) which were toxic to P . putida; and/or the presence of a potential growth substrate . Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs . The mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH . Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P . putida from toxic compounds in its growth environment . Other mutations partially restored P . putida's dehalogenating capability under conditions where toxic substrates were absent . Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment.

J Bacteriol, 1992 Jan, 174(1), 78 - 83
Iron uptake and molecular recognition in Pseudomonas putida: receptor mapping with ferrichrome and its biomimetic analogs; Jurkevitch E et al.; The presence of an Fe(3+)-ferrichrome uptake system in fluorescent Pseudomonas spp . was demonstrated, and its structural requirements were mapped in Pseudomonas putida with the help of biomimetic ferrichrome analogs . Growth tests, 55Fe3+ uptake, and competition experiments demonstrated that the synthetic L-alanine derivative B5 inhibits the action of ferrichrome but does not facilitate Fe3+ transport, while the enantiomeric D-Ala derivative B6 fails to compete with ferrichrome . Contraction of the molecule's envelope by replacing L-Ala by glycine provided a synthetic carrier, B9, which fully simulates ferrichrome as a growth promoter . Sodium azide inhibited 55Fe3+ uptake of the Gly derivative B9, suggesting an active transport process . These data demonstrate the chiral discrimination of the ferrichrome receptor and its sensitivity to subtle structural changes . They further confirm that receptor binding is a necessary but not sufficient condition for Fe3+ uptake to occur and suggest that binding to the receptor and transport proteins might rely on different recognition patterns.

Schriftenr Ver Wasser Boden Lufthyg, 1992, 89, 255 - 68
{Use of bacterial toximeters with separate cell cultures for continuous water monitoring}; Fritz-Langen H et al.; Two commercially available bacterial toxicity monitors are compared . As test organisms pure cultures of Pseudomonas putida are used in both systems . The bacteria are grown continuously in turbidostatic ("Toxalarm", LAR, Berlin, Germany) or chemostatic ("Stiptox-norm", Siepmann und Teutscher, Gross-Umstadt, Germany) regulated cultures in a selective culture medium in nonsterile devices . Toxic substances can be detected by continuously working bacterial respiration tests . Oxygen consumption is the measuring parameter . The bacterial test suspension is mixed continuously in a fixed proportion with air-saturated test water in a measuring cell . The separate culturing of the bacteria and the carrying out of the tests ensures that neither the bacterial culture is endangered by toxic substances nor the sensitivity can be changed by poison adaptation . Results of "Toxalarm"-tests with several chemicals (e.g . Atrazine, Lindane, 2-Nitro-phenol, Sodiumpentachlorophenolate) are presented . The registration of an alarm event (River Rhine) by "Stiptox-norm" is shown.

Eur J Biochem, 1991 Dec 5, 202(2), 231 - 40
Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida; Palmer JA et al.; The third lipoamide dehydrogenase structural gene of Pseudomonas putida, lpd3, was isolated from a library of P . putida PpG2 DNA cloned in Escherichia coli TB1 . The nucleotide sequence of lpd3 and its flanking regions indicate that lpd3 is not part of an operon, which is unique for a prokaryotic lipoamide dehydrogenase . An open reading frame was found 207 bases upstream from the start of transcription, but is encoded on the strand opposite lpd3 . There is no evidence of an open reading frame immediately downstream from lpd3 . The coding region of lpd3 consists of 1401 bp, providing for 466 amino acids plus a stop codon with a G/C content of 62.4% . The transcriptional start site was located 33-bp upstream from the start of translation . The third lipoamide dehydrogenase (LPD-3) shares amino acid identity with the other two lipoamide dehydrogenases of P . putida, 45% with that of the 2-oxoglutarate dehydrogenase and pyruvate multienzyme complexes, and 45.9% with the lipoamide dehydrogenase of the branched-chain oxoacid complex . LPD-3 is more closely related to eukaryotic lipoamide dehydrogenases since it has 53.6% amino acid sequence identity with pig and human lipoamide dehydrogenases and 51.1% identity with yeast lipoamide dehydrogenase . LPD-3 was not produced in wild-type P . putida PpG2 under a variety of growth conditions . However, LPD-3 was produced in P . putida PpG2 carrying pSP14, a pKT240-based clone with the entire lpd3 gene plus 104 bases of the leader . The only demonstrated role of LPD-3 in P . putida is as a substitute for lipoamide dehydrogenase of the 2-oxoglutarate dehydrogenase and pyruvate multienzyme complexes when the latter is inactive or missing.

J Gen Microbiol, 1991 Dec, 137 ( Pt 12), 2911 - 8
Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways; Tricot C et al.; Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both . However, mutants affected in one of the pathways still grow on arginine as sole carbon source . Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route . Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism . Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine . Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline . These results, together with induction of ornithine cyclodeaminase when wild-type P . putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate . Thus in P . putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.

J Gen Microbiol, 1991 Dec, 137 ( Pt 12), 2769 - 79
Phenotypic variation of Pseudomonas putida and P . tolaasii affects attachment to Agaricus bisporus mycelium; Rainey PB; The effect of phenotypic variation on attachment of Pseudomonas tolaasii and P . putida to Agaricus bisporus mycelium was investigated . Quantitative studies demonstrated the ability of each isolate to attach rapidly and firmly to A . bisporus mycelium and significant differences in attachment of wild-type and phenotypic variant strains were observed . This was most pronounced in P . tolaasii, where the percentage attachment of the wild-type form was always greater than that of the phenotypic variant . The medium upon which the bacteria were cultured, prior to conducting an attachment assay, had a significant effect on their ability to attach . Attachment of the wild-type form of P . putida was enhanced when the assay was performed in the presence of CaCl2, suggesting the involvement of electrostatic forces . No correlation was observed between bacterial hydrophobicity and ability to attach to A . bisporus mycelium . Scanning electron microscopy confirmed the results obtained from the quantitative studies and provided further evidence for marked differences in the ability of the pseudomonads to attach to mycelium . Fibrillar structures and amorphous material were frequently associated with attached cells and appeared to anchor bacteria to each other and to the hyphal surface . A time-course study of attachment using transmission electron microscopy revealed the presence of uneven fibrillar material on the surface of cells . This material stained positive for polysaccharide and may be involved in ensuring rapid, firm attachment of the cells.

J Gen Microbiol, 1991 Dec, 137 ( Pt 12), 2761 - 8
Phenotypic variation of Pseudomonas putida and P . tolaasii affects the chemotactic response to Agaricus bisporus mycelial exudate; Grewal SI et al.; The chemotactic response of wild-type Pseudomonas putida and P . tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined . Both P . putida, the bacterium responsible for initiating basidiome development of A . bisporus, and P . tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A . bisporus mycelial exudate . The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants . Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10-20 min, whereas the wild-types took 45-60 min . The differences are partly explained by the more rapid swimming speed of the phenotypic variants . Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates . Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (Mr less than 2000) thermostable compounds . Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate.

Biol Chem Hoppe Seyler, 1991 Dec, 372(12), 1081 - 8
Microbial metabolism of quinoline and related compounds . XII . Isolation and characterization of the quinoline oxidoreductase from Rhodococcus spec . B1 compared with the quinoline oxidoreductase from Pseudomonas putida 86; Peschke B et al.; Quinoline oxidoreductase from Rhodococcus spec . B1 was purified 39-fold to apparent homogeneity in a 5-step procedure with a recovery of 26% . The Mr of the native enzyme as determined by gel chromatography was 300,000 . SDS polyacrylamide gel electrophoresis of the enzyme revealed 3 protein bands corresponding to Mr 82,000, 32,000, and 18,000 . The enzyme contains 1.3 atoms of molybdenum, 8 atoms of iron, 8 atoms of acid-labile sulphur, 2 molecules of FAD and 2 molecules of molybdopterin cytosine dinucleotide . Cyanide, 4-hydroxymercuribenzoate and methanol were effective as inhibitors . The amino-terminal protein sequences of the 3 subunits of quinoline oxidoreductase from Rhodococcus B1 compared to those of quinoline oxidoreductase from Pseudomonas putida 86 revealed no difference among 71 amino acids examined.

Appl Environ Microbiol, 1991 Nov, 57(11), 3361 - 6
Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111; Hernandez BS et al.; Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates . However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested . When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone . Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate . In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol . Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells . Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols . In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.

Appl Environ Microbiol, 1991 Nov, 57(11), 3156 - 62
Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways; Haigler BE et al.; Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group . We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene . Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry . Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene . Cells of Pseudomonas putida F1 and Pseudomonas sp . strain JS150 converted nitrobenzene to 3-nitrocatechol . Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism . P . putida 39/D, a mutant strain of P . putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene . This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150 . Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively) . Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol . The nitrocatechols were slowly degraded to unidentified metabolites . Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation . These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.

J Antibiot (Tokyo), 1991 Nov, 44(11), 1252 - 8
"In vitro" synthesis of different naturally-occurring, semisynthetic and synthetic penicillins using a new and effective enzymatic coupled system; Martinez-Blanco H et al.; Forty-seven different penicillins, including some of great clinical importance, have been synthesized "in vitro" by coupling the newly described enzyme phenylacetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase (AT) from Penicillium chrysogenum . Incubations were carried out at 30 degrees C in 50 mM HCl-Tris buffer pH 8.0 . The reaction mixtures contained 6-APA, CoA, ATP, dithiothreitol, Mg2+ and the corresponding penicillin side-chain precursor . This is the first description of the enzymatic synthesis of all the natural penicillins known, many of the semisynthetic until now reported, and some penicillins that could only be currently obtained by chemical synthesis . The efficiency of this prokaryotic-eukaryotic enzymatic-coupled system and its application to the synthesis of different beta-lactam antibiotics are discussed.

J Bacteriol, 1991 Nov, 173(21), 6811 - 9
Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane; Imai R et al.; Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane (gamma-HCH) . A genomic library of P . paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13 . After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected . A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca . 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene . Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment . The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences . The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

J Bacteriol, 1991 Nov, 173(22), 7077 - 83
Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp . strain P51; van der Meer JR et al.; Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp . strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280 . Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position . When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy . The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei . The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.

J Bacteriol, 1991 Nov, 173(21), 6849 - 58
Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus; Wong HC et al.; The xylose isomerase (xylA) and the xylulose kinase (xylB) genes from Streptomyces rubiginosus were isolated, and their nucleotide sequences were determined . The xylA and xylB genes encode proteins of 388 and 481 amino acids, respectively . These two genes are transcribed divergently from within a 114-nucleotide sequence separating the coding regions . Regulation of the xyl genes in S . rubiginosus was examined by fusing their promoters to the Pseudomonas putida catechol dioxygenase gene and integrating the fusions into the minicircle integration site on the S . rubiginosus chromosome . The expression of catechol dioxygenase was then measured under a variety of conditions . The results indicated that transcription of the xyl genes was induced by D-xylose and repressed by glucose . Data from quantitative S1 mapping were consistent with this conclusion and suggested that xylA had one and xylB had two transcription initiation sites . The transcription initiation site of xylA was 40 bp upstream of the coding region . The two transcription initiation sites of xylB were 20 and 41 bp 5' of its translation initiation codon . Under control of appropriate regulatory elements, the cloned xyl genes are capable of complementing either Escherichia coli xylose isomerase- or xylulose kinase-deficient strains . The deduced amino acid sequence of the S . rubiginosus xylA protein is highly homologous to sequences of other microbial xylose isomerases.

Appl Microbiol Biotechnol, 1991 Nov, 36(2), 222 - 7
Monitoring a genetically engineered bacterium in a freshwater environment by rapid enzymatic amplification of a synthetic DNA "number-plate"; Amici A et al.; In order to set up a sensitive and reliable detection method to monitor environmentally released genetically engineered microorganisms (GEMs) a 72-bp, double-stranded DNA fragment has been built by annealing and ligating four synthetic oligonucleotides . Binding sites for two 20-mer oligonucleotides are situated inside the DNA fragment, flanking the centre . Into the central part of the construction a 30-nucleotide identification sequence has been fitted . Thanks to the presence of the two oligonucleotide binding sites, the synthetic construction ("number-plate") can be submitted to enzymatic amplification using the polymerase chain reaction (PCR), thus enabling the identification system to take advantage of the outstanding sensitivity of this technique . When released into a freshwater microcosm, cells of Pseudomonas putida carrying a "number-plated" chromosome could be easily and rapidly detected merely by submitting boiled cell sediments to PCR amplification.

Mol Microbiol, 1991 Oct, 5(10), 2459 - 74
DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism; Horn JM et al.; The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates . We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols . Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways . The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa . The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e . xylC and alkH . Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members . The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis . However glutamine-487, also thought to be critical, is less well conserved . The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange . The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa . Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism . Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein . Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway . Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified . The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetika, 1991 Oct, 27(10), 1697 - 704
{Cloning of genes degrading 3-chlorobenzoate from Pseudomonas putida strain 87}; Kulakova AN et al.; The ability of Pseudomonas putida strain 87 to catabolize 3-chlorobenzoate was shown to be mediated by genes of pBS109 plasmid . The plasmid may be transferred by conjugation into P . aeruginosa PAO2175 . It seems possible that the pBS109 plasmid codes for pyrocatechase II specific for halogenated catechol, but not catechol . The genes specifying utilization of 3-chlorobenzoate from pBS109 plasmid were cloned in the 5.5 kb BgIII fragment by using broad-host cloning system . The resulting pBS110 plasmid was transferred into P . putida, which results in utilization of 3-chlorobenzoate by transconjugants.

Appl Environ Microbiol, 1991 Oct, 57(10), 2880 - 7
Subcloning of bph genes from Pseudomonas testosteroni B-356 in Pseudomonas putida and Escherichia coli: evidence for dehalogenation during initial attack on chlorobiphenyls; Ahmad D et al.; The bphA, -B, -C, and -D genes from Pseudomonas testosteroni B-356 were mapped to a 5.5-kb DNA fragment of cloned plasmids pDA1 and pDA2 by use of deletion and insertion mutants of these plasmids . The expression of each of these genes was evaluated in Escherichia coli and in Pseudomonas putida, and it was found that the bphC and bphD genes are well expressed in both E . coli and P . putida cells while the bphA and bphB genes are very poorly expressed in E . coli, even when placed downstream of a tac promotor . P . putida clones carrying the bphA gene were used to study the metabolites produced from 4,4'-dichlorobiphenyl, 2,2'-dichlorobiphenyl, and 2,4'-dichlorobiphenyl . It was shown that dehalogenation of 4-Cl and 2-Cl occurs in the course of the initial oxygenase attack on these molecules, which always occurs on carbons 2 and 3, independently of the positions of the chlorine atoms . Our data also suggest that in the case of polychlorobiphenyl congeners carrying chlorine atoms on both rings, it appears that, depending on the chlorine positions, dioxygenation will occur predominantly on one ring over the other . However, attack of the more resistant ring is not excluded, resulting in multiple conversion pathways.

FEMS Microbiol Lett, 1991 Sep 15, 67(1), 1 - 5
Synthesis of 3-furylmethylpenicillin using an enzymatic procedure; Ferrero O et al.; 3-Furylmethylpenicillin was synthesized in vitro from 3-furylacetic acid, 6-aminopenicillanic acid (6-APA), CoA, ATP and Mg2+ . The reaction was catalyzed in two steps by the enzymes phenyl-acetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum . PCL catalyzes the activation of 3-furylacetic acid to 3-furylacetyl-CoA (3-F-CoA) and AT acylates the amino group of 6-APA with the 3-furylacetyl moiety of 3-F-CoA, releasing CoA and 3-furylmethylpenicillin.

Can J Microbiol, 1991 Sep, 37(9), 682 - 91
Ecologically significant effects of Pseudomonas putida PPO301(pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, on microbial populations and processes in soil; Doyle JD et al.; Pseudomonas putida PPO301 (pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, affected microbial populations and processes in a nonsterile xeric soil . In soil amended with 2,4-dichlorophenoxyacetate (500 micrograms/g soil) and inoculated with PPO301 (pRO103), the rate of evolution of carbon dioxide was retarded for approximately 35 days; there was a transient increase in dehydrogenase activity; and the number of fungal propagules decreased below detection after 18 days . In unamended soil inoculated with PPO301(pRO103), the rate of evolution of carbon dioxide and the dehydrogenase activity were unaffected, and the numbers of fungal propagules were reduced by about two orders of magnitude . The numbers of total, spore-forming, and chitin-utilizing bacteria were reduced transiently in soil either amended or unamended with 2,4-dichlorophenoxyacetate and inoculated with PPO301(pRO103) . The activities of arylsulfatases and phosphatases in soil were not affected by the presence of PPO301(pRO103), either in the presence or absence of 2,4-dichlorophenoxyacetate . In soil amended with 2,4-dichlorophenoxyacetate and inoculated with the parental strain (PPO301) or not inoculated, the evolution of carbon dioxide, the numbers of fungal propagules and of total, spore-forming, and chitin-utilizing bacteria, and the dehydrogenase activity were not affected as in soil inoculated with PPO301(pRO103) . These results demonstrated that a genetically engineered microorganism, in the presence of the substrate on which its novel genes can function, is capable of inducing measurable ecological effects in soil.

J Bacteriol, 1991 Sep, 173(17), 5328 - 35
Construction of cloning cartridges for development of expression vectors in gram-negative bacteria; Yen KM; A cloning cartridge was constructed that can be inserted into a plasmid of choice to form an expression vector in which gene expression is inducible with an inexpensive inducer, sodium salicylate, at low concentrations . This cartridge consists of a 3.6-kb restriction fragment which contains the positive regulatory gene nahR from plasmid NAH7, a promoter, PG, that nahR regulates, a multiple cloning site, a transcription terminator, and a gene conferring tetracycline resistance . Within promoter PG of the cloning cartridge, a sequence of three nucleotides upstream of the ATG sequence encoding the initiation codon was altered to create an NdeI recognition site (CATATG) for cloning of the 5' end of a gene without affecting the distance between the transcription start site and the gene coding region . In addition, the 5' end of a gene can be converted into an NdeI recognition site without altering the amino acid sequence it encodes and then cloned into this cartridge for regulated expression . Several other synthetic restriction sites were also inserted downstream of the NdeI site for accepting the 3' end of a cloned gene . A derivative of this cloning cartridge lacking the NdeI sequence was also constructed for cloning and expression of a restriction fragment containing a gene(s) of unknown sequence . Use of the cloning cartridges in a broad-host-range plasmid has allowed successful cloning and inducible expression of several genes in all of the gram-negative bacterial tested to date . Protein production to at least 10% of the total soluble cell proteins was observed from a cloned gene expressed in Pseudomonas putida.

J Biol Chem, 1991 Aug 25, 266(24), 15832 - 8
Activation of the Pseudomonas TOL plasmid upper pathway operon . Identification of binding sites for the positive regulator XylR and for integration host factor protein; Abril MA et al.; Expression of the Pseudomonas putida TOL plasmid upper pathway operon requires a promoter that belongs to the -12/-24 class . Stimulation of transcription from this promoter is positively controlled by the effector-activated XylR protein and requires a form of RNA-polymerase holoenzyme containing the RpoN-encoded sigma factor, sigma 54 . XylR-dependent stimulation of transcription from the Pseudomonas TOL upper pathway promoter was examined using deletions, insertions, and in vivo dimethyl sulfate footprinting . Two upstream activator sequences were identified in the -160 (UAS1) and -130 (UAS2) regions . Deletion of these two regions abolished transcription activation, although conservation of the UAS2 element alone allowed limited transcription stimulation . Separation of UAS1 from UAS2 by half a turn or a full turn significantly reduced XylR stimulation of transcription from the upper pathway operon promoter . An inverted repeated ATTTGN2CAAAT (where N is any nucleoside), which most likely represented the XylR recognition sequence, was identified . Binding of XylR was observed in vivo in the absence of effector, but changes in the binding pattern were induced in the presence of m-methylbenzyl alcohol, a XylR effector . In vivo footprinting analysis revealed that changes in the methylation pattern of G and T also occurred in the -50 to -90 region, which is probably occupied by integration host factor (IHF) protein . IHF was required for maximal expression from the TOL upper pathway operon promoter in Escherichia coli . Separation of the IHF site from UAS2 by a full helix turn did not significantly affect stimulation of transcription, which is consistent with this region playing a conformational role, rather than a regulatory one, in promoter function.

J Bacteriol, 1991 Aug, 173(16), 4970 - 6
Purification of glucose-inducible outer membrane protein OprB of Pseudomonas putida and reconstitution of glucose-specific pores; Saravolac EG et al.; A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida . OprB was surface expressed in whole cells, had a high beta-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation . Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (KS = 110 mM) and other carbohydrates . However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.

J Bacteriol, 1991 Aug, 173(15), 4717 - 24
Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting; Rothmel RK et al.; CatR, a LysR family protein, positively regulates the Pseudomonas putida catBC operon, which is required for growth on benzoate as a sole carbon source . Transcriptional studies show that the catR and catBC promoters are divergent and overlapping by 2 bp . A beta-galactosidase promoter probe vector was constructed to analyze expression from the catR and catBC promoters under induced and uninduced conditions . As predicted, the catBC promoter is expressed only under induced conditions, while the catR promoter is constitutive . CatR has been shown to specifically bind the catRBC promoter region, and this property was used to devise a purification protocol for CatR . Linear M13 DNA containing the catRBC control region was covalently bound to cyanogen bromide-activated Sepharose in order to construct a DNA affinity column . Crude extracts containing hyperproduced CatR protein were then incubated with the affinity resin under binding conditions, and the CatR protein was eluted with 1 M NaCl . CatR was also purified by heparin-agarose chromatography . This highly purified protein was used for gel retardation and hydroxyl-radical footprinting studies . From this analysis, it was shown that CatR binds upstream of the catBC promoter within the transcribed region of catR.

Gene, 1991 Jul 31, 104(1), 91 - 4
Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1; Menn FM et al.; The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes . The latter form part of the tod operon and encode the enzymes responsible for the initial reactions in toluene degradation . The nucleotide (nt) sequence of todF was determined and the deduced amino acid (aa) sequence revealed that the hydrolase contains 276 aa with a Mr of 30,753 . The deduced aa sequence was 63.5% homologous to that reported for 2-hydroxymuconic semialdehyde hydrolase which is involved in phenol degradation by Pseudomonas CF600.

FEBS Lett, 1991 Jul 29, 286(1-2), 55 - 7
Cloning and sequencing the urocanase gene (hutU) from Pseudomonas putida; Fessenmaier M et al.; A clone harbouring the entire urocanase gene (hutU) was obtained from a genomic library of Pseudomonas putida using oligonucleotide probes synthesised on the basis of known flanking sequences . One subunit of urocanase consists of 556 amino acids and has a molecular mass of 60,771 Da.

Biochemistry, 1991 Jul 23, 30(29), 7349 - 58
Overproduction, purification, and characterization of chlorocatechol dioxygenase, a non-heme iron dioxygenase with broad substrate tolerance; Broderick JB et al.; We show here that purified chlorocatechol dioxygenase from Pseudomonas putida is able to oxygenate a wide range of substituted catechols with turnover numbers ranging from 2 to 29 s-1 . This enzyme efficiently cleaves substituted catechols bearing electron-donating or multiple electron-withdrawing groups in an intradiol manner with kcat/KM values between 0.2 x 10(7) and 1.4 x 10(7) M-1 s-1 . These unique catalytic properties prompted a comparison with the related but highly specific enzymes catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase . The chlorocatechol dioxygenase gene (clcA) from the Pseudomonas plasmid pAC27 was subcloned into the expression vector pKK223-3, allowing production of chlorocatechol dioxygenase to approximately 7-8% of total cellular protein . An average of 4 mg of purified enzyme has been obtained per gram of wet cells . Protein and iron analyses indicate an iron stoichiometry of 1 iron/57.5-kDa homodimer, alpha 2Fe . The electronic absorption spectrum contains a broad tyrosinate to iron charge transfer transition centered at 430 nm (epsilon = 3095 M-1 cm-1 based on iron concentration) which shifts to 490 nm (epsilon = 3380 M-1 cm-1) upon catechol binding . The resonance Raman spectrum of the native enzyme exhibits characteristic tyrosine ring vibrations . Electron paramagnetic resonance data for the resting enzyme (g = 4.25, 9.83) is consistent with high-spin iron (III) in a rhombic environment . This similarity between the spectroscopic properties of the Fe(III) centers in chlorocatechol dioxygenase and the more specific dioxygenases suggests a highly conserved catalytic site . We infer that the unique catalytic properties of chlorocatechol dioxygenase are due to other characteristics of its substrate binding pocket.

J Biol Chem, 1991 Jul 15, 266(20), 12921 - 31
Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum; Van Beeumen JJ et al.; The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ . The sequence is the first example of a diheme cytochrome in a flavocytochrome complex . Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome . The two halves also require a single residue internal deletion for alignment . The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit . The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT . It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits . The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase . There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution . This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas . We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R . K., Terpstra, P., and Hol, W . G . J . (1986) J . Mol . Biol . 187, 101-107) for nucleotide binding sites.

Biochim Biophys Acta, 1991 Jul 12, 1078(3), 321 - 5
Binding sites of pyridine in cytochrome P-450cam; Ristau O et al.; Careful titration of oxidized cytochrome P-450cam from Pseudomonas putida with pyridine revealed deviations of the Eadie plot from linearity in the substrate-bound as well as in the substrate-free protein . A binding model which assumes two binding sites for pyridine--the iron and the camphor binding site--is able to describe completely the nonlinear Eadie plot.

FEBS Lett, 1991 Jul 8, 285(1), 85 - 8
Divergent evolution of chloroplast-type ferredoxins; Harayama S et al.; The TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes required for the oxidation of toluene to Krebs cycle intermediates . The structural genes for these enzymes are encoded in two operons which comprise the xylCMABN and xylXYZLTEGFJQKIH genes, respectively . The function of the xylT gene has not yet been identified . The nucleotide sequence of xylT was determined in this study and putative gene product was shown to contain a sequence characteristic for chloroplast-type ferredoxins . The nahT gene, the homologue of xylT, present on NAH plasmid NAH7 encoding naphthalene-degrading enzymes, was also sequenced . The sequence conservation between xylT and nahT strongly suggests that both gene products have some physiological function . Chloroplast-type ferredoxins have been discovered in photosynthetic organisms (plants, algae, cyanobacteria and Rhodobacter) and Halobacterium species . Furthermore, chloroplast-type ferredoxin-like sequences have been found in the electron-transfer components of some oxygenases . The sequences of XylT and NahT were compared with those of the previously identified chloroplast-type ferredoxins, in order to examine their evolutionary relationships.

Appl Environ Microbiol, 1991 Jul, 57(7), 1905 - 13
Use of a xylE marker gene to monitor survival of recombinant Pseudomonas putida populations in lake water by culture on nonselective media; Winstanley C et al.; IncQ marker plasmids were previously constructed to enable the analysis of the survival of populations of Pseudomonas putida released into lake water (C . Winstanley, J . A . W . Morgan, R . W . Pickup, J . G . Jones, and J . R . Saunders, Appl . Environ . Microbiol . 55:771-777, 1989) . We constructed equivalent IncP plasmids, pLV1016 and pLV1017, to provide conjugative alternative systems . Detection of the xylE gene carried by marker plasmids was found to be a valid indicator to use for studying the survival of released populations by culturing on nonselective media . These plasmids were used to study the survival of populations of Pseudomonas putida in both sterile and untreated lake water . The effects of inoculum size, the metabolic burden imposed on the cell by the unregulated expression of xylE, and an auxotrophic mutation carried by the host strain were studied . We also assessed the reproducibility and hence the predictability of the survival of released populations . Model systems with a single lake water sample and model systems with three different lake water samples, taken from the same site in consecutive months, were used to analyze variability between replicates and to assess differences caused by host strain or water sample . A large variability was found depending on which water sample was used . These findings imply that it will be difficult to predict accurately the survival of released populations in the natural environment.

J Biol Chem, 1991 Jun 25, 266(18), 11939 - 46
Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis; Graham-Lorence S et al.; The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase . As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template . Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate . To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450) . When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type . These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket . The next set of mutants were designed to determine the importance of Glu302 in catalysis . Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined . It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity . On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax . These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.

Gene, 1991 Jun 15, 102(1), 13 - 8
Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp . EST1001: expression in Escherichia coli and Pseudomonas putida; Nurk A et al.; The plasmid pEST1412 contains the genes, pheA and pheB, encoding phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12}), respectively . Thse were originally cloned from the plasmid DNA of Pseudomonas sp . EST1001 {Kivisaar et al., Plasmid 24 (1990) 25-36} . Although pheA and pheB are cotranscribed using the promoter sequences derived from Tn4652 and the level of expression of C120 activities from pEST1412 was equal both in Escherichia coli and in Pseudomonas putida, the level of PMO activity measured in the cell-free extracts of E . coli was lower than that in P . putida . The nucleotide sequence of the 2.0-kb PstI-HindIII fragment of pEST1412 carrying pheA was determined . A 1821-bp ORF was found in this DNA . The structural gene (tfdB) encoding 2,4-dichlorophenol hydroxylase from pJP4 has been sequenced {Perkins et al., J . Bacteriol . 172 (1990) 2351-2359} . Comparison of the deduced amino acid sequences of tfdB and pheA revealed highly conserved regions in the protein products of these genes.

J Biol Chem, 1991 Jun 5, 266(16), 10246 - 8
Observation of the O-O stretching Raman band for cytochrome P-450cam under catalytic conditions; Egawa T et al.; Dioxygen stretching (voo) Raman band was observed for the oxy form of Pseudomonas putida cytochrome P-450 (P-450cam) generated at room temperature under catalytic conditions, that is, in the presence of D-camphor, beta-NADH, putidaredoxin, and putidaredoxin reductase, by using the mixed flow transient Raman apparatus . At the same time the visible absorption spectra were monitored for the transient species . It was found that the voo frequency is little altered by binding of putidaredoxin to P-450cam, although the reduction rate of the oxy form becomes faster . Another intermediate with an oxygen isotope-sensitive band was not found in a time region until 2 s after mixing of the reduced enzyme with oxygen.

J Bacteriol, 1991 Jun, 173(12), 3700 - 8
Characterization of the Pseudomonas sp . strain P51 gene tcbR, a LysR-type transcriptional activator of the tcbCDEF chlorocatechol oxidative operon, and analysis of the regulatory region; van der Meer JR et al.; Plasmid pP51 of Pseudomonas sp . strain P51 contains two gene clusters encoding the degradation of chlorinated benzenes, tcbAB and tcbCDEF . A regulatory gene, tcbR, was located upstream and divergently transcribed from the chlorocatechol oxidative gene cluster tcbCDEF . The tcbR gene was characterized by DNA sequencing and expression studies with Escherichia coli and pET8c and appeared to encode a 32-kDa protein . The activity of the tcbR gene product was analyzed in Pseudomonas putida KT2442, in which it appeared to function as a positive regulator of tcbC expression . Protein extracts of both E . coli overproducing TcbR and Pseudomonas sp . strain P51 showed specific DNA binding to the 150-bp region that is located between the tcbR and tcbC genes . Primer extension mapping demonstrated that the transcription start sites of tcbR and tcbC are located in this region and that the divergent promoter sequences of both genes overlap . Amino acid sequence comparisons indicated that TcbR is a member of the LysR family of transcriptional activator proteins and shares a high degree of homology with other activator proteins involved in regulating the metabolism of aromatic compounds.

Eur J Biochem, 1991 Jun 1, 198(2), 307 - 15
Primary and secondary structural patterns in eukaryotic cytochrome P-450 families correspond to structures of the helix-rich domain of Pseudomonas putida cytochrome P-450cam . Indications for a similar overall topology; Ouzounis CA et al.; An extensive sequence analysis of the eukaryotic cytochrome P-450 (P-450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P-450cam) . All sequences available on-line were collected, classified and aligned within families . Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi-alignment was constructed . Profile patterns of the most conserved regions were generated and screened against the sequence of P-450cam, the structure of which has been elucidated by X-ray crystallography . While some of these profiles did not map on the P-450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant . Our analysis suggests that the helix-rich domain with the cysteine pocket and the oxygen-binding site is conserved in all P-450 forms . Helices I and L from P-450cam can be easily identified in all eukaryotic P-450 forms . Other helices which seem to exist in all P-450 forms include helices C, D, G and J . K . In the helix-poor domain of P-450cam, only structures b3/b4 seem to have been conserved . The obvious sequence conservation throughout the helix-rich domain of the P-450cam protein might be expected for a molecular class whose overall topology is preserved . Additional support for the conservation of structure between eukaryotic cytochromes P-450 and P-450cam comes from secondary structure prediction of the eukaryotic sequences.

J Gen Microbiol, 1991 Jun, 137 ( Pt 6), 1375 - 85
Bioconversion of 2-hydroxy-6-oxo-6-(4'-chlorophenyl)hexa-2,4-dienoic acid, the meta-cleavage product of 4-chlorobiphenyl; Ahmad D et al.; Bacterial conversion of 4-chlorobiphenyl (4-CB) usually proceeds through a pathway involving an initial oxidation of the unsubstituted ring in the 2,3 position followed by a 1,2 meta-cleavage . The meta-cleavage product (MCP) is converted through a single hydrolysis step into chlorobenzoic acid . However, several other acidic metabolites that were not expected as part of this pathway have already been described . In this paper, we used strains of Pseudomonas putida carrying cloned genes from Pseudomonas testosteroni B-356 that are involved in polychlorinated biphenyl (PCB) degradation to demonstrate that several acidic metabolites found in the culture media of various bacteria grown in the presence of 4-CB result from alternative novel bioconversion pathways of MCP . The degradation products of MCP through these pathways were identified as analogues with saturated or shorter side chains or as 4'-chlorophenyl-2-picolinic acid; pathways leading to their formation are proposed.

J Gen Microbiol, 1991 Jun, 137 ( Pt 6), 1369 - 74
Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture; Duetz WA et al.; Pseudomonas putida mt-2, harbouring the TOL plasmid pWW0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates . The fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and xylene (TOL- cells), was determined . Genetic analysis revealed that all TOL- cells isolated harboured partially deleted plasmids, lacking the TOL catabolic genes . The growth-rate advantage of the TOL- cells was quantified from the kinetics of their increase as a fraction of the total population . At a dilution rate of 0.1 h-1 no growth-rate advantage of TOL- cells was found when phosphate or ammonium were limiting . Under sulphate-limitation, ingrowth of TOL- cells was evident but did not follow a straightforward pattern . Under succinate-limitation the growth-rate advantage was the highest, particularly at low dilution rates (about 50% at D = 0.05 h-1) . In phauxostat culture, at the maximal growth rate, the growth-rate advantage of TOL- cells was less than 1% . The specific activity in TOL+ cells of the plasmid-encoded enzyme catechol 2,3-dioxygenase was relatively high at a low growth rate.

J Gen Microbiol, 1991 Jun, 137 ( Pt 6), 1363 - 8
Mathematical analysis of catabolic function loss in a population of Pseudomonas putida mt-2 during non-limited growth on benzoate; Duetz WA et al.; Pseudomonas putida mt-2, harbouring the TOL plasmid PWW0, was grown continuously on benzoate in a phauxostat at a non-limited rate . The gradual decrease in the population carrying the complete TOL plasmid was caused predominantly by a growth-rate advantage of spontaneous mutants carrying a partially deleted plasmid (TOL- cells) . The growth-rate difference (v) was quantified both by measuring the increase in the dilution rate (from 0.68 to 0.79 h-1; v = 0.11 h-1) and by mathematical analysis of the ingrowth of TOL- cells (v = 0.12 h-1) . The latter procedure also established that the segregation rate was of the order of magnitude 10(-5) h-1 . Similar values for the growth-rate advantage and the segregation rate were found when both benzoate and succinate were present in non-limiting concentrations . It is suggested that the growth-rate disadvantage of the wild-type strain is caused by inhibitory effects of an intermediate in the degradation of benzoate via the plasmid-encoded meta-pathway.

J Bacteriol, 1991 Jun, 173(12), 3756 - 62
Up-promoter mutations in the trpBA operon of Pseudomonas aeruginosa; Han CY et al.; In Pseudomonas aeruginosa, the operon encoding tryptophan synthase (trpBA) is positively regulated by the TrpI protein and an intermediate in tryptophan biosynthesis, indoleglycerol phosphate (InGP) . A gene fusion in which the trpBA promoter directs expression of the Pseudomonas putida xylE gene was constructed . By using a P . putida F1 todE mutant carrying this fusion on a plasmid, three cis-acting mutations that increased xylE expression enough to allow the todE strain to grow on toluene were isolated . The level of xylE transcript from the trpBA promoter was increased in all three mutants . All three mutations are base substitutions located in the -10 region of the trpBA promoter; two of these mutations make the promoter sequence more like the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence . The activities of the wild-type and mutant trpBA promoters, as monitored by xylE expression, were assayed in P . putida PpG1 and in E . coli . The up-regulatory phenotypes of the mutants were maintained in the heterologous backgrounds, as was trpI and InGP dependence . These results indicate that the P . aeruginosa trpBA promoter has the key characteristics of a typical E . coli positively regulated promoter . The results also show that the P . aeruginosa and P . putida trpI activator gene products are functionally interchangeable.

FEMS Microbiol Lett, 1991 Jun 1, 65(1), 1 - 7
Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila; Rivero O et al.; Aeromonas virulence is thought to depend on multigenic functions . The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector . The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx . 68 kDa which is highly inhibited by PMSF . The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2 . We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments . The protein P2 was secreted into the periplasms of both P . putida and E . coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A . hydrophila SO2/2.

Appl Environ Microbiol, 1991 Jun, 57(6), 1790 - 2
Assay for detection and enumeration of genetically engineered microorganisms which is based on the activity of a deregulated 2,4-dichlorophenoxyacetate monooxygenase; King RJ et al.; An assay system was developed for the enumeration of genetically engineered microorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which converts phenoxyacetate (PAA) to phenol . In PAA-amended cultures of Pseudomonas aeruginosa PAO1C(pRO103) and Pseudomonas putida PPO301(pRO103), strains which express a deregulated TFD monooxygenase, phenol production was proportional to cell number . Phenol was reacted, under specific conditions, with a 4-aminoantipyrine dye to form an intensely colored dye-phenol complex (AAPPC), which when measured spectrophotometrically could detect as few as 10(3) cells per ml . This assay was corroborated by monitoring the disappearance of PAA and the accumulation of phenol by high-performance liquid chromatography and gas chromatography . The AAPPC assay was modified for use with plate cultures and clearly distinguished colonies of PPO301(pRO103) and PAO1C(pRO103) from a strain expressing a regulated TFD monooxygenase . Colonies of P . putida PPO301(pRO101) remained cream colored, while colonies of PPO301(pRO103) and PAO1C(pRO103) turned a distinct red.

J Chromatogr, 1991 May 31, 566(2), 493 - 8
Determination of biocatalyst consumption in an aminopeptidase process using automated sample preparation and high-performance liquid chromatography; Duchateau AL et al.; A rapid and sensitive method has been developed for the determination of the biocatalyst consumption in the chemo-enzymic production of optically pure natural and synthetic alpha-H-amino acids . It is based on automated sample preparation from an enzymic reaction mixture, reversed-phase high-performance liquid chromatographic separation, post-column reaction and fluorimetric detection . The assay procedure has been applied to the enzymic conversion of racemic norvaline amide into L-norvaline, catalysed by an L-specific aminopeptidase from Pseudomonas putida . Both norvaline amide and norvaline can be analysed in a single assay in the low nanogram range . The method yields reproducible results and requires 30 min from the time of sampling the enzymic reaction mixture to quantitation . The reaction mixture is automatically sampled and analysed several times during the course of the reaction . With the results obtained a conversion curve can be constructed from which the exact biocatalyst consumption can be calculated . By adaptation of the mobile phase, the method can also be applied to other amino acid amides used as substrates in the aminopeptidase reaction.

EMBO J, 1991 May, 10(5), 1159 - 67
An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid; de Lorenzo V et al.; Transcription from promoter Pu of the upper catabolic operon of the Pseudomonas putida TOL plasmid which specifies conversion of toluene/xylenes to benzoate/toluates is activated by the TOL-encoded regulator XylR protein in the presence of substrates of the catabolic pathway and in conjunction with the sigma 54(NtrA)-containing form of RNA polymerase . This regulatory circuit was faithfully reproduced in Escherichia coli in single copy gene dosage by integrating the corresponding controlling determinants into the chromosomes of several K12 derivatives by means of specialized transposons . In vivo monitoring of the activity of a Pu-lacZ fusion in E . coli strains with different genetic backgrounds demonstrated that integration host factor (IHF) is involved in Pu regulation and that hyperproduction of the XylR protein leads to a decrease of Pu activity in a manner in which deletion of the putative DNA-binding domain of the XylR does not impair its inhibitory effect when hyperproduced . One discrete IHF binding site and two potential XylR sites (consensus sequence 5'-TTGANCAAATC-3'), bracketted by short stretches of DNase I-hypersensitive bonds, were detected upstream of the transcription initiation site . A model accounting for the features found is proposed which includes the IHF-promoted looping of upstream XylR-DNA complexes so that they contact the sigma 54(NtrA)-RNA polymerase bound at -12/-24 positions.

J Biochem (Tokyo), 1991 May, 109(5), 791 - 7
Intermediate and mechanism of hydroxylation of o-iodophenol by salicylate hydroxylase; Suzuki K et al.; Salicylate hydroxylase {EC 1.14.13.1} from Pseudomonas putida catalyzes the hydroxylation of salicylate, and also o-aminophenol, o-nitrophenol, and o-halogenophenols, to catechol . The reactions with these o-substituted phenols comprise oxygenative deamination, denitration, and dehalogenation, respectively . The reaction stoichiometry, as to NADH oxidized, oxygen consumed, and catechol formed, is 2 : 1 : 1, respectively . The mechanisms for the deiodination and oxygenation of o-iodophenol were investigated in detail by the use of I(+)-trapping reagents such as DL-methionine, 2-chlorodimedone, and L-tyrosine . The addition of the traps did not change the molar ratio of catechol formed to NADH oxidized, nor iodinated traps produced were in the incubation mixture . The results suggest that I+ was not produced on the deiodination in the hydroxylation of o-iodophenol . On the other hand, L-ascorbate, L-epinephrine, and phenylhydrazine increased the molar ratio . o-Phenylenediamine decreased it, being converted to phenazine . This suggests that o-benzoquinone is formed in the oxidation of o-iodophenol as a nascent product . The quinone was detected spectrophotometrically by means of the stopped-flow method . Kinetic analysis of the reactions revealed that o-benzoquinone is reduced nonenzymatically to catechol by a second molecule of NADH . A mechanism of elimination for the ortho-substituted groups of substrate phenols by the enzyme is proposed and discussed.

J Bacteriol, 1991 May, 173(10), 3109 - 16
Cloning and sequence analysis of the LPD-glc structural gene of Pseudomonas putida; Palmer JA et al.; Pseudomonas putida is able to produce three lipoamide dehydrogenases: (i) LPD-glc, which is the E3 component of the pyruvate and 2-ketoglutarate dehydrogenase complexes and the L-factor for the glycine oxidation system; (ii) LPD-val, which is the specific E3 component of the branched-chain keto acid dehydrogenase complex and is induced by growth on leucine, isoleucine, or valine; and (iii) LPD-3, which was discovered in a lpdG mutant and whose role is unknown . Southern hybridization with an oligonucleotide probe encoding the highly conserved redox-active site produced three bands corresponding to the genes encoding these three lipoamide dehydrogenases . The complete structural gene for LPD-glc, lpdG, was isolated, and its nucleotide sequence was determined . The latter consists of 476 codons plus a stop codon, TAA . The structural gene for LPD-glc is preceded by a partial open reading frame with strong similarity to the E2 component of 2-ketoglutarate dehydrogenase of Escherichia coli . This suggests that lpdG is part of the 2-ketoglutarate dehydrogenase operon . LPD-glc was expressed in Pseudomonas putida JS348 from pHP4 which contains a partial open reading frame corresponding to the E2 component, 94 bases of noncoding DNA, and the structural gene for lpdG . This result indicates that lpdG can be expressed separately from the other genes of the operon.

Appl Environ Microbiol, 1991 May, 57(5), 1325 - 32
Expression, localization, and functional analysis of polychlorinated biphenyl degradation genes cbpABCD of Pseudomonas putida; Khan AA et al.; Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19 . The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls . These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540 . Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540 . Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E . coli containing the hybrid plasmid pAW540 . These enzyme activities were up to 10-fold higher than those found in P . putida OU83 . These results led us to conclude that cbpABCD genes of P . putida OU83 were encoded on cloned DNA and expressed in E . coli . Whether the expression of cbpABCD genes of P . putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8 . The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E . coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1991 Apr 15, 63(2-3), 291 - 5
A new family of bacterial regulatory proteins; Haydon DJ et al.; A new family of bacterial regulatory proteins has been identified by sequence similarity . The family contains the repressor of the Bacillus subtilis gluconate operon (GntR), the regulators for histidine utilization in Pseudomonas putida (HutCPp) and Klebsiella aerogenes (HutCKa), the repressor (FadR) of fatty acid degradation in Escherichia coli, a regulator involved in the conjugal transfer of the broad host range plasmid pIJ101 (KorA), and three proteins of unidentified function in E . coli (GenA, P30 and PhnF) . The proteins share amino acid sequence similarities in a 69-residue N-terminal region . A helix-turn-helix motif is predicted in the most highly-conserved segment of each protein suggesting that they are members of a new family of helix-turn-helix DNA-binding proteins.

Genetika, 1991 Apr, 27(4), 649 - 56
{Genetic control of tryptophan hypersynthesis in regulatory mutants of Pseudomonas putida}; Olekhnovich IN et al.; The 6-fluorotryptophan resistant MR1 mutant was obtained from Pseudomonas putida M30 (Tyr- Phe-) strain . The mutant was able to excrete tryptophan (60 micrograms/ml) and has derepressed aroF gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase . The mutation isolated was identified as aroR with the help of cloning early aroF gene of P . putida . On the next step of selection, regulatory mutant MR2 was obtained producing 240 micrograms/ml of tryptophan . The MR2 has derepressed unlinked trpE and trpDC genes and represents a mutant of the trpR type . Expression of the trpE gene of P . putida MR2 weakened in the presence of tryptophan excess in the medium, which points to attenuation of this gene . From the prototrophic variant of P . putida MR2 the MRP3 mutant producing 850 micrograms/ml of tryptophan was obtained . This mutant was characterized by twofold increase in the activity of the anthranilate synthase encoded by the trpE gene . The assay of the activity of tryptophanyl-tRNA synthase in P . putida MRP3 demonstrated that the mutant has TrpS+ phenotype.

Genetika, 1991 Apr, 27(4), 589 - 97
{Cloning of the Arthrobacter globiformis fcbA gene for dehalogenase and construction of a hybrid pathway of 4-chlorobenzoic acid degradation in Pseudomonas putida}; Plotnkova EG et al.; The Artrobacter globiformis KZT1 fcbA gene responsible for dehalogenase (4-chlorobenzoate-4-hydroxylase) activity was cloned in Escherichia coli and Pseudomonas putida cells . The character of the fcbA gene expression was studied . Notwithstanding amplification of the gene dose and control of the inducible Plac promoter, the level of substrate dehalogenation by recombinant E . coli strains was lower, as compared with that in the original KZT1 strain . Cloning of the fcbA gene in P . putida KZ6R cells utilizing 4HBA resulted in a recombinant pathway of 4CBA degradation, which proved more effective for substrate consumption, in comparison with the original KZT1 strain.

J Biochem (Tokyo), 1991 Apr, 109(4), 645 - 9
Degradation of L-djenkolate catalyzed by S-alkylcysteine alpha,beta-lyase from Pseudomonas putida; Kamitani H et al.; S-Alkylcysteine alpha, beta-lyase {EC 4.4.1.6} of Pseudomonas putida catalyzes alpha,beta-elimination of L-djenkolate {3,3'-methylenedithiobis(2-aminopropionic acid)} to produce pyruvate, ammonia, and S-(mercaptomethyl)cysteine initially . Secondly, S-(mercaptomethyl)-cysteine, which was identified in the form of S-(mercaptomethyl)cysteine thiolactone and S-(2-thia-3-carboxypropyl)cysteine in the absence and presence of iodoacetic acid, respectively, is decomposed enzymatically to pyruvate, ammonia, and bis(mercapto)methane, or spontaneously to cysteine, formaldehyde, and hydrogen sulfide . Balance studies showed that 1.3 mol each of pyruvate and ammonia and 0.2 mol each of formaldehyde and cysteine were produced with consumption of 1 mol of L-djenkolate . 1,2,4,5-Tetrathiane, 1,2,4-trithiolane, 1,2,4,6-tetrathiepane, and 1,2,3,5,6-pentathiepane, which are derivatives of bis(mercapto)methane, were also produced during the alpha,beta-elimination of L-djenkolate . In addition, a polymer with the general formula of -(CH2S)n- was produced as a white precipitate . When the alpha,beta-elimination of L-djenkolate was carried out in the presence of 20 mM iodoacetic acid, neither formaldehyde, cysteine, hydrogen sulfide, or the polymer were formed . Instead, the S-carboxymethyl derivatives of bis(mercapto)methane and S-(mercaptomethyl)cysteine were produced in addition to pyruvate and ammonia.

J Bacteriol, 1991 Apr, 173(8), 2724 - 8
Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7; Benjamin RC et al.; Detailed restriction and nucleotide sequence analysis of the Pseudomonas putida TOL plasmid pDK1 xylE gene revealed significant homology with isofunctional xylE (81.5%) and nahH (78.0%) genes from the TOL pWW0 and NAH7 plasmids . The highest degrees of nucleotide and apparent amino acid conservation (82.2 and 86.4%, respectively) among all three genes were found to exist within a region comprising 264 nucleotides encoding the C terminus . A comparison of localized regions revealed significantly greater homology between xylEpWW0 and xylEpDK1 within the C-terminal region, whereas xylEpWW0 and nahH showed greater similarity at the N terminus . The possibility that xylEpWW0 may represent a genetic hybrid of xylEpDK1 and nahH is discussed.

Biochem J, 1991 Mar 15, 274 ( Pt 3), 875 - 80
Microbial degradation of the morphine alkaloids . Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10; Bruce NC et al.; The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10 . A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P . putida M10 . Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine . The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine . The Km values for morphine and codeine were 0.46 mM and 0.044 mM respectively . The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1,10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme.

Biochim Biophys Acta, 1991 Mar 4, 1073(2), 431 - 3
Fluorometric determination of phenylacetyl-CoA ligase from Pseudomonas putida: a very sensitive assay for a newly described enzyme; Rodriguez-Aparicio LB et al.; Phenylacetyl-CoA ligase (AMP-forming) from Pseudomonas putida is a newly described enzyme (Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B . and Luengo, J.M . (1990) J . Biol . Chem . 265, 7084-7090) specifically involved in the catabolism of phenylacetic acid . This enzyme catalyzes the formation of phenylacetyl-CoA in the presence of ATP, CoA, Mg2+ and phenylacetic acid . A rapid method of assaying this enzyme in partially purified preparations has been developed by coupling this reaction with adenylate kinase, pyruvate kinase and kinase and lactate dehydrogenase . The rate of phenylacetyl-CoA formation was measured indirectly by monitoring fluorometrically the NADH oxidation at 340 nm (excitation at 340 nm and analysis of the emitted light at 465 nm) . The advantage of this method of assay over others (colorimetric, HPLC and spectrophotometric) is discussed.

J Bacteriol, 1991 Mar, 173(5), 1690 - 5
Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system; Suzuki M et al.; Xylene monooxygenase, encoded by the TOL plasmid of Pseudomonas putida, catalyzes the oxidation of toluene and xylenes and consists of two different subunits encoded by xylA and xylM . In this study, the complete nucleotide sequences of these genes were determined and the amino acid sequences of the xylA and xylM products were deduced . The XylM sequence had a 25% homology with alkane hydroxylase, which catalyzes the omega-hydroxylation of fatty acids and the terminal hydroxylation of alkanes . The sequence of the first 90 amino acids of XylA exhibited a strong similarity to the sequence of chloroplast-type ferredoxins, whereas the rest of the XylA sequence resembled that of ferredoxin-NADP+ reductases . Based on this information, the structure and function of xylene monooxygenase were deduced . XylM may be a catalytic component for the hydroxylation of the carbon side chain of toluene and xylenes and, as is the alkane hydroxylase protein, may be a membrane-bound protein containing ferrous ion as a prosthetic group . XylA may have two domains consisting of an N-terminal region similar to chloroplast-type ferredoxins and a C-terminal region similar to ferredoxin-NADP+ reductases . The ferredoxin portion of XylA may contain a {2Fe-2S} cluster and reduce the oxidized form of the XylM hydroxylase . The activity determined by the C-terminal region of the XylA sequence may be the reduction of the oxidized form of ferredoxin by concomitant oxidation of NADH.

Genetika, 1991 Mar, 27(3), 389 - 98
{Localization of camphor degradative plasmids on the chromosome of Pseudomonas putida strains PaW}; Miae AA et al.; Camphor degradative plasmids (CAM, pRK1) are preferentially situated on chromosomes of Pseudomonas putida strains PaW . After having been transferred into Cam+ strains, the TOL plasmid pWWO dissociates into the cryptic plasmid pWWO-8 and chromosome-borne transposon Tn4651 . The opposite situation, i.e . reconstruction of the TOL plasmid pWWO from the cryptic plasmid pWWO-8 and chromosome-borne catabolic operons of the pWWO plasmid has been described . Cam- derivatives of the CAM plasmid were obtained in vivo which contain the TOL plasmid transposons Tn4651 or Tn4652 as obligatory structural elements . These plasmids as well as pWWO-8 determine conjugational mobilization of chromosome-located cam operons followed by their integration into the chromosome of recipient.

Biol Chem Hoppe Seyler, 1991 Mar, 372(3), 203 - 11
Microbial metabolism of quinoline and related compounds . VIII . Xanthine dehydrogenase from a quinoline utilizing Pseudomonas putida strain; Hettrich D et al.; The xanthine dehydrogenase from Pseudomonas putida 86 was purified 68-fold to homogeneity with 47% recovery . SDS-polyacrylamide gel electrophoresis of the enzyme revealed two protein bands corresponding to an Mr of 87,000 and 52,000 . The Mr of the native enzyme was calculated to 550,000 by gel chromatography . The enzyme contained 4 atoms of molybdenum, 16 atoms of iron, 16 atoms of acidlabile sulphur and 4 molecules of FAD . Due to the composition of the cofactors the xanthine dehydrogenase belongs to the class of molybdo-iron/sulphur-flavoproteins . Form A, an oxidation product of the molybdenum cofactor, was identified . Methanol and cyanide were effective inhibitors.

Mol Microbiol, 1991 Mar, 5(3), 647 - 55
The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein; Bitter W et al.; The initial step in the uptake of iron via ferric pseudobactin by the plant-growth-promoting Pseudomonas putida strain WCS358 is binding to a specific outer-membrane protein . The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined . It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N-terminal signal sequence of 47 amino acids . Significant homology, concentrated in four boxes, was found with the TonB-dependent receptor proteins of Escherichia coli . The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55Fe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all . Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins . It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.

Biochemistry, 1991 Feb 12, 30(6), 1635 - 41
Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region; You IS et al.; Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase . This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism . DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues . The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase {You, I.-S., Murray, R . I., Jollie, D., & Gunsalus, I . C . (1990) Biochem . Biophys . Res . Commun . 169, 1049-1054} . The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function . The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins . A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase . The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.

J Bacteriol, 1991 Feb, 173(3), 1125 - 38
Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN); Kullik I et al.; Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, sigma 54 (RpoN NtrA GlnF) . In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoN1 and rpoN2) were identified and sequenced . The two predicted B . japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to the RpoN proteins of other bacteria . Downstream of rpoN2 (but not of rpoN1), two additional open reading frames were identified that corresponded to open reading frames located at similar positions in Klebsiella pneumoniae and Pseudomonas putida . Both B . japonicum rpoN genes complemented the succinate- and nitrate-negative phenotypes of a Rhizobium meliloti rpoN mutant . B . japonicum strains carrying single or double rpoN mutations were still able to utilize C4-dicarboxylates as a carbon source and histidine, proline, or arginine as a nitrogen source, whereas the ability to assimilate nitrate required expression of at least one of the two rpN genes . In symbiosis both rpoN genes could replace each other functionally . The rpoN1/2 double mutant induced about twice as many nodules on soybeans as did the wild type, and these nodules lacked nitrogen fixation activity completely . Transcription of a nifH'-'lacZ fusion was not activated in the rpoN1/2 mutant background, whereas expression of a fixR'-'lacZ fusion in this mutant was affected only marginally . By using rpoN'-'lacZ fusions, rpoN1 expression was shown to be activated at least sevenfold in microaerobiosis as compared with that in aerobiosis, and this type of regulation involved fixLJ . Expression of rpoN2 was observed under all conditions tested and was increased fivefold in an rpoN2 mutant . The data suggested that the rpoN1 gene was regulated in response to oxygen, whereas the rpoN2 gene was negatively autoregulated.

J Biochem (Tokyo), 1991 Feb, 109(2), 348 - 53
Hydroxylation of o-halogenophenol and o-nitrophenol by salicylate hydroxylase; Suzuki K et al.; Salicylate hydroxylase {EC 1.14.13.1} from Pseudomonas putida catalyzed the formation of catechol from substrate analogues such as o-nitro-, o-amino-, o-iodo-, o-bromo-, and o-chloro-phenol by removing the ortho-substituted groups . They are converted into nitrite, ammonia, and halide ions, respectively . Kinetic parameters of these reactions were determined by spectrophotometric and polarographic methods . Hydroxylation of o-nitro- or o-iodophenol proceeds with the unusual stoichiometry of 2:1:1 for consumed NADH, O2-uptake, and catechol formed . Other ortho-substituted phenols examined also gave the same results . Like salicylate, these substrates perturb the absorption spectrum of salicylate hydroxylase in the visible region, indicating the formation of enzyme.substrate complexes . Titration experiments with ortho-substituted phenols gave the dissociation constants of the complexes . The complexes were quantitatively reduced with NADH or dithionite without detectable formation of the intermediates . The fact that one atom of 18O2 was incorporated into the produced catechol in hydroxylation of o-nitrophenol indicates that the reaction is of monooxygenase nature . It is concluded that salicylate hydroxylase cleaves the C-N and C-X bonds of ortho-substituted phenols.

Protein Eng, 1991 Feb, 4(3), 271 - 82
A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity; Zvelebil MJ et al.; A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida . As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building . Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam . The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added . The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface . The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment . Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity . A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity . Residues that are likely to interact with the haem were identified . The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family . Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.

Biochemistry, 1991 Jan 8, 30(1), 238 - 47
Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-A resolution; Mathews FS et al.; p-Cresol methylhydroxylase (PCMH) isolated from Pseudomonas putida is an alpha 2 beta 2 tetramer of approximate subunit Mr 49,000 and 9,000 . It is a flavocytochrome c containing covalently bound FAD in the larger subunit and covalently bound heme in the smaller . Crystals in space group P2(1)2(1)2(1) with unit-cell parameters a = 140.3 A, b = 130.6 A, and c = 74.1 A contain one full molecule per asymmetric unit and diffract anisotropically to about 2.8-A resolution in two directions and to about 3.3-A resolution in the third . An electron density map has been computed at a nominal resolution of 3.0 A by use of area detector data from native crystals and from two derivatives . The phases were improved with the B.C . Wang solvent leveling procedure, and the map was averaged about the noncrystallographic 2-fold axis . The cytochrome subunit, whose amino acid sequence is known, has been fitted to the electron density on a graphics system . The course of the polypeptide chain of the flavoprotein subunit, whose sequence is mostly unknown, has been traced in a minimap and a model of polyalanine fitted to the electron density on the graphics system . The flavoprotein subunit consists of three domains in close contact . The N-terminal domain consists largely of beta-structure and contains most of the FAD binding site . The second domain contains a seven-stranded antiparallel beta-sheet of unusual topology connected by antiparallel alpha-helices on one side . The flavin ring lies at the juncture of the first two domains . The third domain lies against the first domain and helps cover the rest of the FAD chain . The cytochrome subunit resembles other small cytochromes such as c-551 and c5 and fits into a depression on the surface of the large flavoprotein subunit . The flavin and heme planes are nearly perpendicular, the normals to the planes being approximately 65 degrees apart . The two groups are separated by about 8 A, the distance from one of the vinyl methylene carbon atoms of the heme to the 8 alpha-methyl group of the flavin ring.

Gene, 1991 Jan 2, 97(1), 39 - 47
A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants; Morales VM et al.; A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH . Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation . The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains . They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point . These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor . The expression of these genes in E . coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated . The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E . coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively . The taclac promoter, however, proved to be useful for the cloning in E . coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number . Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction . Two subunits of the E . coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.

Mikrobiol Zh, 1991 Jan-Feb, 53(1), 63 - 7
{The effect of amino acids in the asparagine family on the aspartate kinase and homoserine dehydrogenase of ethionine-resistant mutants of Pseudomonas putida}; Polodienko OB et al.; The activity of aspartate kinase and homoserin dehydrogenase from ethionine resistant mutants Pseudomonas putida 25 and 6 have been studied as affected by amino acids from the family of asparagine . They are characterized by a capacity to the surplus synthesis of methionine . It is shown that mutants have negative regulation of the level of activity of the studied enzymes . It is supposed that the mutations (or mutation) could take place which affected properties of enzymes, which participated directly in the biosynthesis of methionine, in the analogue resistant clones 25 and 6.

Arch Microbiol, 1991, 155(2), 164 - 9
Molybdenum-dependent degradation of quinoline by Pseudomonas putida Chin IK and other aerobic bacteria; Blaschke M et al.; Eighteen different aerobic bacteria were isolated which utilized quinoline as sole source of carbon, nitrogen, and energy . Attempts were unsuccessful at isolating anaerobic quinoline-degrading bacteria . The optimal concentration of quinoline for growth was in the range of 2.5 to 5 mM . Some organisms excreted 2-hydroxyquinoline as the first intermediate . Hydroxylation of quinoline was catalyzed by a dehydrogenase which was induced in the presence of quinoline or 2-hydroxyquinoline . Quinoline dehydrogenase activity was dependent on the availability of molybdate in the growth medium . Growth on quinoline was inhibited by tungstate, an antagonist of molybdate . Partially purified quinoline dehydrogenase from Pseudomonas putida Chin IK indicated the presence of flavin, iron-sulfur centers, and molybdenum-binding pterin . Mr of quinoline dehydrogenase was about 300 kDa in all isolates investigated.

Genetika, 1991 Jan, 27(1), 39 - 50
{Phage-stable mutants of Pseudomonas putida: new types of stability}; Al'nikin AF et al.; More than 170 phage-resistant mutants (PRM) of the first order of Pseudomonas putida strain PpG1 were obtained using newly isolated and previously described bacteriophages specific for this strain . According to the results of analysis of resistance of the mutants to each of 31 phages of PpG1 strain and 8 phages of the PpN strain, the PRM strains were distributed into 20 groups . In most cases, the reason for resistance is loss of absorption capacity of bacteria . However, no direct relation between the level of absorption and efficiency of phage plating was detected . It was shown that some of the PRM of P . putida PpG1 strains acquired the ability to maintain the growth of phages specific for the other P . putida strain, PpN . Frequencies of isolating mutants of various resistance types depend on the concrete phage used . In accordance with their absorption specificity, all phages were distributed into 23 groups, and a tridimensional formal scheme of receptor sites for these phages on the PpG1 strain was drawn . In the process of selection of the PpG1 clones resistant to non-lysogenizing mutant of temperate PP71 phage, a variant of this strain manifesting the phenomenon of "auto-plaquing" was found . These results support the mutational origin of this phenomenon in some cases.

Appl Environ Microbiol, 1991 Jan, 57(1), 260 - 6
Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid; Ramos JL et al.; Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate . The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil . The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate . The addition to soils of aromatics that are cometabolized by P . putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival . Survival of P . putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C . The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.

J Invertebr Pathol, 1991 Jan, 57(1), 66 - 70
Construction of tracer plasmids for Bacillus sphaericus 1593 utilizing the xylE gene from Pseudomonas putida; Taylor LD et al.; Genetically engineered microorganisms (GEMS) released into the environment must be traceable in order to accurately assess their impact on the area of release . Tracer genes other than those that introduce antibiotic resistance are preferred for use in identifying genetically engineered strains . In this study, we describe the construction of a series of tracer plasmids for use in Bacillus sphaericus using the xylE gene from the Pseudomonas putida TOL plasmid . This gene codes for the enzyme catechol 2,3-dioxygenase which converts the colorless substance catechol to 2-hydroxymuconic semialdehyde, a yellow product which is easily detected . Colonies of cells which express the xylE gene turn yellow shortly after being exposed with a solution of catechol.

J Bacteriol, 1991 Jan, 173(2), 463 - 71
Molecular characterization and expression analysis of the anthranilate synthase gene of Pseudomonas syringae subsp . savastanoi; da Costa e Silva O et al.; The trpE gene, which encodes the large component of the enzyme anthranilate synthase, was isolated from a Pseudomonas syringae subsp . savastanoi (P . savastanoi) cosmid library . Cosmids that complemented an Escherichia coli trpE mutation contained a gene whose product is 86% homologous at the deduced amino acid level to TrpE of Pseudomonas aeruginosa and Pseudomonas putida . Amino acid sequence comparison with other TrpE sequences revealed the existence of conserved regions between the procaryotic and eucaryotic polypeptide sequences analyzed, regions that might be of functional importance . We also report on studies on the expression pattern of this gene . We analyzed the promoter activity of a trpE::lacZ transcriptional fusion, the relative amount of trpE steady-state mRNA, and the activity of anthranilate synthase from cells grown in minimal medium with or without exogenously added tryptophan and in complete medium . We concluded that under the conditions tested, expression of the trpE gene of P . savastanoi is independent of the concentration of tryptophan in the culture medium . Implications of such an expression pattern on the virulence of this bacterium are discussed.

Nucleic Acids Symp Ser, 1991, (25), 115 - 6
Highly conserved coding sequences in polychlorinated biphenyl (PCB)-degraders of Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715; Furukawa K et al.; Sixteen strains that utilize biphenyl or polychlorinated biphenyl (PCB) as the sole source of carbon and energy have previously been isolated . Nucleotide sequence of the bphABCXD operon (11 kb) of Pseudomonas pseudoalcaligenes KF707 has now been determined . When bphD region is compared with the previously reported bphD sequence of another PCB-degrader, Pseudomonas putida KF715, homologies at the level of nucleotides as well as amino acids are as high as 96% . Moreover, both bphABCXD operon (KF707) and bphABCD operon (KF715) share the identical transcriptional initiation site . Since these aromatic hydrocarbon-degraders are coded on chromosomes, whereas other majorities of hydrocarbon-degraders are coded on plasmids, there must be a mechanism governing the chromosomal gene-shuffling.

J Bacteriol, 1990 Dec, 172(12), 6651 - 60
Growth-phase-dependent expression of the Pseudomonas putida TOL plasmid pWW0 catabolic genes; Hugouvieux-Cotte-Pattat N et al.; Pseudomonas putida TOL plasmid pWW0 catabolic genes are clustered into two operons . The first, the upper operon, is controlled by the xylR regulatory gene, whereas the second, the meta operon, is controlled by the xylS regulatory gene . The xylS gene itself is subjected to control by xylR . In this study, we show that the TOL catabolic operons were poorly induced in cells growing at the early-exponential-growth phase but strongly induced in cells at late-exponential-growth phase . We constructed fusions of four TOL promoters, Pm (the promoter of the meta operon), Pu (the promoter of the upper operon), Ps (the promoter of the xylS regulatory gene), and Pr (the promoter of the xylR regulatory gene) with lacZ and examined, in Escherichia coli and P . putida, the expression of these promoters in relation to the growth phase . Expression from Pm, Pu, Ps, and Pr was almost constant if the host cells did not carry either xylS or xylR . Similarly, expression of Pm and Pu in P . putida in the absence of XylS and XylR was constant during the growth of the cells . XylS-dependent transcription of Pm and XylR-dependent transcription of Ps and Pu, in contrast, varied with the growth phase . This observation suggested that the interaction of XylS and XylR with target promoters or with RNA polymerases was influenced by the growth phase . The nature of the signal which triggers the growth-phase-dependent regulation was not clear . A change in the oxygen partial pressure was not responsible for the regulation . E . coli mutants defective in relA, crp, and cya exhibited growth-phase-dependent expression of the TOL catabolic genes, indicating that cyclic AMP and relA-dependent synthesis of ppGpp are not involved in this phenomenon.

Biol Chem Hoppe Seyler, 1990 Dec, 371(12), 1137 - 44
Microbial metabolism of quinoline and related compounds . VII . Quinoline oxidoreductase from Pseudomonas putida: a molybdenum-containing enzyme; Bauder R et al.; The quinoline oxidoreductase from Pseudomonas putida was purified 50-fold to homogeneity with 21% recovery, using ammonium sulfate precipitation, hydrophobic interaction-, anion exchange-, and gel chromatography . The Mr of the native enzyme was calculated to be 300,000 by gel filtration . SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to Mr 85,000, 30,000 and 20,000 . The enzyme contained 8 atoms of iron, 8 atoms of acid-labile sulfide, 2 molecules of FAD, and the molybdenum cofactor, molybdopterin . Besides quinoline, the quinoline oxidoreductase also catalysed the conversion of 5-, 6-, 7- and 8-hydroxyquinoline and 8-chloroquinoline to the corresponding 2-oxo compounds . The incorporated oxygen atom was derived from water . Cyanide and methanol were effective inhibitors.

Appl Environ Microbiol, 1990 Dec, 56(12), 3664 - 70
Degradation of the metal-cyano complex tetracyanonickelate(II) by cyanide-utilizing bacterial isolates; Silva-Avalos J et al.; Ten bacterial isolates capable of growth on tetracyanonickelate(II) {K2{Ni(CN)4}} (TCN) as the sole nitrogen source were isolated from soil, freshwater, and sewage sludge enrichments . Seven of the 10 were identified as pseudomonads, while the remaining 3 were classified as Klebsiella species . A detailed investigation of one isolate, Pseudomonas putida BCN3, revealed a rapid growth rate on TCN (generation time, 2 h), with substrate removal and growth occurring in parallel . In addition to TCN, all isolates were able to utilize KCN, although the latter was significantly more toxic; MICs ranged from 0.2 to 0.8 mM for KCN and greater than or equal to 50 mM for TCN . While growth occurred over a wide range of TCN concentrations (0.25 to 16 mM), degradation was most substantial under growth-limiting conditions and did not occur when ammonia was present . In addition, cells grown on TCN were found to accumulate nickel cyanide {Ni(CN)2} as a major biodegradation product . The results show that bacteria capable of growth on TCN can readily be isolated and that degradation (i) appears to parallel the capacity for growth on KCN, (ii) does not occur in the presence of ammonia, and (iii) proceeds via the formation of Ni(CN)2 as a biological metabolite.

Biochim Biophys Acta, 1990 Nov 30, 1087(3), 309 - 15
Nucleotide sequence and expression of the isoamylase gene from an isoamylase-hyperproducing mutant, Pseudomonas amyloderamosa JD210; Chen JH et al.; The isoamylase gene (ISO) of Pseudomonas amyloderamosa JD210, an isoamylase-hyperproducing mutant, was cloned in an isoamylase-deficient and transformable mutant strain K31 . By deletion analysis, the ISO gene was found to be located within a 3.3 kilobases BamHI fragment . Its nucleotide sequence contained an open reading frame of 2328 nucleotides (776 amino acids) encoding a secreted isoamylase precursor . The ISO gene fragment was inserted into plasmids pKT230 and pBR 322 in opposite orientations . The expression of the ISO gene in the constructed plasmids was compared in P . amyloderamosa K31, Pseudomonas aeruginosa PAO1-161, Pseudomonas putida mt-2 and Escherichia coli HB101 . In all transformed cells, the majority of the isoamylase produced was secreted and higher isoamylase activities were obtained in transformats with the transcriptional direction of the ISO gene similar to the nearby drug-determinant gene of the vector.

J Mol Biol, 1990 Nov 20, 216(2), 251 - 60
Upstream regulatory sequence for transcriptional activator XylR in the first operon of xylene metabolism on the TOL plasmid; Inouye S et al.; Transcription of the first operon coding for m-xylene-degrading enzymes on the TOL plasmid of Pseudomonas putida is activated by the xylR gene product in the presence of m-xylene . The operon has the consensus sequence of the ntr/nif promoters at -24 and -12 regions, and the transcription is dependent on an RNA polymerase containing a sigma factor NtrA (RpoN or sigma 54) . Deletion derivatives of the upstream sequence of the operon promoter were made in vitro and connected with the xylE gene on a plasmid . Their promoter activities were analyzed in Escherichia coli by monitoring catechol 2,3-dioxygenase activity, the xylE gene product . A cis-acting DNA element was identified, which is required for activation of the operon promoter by XylR protein in the presence of the inducer . This regulatory sequence of about 40 base-pairs in length was located 150 base-pairs upstream from the transcription start site . Analysis of the mutants containing insertions between the upstream regulatory sequence and the promoter sequence demonstrated strong dependence of the activation upon helical periodicity of DNA . The regulatory sequence functioned in the inverse orientation or at a distance of more than 1 x 10(3) base-pairs upstream from the promoter though less efficient . These results indicated that this upstream regulatory sequence might be the binding site for XylR protein . DNA-loop formation through protein-protein interaction between XylR protein attached to the upstream sequence and the NtrA-containing RNA polymerase bound by the promoter sequence was suggested for activation of the operon transcription . A sequence similar to the regulatory sequence of the first operon of xylene metabolism was found in the upstream region of the xylS gene, which is also activated by XylR protein in the presence of m-xylene.

Arch Biochem Biophys, 1990 Nov 1, 282(2), 248 - 53
Different types of formaldehyde-oxidizing dehydrogenases in Nocardia species 239: purification and characterization of an NAD-dependent aldehyde dehydrogenase; Van Ophem PW et al.; Three different dehydrogenases able to oxidize formaldehyde were found in the Gram-positive methylotroph, Nocardia sp . 239: an NAD-dependent aldehyde dehydrogenase (NA-ADH), and NAD- and factor-dependent formaldehyde dehydrogenase (FD-FDH), and a dye-linked aldehyde dehydrogenase (DL-ADH) . The ratio of the activities observed for the two NAD-linked enzymes varied with growth conditions: batch-wise grown cells had nearly the same activities for both enzymes; in fed batch-wise grown cells (methanol limitation) only FD-FDH was detected . The latter is clearly involved in formaldehyde oxidation, since the enzyme and the factor were found only in methanol-grown cells and the enzyme is specific for formaldehyde . In contrast, the two aldehyde dehydrogenases may have significance for aldehyde dissimilation in general, since both activities could also be demonstrated in ethanol-grown cells (but not in glucose-grown cells) and higher aldehydes are even better substrates than formaldehyde . NA-ADH was purified to homogeneity . The enzyme seems to be a homotetramer since it showed a relative molecular mass of 200,000 and the denaturated form of 55,000 . Other characteristics are as follows: the enzyme showed substrate inhibition for the aldehydes tested; optimal activity was found at pH 9.2; the reverse reaction was not observed; the enzyme was specific for NAD; GSH, K+, or NH4+ addition did not stimulate formaldehyde oxidation; the order of NAD and substrate addition to the enzyme was not important; several compounds able to block SH groups were inhibitory . Comparison with NAD-linked aldehyde dehydrogenases from Gram-negative bacteria showed that the Nocardia enzyme is distinct from the enzyme of Pseudomonas putida (EC 1.2.1.46) and of Hyphomicrobium X.

Genetics, 1990 Nov, 126(3), 497 - 503
Isolation of high frequency of recombination donors from Tn5 chromosomal mutants of Pseudomonas putida PPN and recalibration of the genetic map; Strom AD et al.; A Tn5 loaded derivative of the IncP-10 plasmid R91-5 (pMO75) was used as a suicide vector to generate random chromosomal insertion mutations in Pseudomonas putida PPN . Reintroduction of pMO75 into such mutants resulted in integration of the plasmid at the site of Tn5 insertion, giving rise to two classes of high frequency of donors recombination (Hfr) donors, transferring chromosome at high frequency (greater than 10(-1) per donor cell) in opposite directions . Consequently, Tn5 induced auxotrophic mutations could be equated with or distinguished from previously mapped mutations, and closely linked markers ordered, on the basis of marker recovery using the two classes of Hfr donor . The isolation of many new transfer origins allowed more accurate time-of-entry analysis than previously possible and resulted in the reduction of the genetic map from 103 min to 88 min.

J Bacteriol, 1990 Nov, 172(11), 6355 - 62
Genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in Pseudomonas putida TMB; Polissi A et al.; The catabolic pathway for the degradation of aromatic hydrocarbons encoded by Pseudomonas putida TMB differs from the TOL plasmid-encoded pathway as far as regulation of the upper pathway is concerned . We found, by analyzing Tn5-induced mutants and by Southern blot hybridization with appropriate probes derived from the TOL plasmid pWW0, that the catabolic genes of strain TMB were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain . The catabolic genes of TMB and pWW0 had sequence homology, as shown by Southern blot hybridization, but differed significantly in their restriction patterns . The analysis of the mutants suggests that a regulatory mechanism similar to that present in pWW0 coexists in TMB with a second mode of regulation which is epistatic on the former and that the chromosomal region carrying the catabolic genes is prone to rearrangements and deletions.

FEMS Microbiol Lett, 1990 Nov, 60(3), 259 - 64
Cloning and expression of the plasmid-encoded benzene dioxygenase genes from Pseudomonas putida ML2; Tan HM et al.; Hybridization using heterologous dioxygenase gene probes indicated the presence of the genes encoding the enzyme benzene dioxygenase on a 112 kb plasmid from Pseudomonas putida ML2 . They were identified as benzene dioxygenase genes (bed ABC1C2) by cloning in Escherichia coli and analysis of expression by Western blotting using antibodies raised to the four polypeptides of purified benzene dioxygenase.

J Biotechnol, 1990 Nov, 16(3-4), 199 - 209
Immobilized and free cell continuous cultures of a recombinant E . coli producing catechol 2,3-dioxygenase in a two-stage chemostat: improvement of plasmid stability; Berry F et al.; The immobilization of recombinant strains of E . coli W3110/pTG205 in K-carrageenan gel beads improves the plasmid stability during continuous cultures in the absence of selection pressure . Since, xyl E gene (which encodes catechol 2,3-dioxygenase from Pseudomonas putida) transcription is controlled by the trp promoter, the effects of tryptophan (repressor) and 3 beta-indolyl acrylic acid (derepressor) on pTG 205 stability and enzyme production have been studied in both free and immobilized cell cultures . A two-stage continuous culture system running for 150 h is described . In the first stage an immobilized culture is performed in the presence of tryptophan with a significant plasmid stability . The cells released from the gel beads are continuously transferred in the second stage reactor where expression is induced by 3 beta-indolyl acrylic acid . In these conditions an efficient production of catechol 2,3-dioxygenase is observed.

J Biol Chem, 1990 Oct 25, 265(30), 18192 - 9
Cloning and expression of rat histidase . Homology to two bacterial histidases and four phenylalanine ammonia-lyases; Taylor RG et al.; Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid . Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site . The amino site precursor and the mechanism of formation of dehydroalanine are not known . As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum . The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da . The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells . Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA . COS cells transfected with a histidase expression vector produce active histidase . The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism . Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively . Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase . On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.

Biochemistry, 1990 Oct 23, 29(42), 9856 - 62
Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli; Tsou AY et al.; The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned . The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase {Ransom, S . C., Gerlt, J . A., Powers, V . M., & Kenyon, G . L . (1988) Biochemistry 27, 540} are organized in an operon (mdlCBA) . Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P . putida . (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast . Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor . These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways . The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.

Nature, 1990 Oct 18, 347(6294), 692 - 4
Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous; Neidhart DJ et al.; Mandelate racemase (MR) and muconate lactonizing enzyme (MLE) catalyse separate and mechanistically distinct reactions necessary for the catabolism of aromatic acids by Pseudomonas putida . The X-ray crystal structure of MR, solved at 2.5 A resolution, reveals that the secondary, tertiary and quaternary structures of MR and MLE are remarkably similar; also, MR and MLE are about 26% identical in primary structure . However, MR has no detectable MLE activity and vice versa . Thus, MR and MLE constitute the first example of enzymes that catalyse different reactions, as opposed to mechanistically identical reactions on different substrates, yet possess sufficient structural and sequence identity that they are likely to have evolved from a common ancestor . The discovery that MR and MLE catalyse different reactions but share a common structural framework has broad implications for the natural evolution of enzymes and metabolic pathways, as well as for the rational modification of enzyme activities.

Genetika, 1990 Oct, 26(10), 1713 - 9
{Cloning genes for biosynthesis of Pseudomonas putida tryptophan in Escherichia coli cells}; Olekhnovich IN et al.; The trpE, trpC and trpIBA genes of Pseudomonas putida were cloned by complementation of the corresponding auxotrophic mutations of Escherichia coli using pBR322 as a vector . With the exception of trpE, transcription of all genes in new host takes place under control of their own promoters . Expression of the trpD gene linked to trpC was not registered in E . coli . Repressible trpC enzyme was synthesized constitutively in E . coli . Characteristic regulation of P . putida trpBA genes via induction by indolglycerol phosphate is retained in E . coli . The activator gene trpI and tryptophan synthase genes were closely linked in the trpIBA sequence.

Wei Sheng Wu Xue Bao, 1990 Oct, 30(5), 397 - 9
{The preparation and properties of catechol-1,2-dioxygenase from Pseudomonas putida}; Kou X et al.; Catechol-1,2-dioxygenase (EC 1.13.11.1) catalyzes the degradation of catechol to cis, cis-muconic acid . The biochemical properties of catechol-1,2-dioxygenase from Pseudomonas putida 84103 were investigated . The optimum pH and temperature is 7.5-8.0 and 25-30 degrees C, respectively . Cu2+, Zn2+ inhibit the enzyme activity . The paper chromatograph and UV absorption spectrum of enzymatic reaction product are accordance with those of the standard muconic acid.

J Bacteriol, 1990 Oct, 172(10), 5655 - 63
Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon; Madhusudhan KT et al.; Branched-chain keto acid dehydrogenase is a multienzyme complex produced by Pseudomonas putida when it is grown in a minimal medium containing branched-chain amino acids . A 1.87-kilobase (kb) DNA fragment was cloned and sequenced which contained 0.24 kb of the E1 alpha structural gene and 1.6 kb of upstream DNA . There were 854 base pairs (bp) of noncoding DNA upstream of bkdA1, the first gene of the bkd operon, and 592 bp between the transcriptional and translational starts . The G + C content of the noncoding region was 56.7% compared with 65.2% for all the structural genes of the operon . A partial open reading frame was found on the strand opposite that of the bkd operon beginning at base 774 . When the bkd promoter was cloned into the promoter probe vector pKT240, streptomycin resistance was obtained in P . putida but not Escherichia coli with the promoter in both orientations, which indicates either that the bkd promoter is bidirectional or that there are two promoters in this region . A series of ordered deletions on both sides of the proposed site of the start of transcription revealed that almost 700 bp upstream of the start of translation were required for expression . Streptomycin resistance was also obtained in an rpoN mutant of P . putida KT2440 containing constructs with the intact bkd promoter, indicating that the bkd operon does not require the rpoN sigma factor for expression . Another construct containing the bkd promoter, bkdA1, and bkdA2 in pKT240 was used to transform P . putida JS113, a mutant which was unable to produce the E1 subunits of the branched-chain keto acid dehydrogenase . In this case, very high inducible expression of the bkd operon was obtained.

J Bacteriol, 1990 Oct, 172(10), 5563 - 74
Sequence and analysis of the rpoN sigma factor gene of rhizobium sp . strain NGR234, a primary coregulator of symbiosis; van Slooten JC et al.; We report the nucleotide sequence of the rpoN gene from broad-host-range Rhizobium sp . strain NGR234 and analyze the encoded RPON protein, a sigma factor . Comparative analysis of the deduced amino acid sequence of RPON from NGR234 with sequences from other gram-negative bacteria identified a perfectly conserved RPON box unique to RPON sigma factors . Symbiotic regulatory phenotypes were defined for a site-directed internal deletion within the coding sequence of the rpoN gene of Rhizobium strain NGR234: they included quantitative nodulation kinetics on Vigna unguiculata and microscopic analysis of the Fix- determinate nodules of V . unguiculata and Macroptilium atropurpureum . RPON was a primary coregulator of nodulation and was implicated in establishment or maintenance of the plant-synthesized peribacteroid membrane . Phenotypes of rpoN in Rhizobium strain NGR234 could be grouped as symbiosis related, rather than simply pleiotropically physiological as in free-living bacteria such as Klebsiella pneumoniae and Pseudomonas putida.

FEMS Microbiol Lett, 1990 Oct, 60(1-2), 113 - 6
Design of an enzymatic hybrid system: a useful strategy for the biosynthesis of benzylpenicillin in vitro; Martinez-Blanco H et al.; A hybrid (prokaryotic-eukaryotic) enzyme system leading to the production of benzylpenicillin has been developed . In vitro synthesis of penicillin G was achieved by incubating 6-aminopenicillanic acid, CoA, phenylacetic acid, homogeneously pure phenylacetyl-CoA ligase (PA-CoA ligase) from Pseudomonas putida and acyl-CoA:6-APA acyltransferase (AT) from Penicillium chrysogenum . Benzylpenicillin was also obtained when AT was coupled with PA-CoA ligase and isopenicillin N-synthetase (IPNS) . This is the first description of an in vitro assay that, using enzymes of different microbial origin, mimics the three last enzymatic steps leading to the biosynthesis of penicillin G in P . chrysogenum.

J Bacteriol, 1990 Oct, 172(10), 5999 - 6009
Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition; Koenig K et al.; The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with {185W}tungstate . This labeling was used as a reporter to purify both labile proteins . The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps . Both radioactive proteins were separated and purified to homogeneity . Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively . With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis . Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity . The latter parameters showed a perfect correlation . This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase . The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000 . The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme . The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined . The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases.

Biol Chem Hoppe Seyler, 1990 Oct, 371(10), 999 - 1003
Microbial metabolism of quinoline and related compounds . V . Degradation of 1H-4-oxoquinoline by Pseudomonas putida 33/1; Bott G et al.; A bacterial strain was isolated with the ability to use 1H-4-oxoquinoline as the sole source of carbon, nitrogen and energy . On the basis of its physiological properties, this isolate was classified as Pseudomonas putida . 1H-3-Hydroxy-4-oxoquinoline, N-formylanthranilic acid, anthranilic acid and catechol were identified as intermediates in the degradation pathway . The latter was further degraded by ortho-cleavage . The enzymatic conversion of 1H-4-oxoquinoline into 1H-3-hydroxy-4-oxoquinoline requires oxygen and NADH . Experiments with 18O2 showed that the oxygen consumed in this enzymatic reaction is derived from the atmosphere.

Biochim Biophys Acta, 1990 Sep 27, 1040(3), 327 - 36
Functional modification of an arginine residue on salicylate hydroxylase; Suzuki K et al.; Salicylate hydroxylase from Pseudomonas putida (EC 1.14.13.1, salicylate, NADH:oxygen oxidoreductase) is an FAD-containing monooxygenase, which catalyzes decarboxylative hydroxylation of salicylate to produce catechol in the presence of NADH and O2 . By chemical treatment of the enzyme with dicarbonyl reagents, such as glyoxal, the original oxygenase activity was converted to the salicylate-dependent NADH-dehydrogenase activity with free FAD as electron acceptor . One of twenty arginine residues of this enzyme is concerned with this alteration of activity, as shown by the result of its modification at pH 6.9 . This result is further supported by the isolation of one arginine-modified enzyme by chromatographic methods on DEAE-Sephadex, A-50 columns . It exhibits the dehydrogenase activity predominantly . This modified enzyme is spectrophotometrically and electrophoretically characterized by a minute conformational change around the active site, and kinetically by a 7-fold increase in an apparent Km for NADH and a decrease of more than 5-fold in an apparent Km for FAD as electron acceptor, with an apparent Vmax of 22 s-1 for the dehydrogenase activity . Flow kinetics also showed a marked decrease in the rate for oxygenation of the reduced enzyme-salicylate complex from 21 s-1 (native enzyme) to 3.3 s-1 (modified enzyme) . These facts suggest that one arginine residue of the enzyme is responsible for the NADH binding site, and chemical modification of one arginine residue of the enzyme induces some conformational change around the active site to alter the catalytic activity from oxygenation to dehydrogenation.

Eur J Biochem, 1990 Sep 24, 192(3), 669 - 76
Mechanism of action of urocanase . Specific 13C-labelling of the prosthetic NAD+ and revision of the structure of its adduct with imidazolylpropionate; Klepp J et al.; 1 . {4-13C}Nicotinate was synthesised and used to support the growth of a nicotinate auxotrophic mutant of Pseudomonas putida . 13C-NMR spectroscopy of the isolated urocanase confirmed the efficient incorporation of 13C into C4 of the nicotinamide ring of the tightly bound NAD+ cofactor . 2 . beta-{( 2'-13C}Imidazol-4-yl)propionate was synthesised according to known procedures and used for inhibition of the 13C-labelled urocanase . An increase in the absorbance at 330 nm indicated adduct formation between enzyme-bound NAD+ and inhibitor . The adduct was stabilised by oxidation with phenazine methosulfate and isolated using a slight modification of the procedure of Matherly et al . {Matherly, L . H., DeBrosse, C . W . & Phillips, A . T . (1982) Biochemistry 21, 2789-2794} . 3 . The 13C-NMR spectrum of the doubly labelled adduct, {4-13C}NAD-{2'-13C}imidazolylpropionate, showed no one-bond 13C-13C coupling between labelled sites . The 1H-NMR spectrum of this adduct in 2H2O showed only one imidazole signal, which appeared as a doublet (1JC-H = 212 Hz), confirming the presence of a proton at the labelled C2' . The lack of a C5' signal and further NMR data provide evidence for a C-C bond between C4 of the nicotinamide and C5' of the imidazole ring . 4 . The revised structure for the enzymatically formed addition complex suggests a novel mechanism for the urocanase reaction which is not only chemically plausible but also explains the previously observed urocanase-catalysed exchange of the C5 proton of urocanate and of beta-(imidazol-4-yl)propionate.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 179 - 85
Degradation of 3-chlorobiphenyl by in vivo constructed hybrid pseudomonads; Mokross H et al.; 3-Chlorobiphenyl-degrading bacteria were obtained from the mating between Pseudomonas putida strain BN10 and Pseudomonas sp . strain B13 . Strains such as BN210 resulted from the transfer of the genes coding the enzyme sequence for the degradation of chlorocatechols from B13 into BN10, whereas B13 derivatives such as B131 have acquired the biphenyl degradation sequence from BN10 . During growth of the hybrid strains on 3-chlorobiphenyl 90% chloride was released . Activities of phenylcatechol 2,3-dioxygenase, benzoate dioxygenase, catechol 1,2-dioxygenase, chloromuconate cyloisomerase and 4-carboxymethylenebut-2-en-4-olide hydrolase were found in 3-chlorobiphenyl-grown cells . The hybrid strains were found to convert some congeners of the Aroclor 1221 mixture such as mono- and dichloro-substituted biphenyls.

J Bacteriol, 1990 Sep, 172(9), 5477 - 81
Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes; Schwacha A et al.; The hutC gene of Klebsiella aerogenes encodes a repressor that regulates expression of the histidine utilization (hut) operons . The DNA sequence of a region known to contain hutC was determined and shown to contain two long rightward-reading open reading frames (ORFs) . One of these ORFs was identified as the 3' portion of the hutG gene . The other ORF was the hutC gene . The repressor predicted from the hutC sequence contained a helix-turn-helix motif strongly similar to that seen in other DNA-binding proteins, such as lac repressor and the catabolite gene activator protein . This motif was located in the N-terminal portion of the protein, and this portion of the protein seemed to be sufficient to allow repression of the hutUH operon but insufficient to allow interaction with the inducer . The presence of a promoterlike sequence and a ribosome-binding site immediately upstream of the hutC gene explained the earlier observation that hutC can be transcribed independently of the other hut operon genes . The predicted amino acid sequence of hut repressor strongly resembled that of the corresponding protein from Pseudomonas putida (S . L . Allison and A . T . Phillips, J . Bacteriol . 172:5470-5476, 1990) . An unexpected, leftward-reading ORF extending from about the middle of hutC into the preceding (hutG) gene was also detected . The deduced amino acid sequence of this leftward ORF was quite distinct from that of an unexpected ORF of similar size found immediately downstream of the P . putida hutC gene . The nonstandard codon usage of this leftward ORF and the expression of repressor activity from plasmids with deletions in this region made it unlikely that this ORF was necessary for repressor activity.

J Bacteriol, 1990 Sep, 172(9), 5470 - 6
Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Pseudomonas putida; Allison SL et al.; The hutC gene of Pseudomonas putida encodes a repressor which, in combination with the inducer urocanate, regulates expression of the five structural genes necessary for conversion of histidine to glutamate, ammonia, and formate . The nucleotide sequence of the hutC region was determined and found to contain two open reading frames which overlapped by one nucleotide . The first open reading frame (ORF1) appeared to encode a 27,648-dalton protein of 248 amino acids whose sequence strongly resembled that of the hut repressor of Klebsiella aerogenes (A . Schwacha and R . A . Bender, J . Bacteriol . 172:5477-5481, 1990) and contained a helix-turn-helix motif that could be involved in operator binding . The gene was preceded by a sequence which was nearly identical to that of the operator site located upstream of hutU which controls transcription of the hutUHIG genes . The operator near hutC would presumably allow the hut repressor to regulate its own synthesis as well as the expression of the divergent hutF gene . A second open reading frame (ORF2) would encode a 21,155-dalton protein, but because this region could be deleted with only a slight effect on repressor activity, it is not likely to be involved in repressor function or structure.

Eur J Biochem, 1990 Aug 17, 191(3), 705 - 14
Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida; Shaw JP et al.; Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized . Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible . Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible . Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes . The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM . Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide . Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa . Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.

Biochemistry, 1990 Aug 7, 29(31), 7348 - 56
Purification, characterization, and structure of pseudobactin 589 A, a siderophore from a plant growth promoting Pseudomonas; Persmark M et al.; Under conditions of low-iron stress the plant growth promoting bacterium Pseudomonas putida 589 (DSM 50202) produced a yellow-green fluorescent iron-binding peptide siderophore, which was designated pseudobactin 589 A and had an affinity constant toward Fe3+ of 10(25) at pH 7 . Protonated pseudobactin 589 A had the molecular formula C54H78O26N15 and a nominal mass spectral molecular mass of 1353 g/mol . Its structure was determined by a combination of nuclear magnetic resonance, fast atom bombardment mass spectrometry, and Edman degradation . Pseudobactin 589 A consisted of a nonapeptide with the amino acid sequence L-Asp-L-Lys-(D)-beta-OH-Asp-D(L)-Ser-L-Thr-D-Ala-D-Glu-L(D)-Ser-L-N delta-OH- Orn, in which lysine was amide bonded via the carboxy and the N epsilon-amino groups . A quinoline-derived chromophore was connected via an amide bond to the alpha-amino nitrogen of aspartic acid and an L-malamide residue was attached to the chromophore . The three bidentate Fe3+ binding ligands consisted of an o-dihydroxy aromatic group from the quinoline derivative, beta-hydroxyaspartic acid, and an internally cyclized N delta-hydroxyornithine . The structure of pseudobactin 589 A is unique but strikingly similar to that of other pseudobactin-type siderophores from other plant growth promoting and plant deleterious pseudomonads.

Bioorg Khim, 1990 Aug, 16(8), 1134 - 7
{Rare initiation codons are regulators of expression of the rpoC gene}; Boni IV et al.; Translation of the rpoC genes in Escherichia coli and Salmonella typhimurium is known to start from the GUG codon . Now, using toeprint analysis we have shown UUG to be the initiation codon of the Pseudomonas putida rpoC gene . IF3 does not seem to proofread initiation at the UUG codon . The rpoC genes of P . putida, E . coli, and S . typhimurium, which use rare start codons, have strong SD-domains AGGAGG (P . p.) and GGGAG (E . c., S . t.), optimal seven-nucleotide spacing between SD and start codons, and good second codon AAA . We suggest that rpoC presents an infrequent case of the regulation of translation initiation by selecting the start codon.

J Bacteriol, 1990 Aug, 172(8), 4448 - 55
Cloning and expression of the ponB gene, encoding penicillin-binding protein 1B of Escherichia coli, in heterologous systems; Pla J et al.; A fragment from the ponB region of the Escherichia coli chromosome comprising the promoterless sequence encoding penicillin-binding protein 1B (PBP 1B) has been cloned in a broad-host-range expression vector under the control of the kanamycin resistance gene promoter present in the vector . The hybrid plasmid (pJP3) was used to transform appropriate strains of Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa . In all instances, the coding sequence was expressed in the heterologous hosts, yielding a product with electrophoretic mobility, protease accessibility, membrane location, and beta-lactam-binding properties identical to those of native PBP 1B in E . coli . These results indicated that PBP 1B of E . coli is compatible with the cytoplasmic membrane environment of unrelated bacterial species and support the idea that interspecific transfer of mutated alleles of genes coding for PBPs could potentially be an efficient spreading mechanism for intrinsic resistance to beta-lactams.

J Nutr Sci Vitaminol (Tokyo), 1990 Aug, 36(4), 339 - 47
Purification and characterization of S-alkylcysteine alpha,beta-lyase from Pseudomonas putida; Kamitani H et al.; S-Alkylcysteine alpha,beta-lyase {EC 4.4.1.6} was purified to more than 90% homogeneity from the cell extract of Pseudomonas putida ICR 3640 . The enzyme has a molecular weight of about 195,000, and is composed of six subunits identical in molecular weight (37,000) . Pyridoxal 5'-phosphate is required as a cofactor . The enzyme catalyzes the alpha,beta-elimination of S-methyl-L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, Se-methyl-DL-selenocysteine, and O-methyl-L-serine . However, S-methyl-D-cysteine, L-methionine, and L-norvaline were inert . The enzyme catalyzes also the beta-replacement reaction of the thiomethyl group of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines . In addition to S-methyl-L-cysteine, Se-methyl-DL-selenocysteine and O-methyl-L-serine also serve as substrates in the beta-replacement reaction.

Biochem J, 1990 Aug 1, 269(3), 815 - 9
Stereochemical aspects of the oxidation of 4-ethylphenol by the bacterial enzyme 4-ethylphenol methylenehydroxylase; Reeve CD et al.; The O2-independent hydroxylase 4-ethylphenol methylenehydroxylase (4EPMH) from Pseudomonas putida JD1 catalysed the complete conversion of 4-ethylphenol into 1-(4-hydroxyphenyl)ethanol together with a small amount of 4-hydroxyacetophenone, but with no formation of the side product 4-vinylphenol reported to be formed when the similar enzyme p-cresol methylhydroxylase (PCMH) catalyses this reaction . The enantiomer of 1-(4-hydroxyphenyl)ethanol produced by 4EPMH was R(+) when horse heart cytochrome c or azurin was used as electron acceptor for the enzyme . PCMHs from various bacterial strains produced the S(-)-alcohol . Both enantiomers of 1-(4-hydroxyphenyl)ethanol were substrates for conversion into 4-hydroxyacetophenone by 4EPMH, but the S(-)-isomer was preferred . The Km and kcat . were 1.2 mM and 41 s-1 respectively for the S(-)-alcohol and 4.7 mM and 22 s-1 for the R(+)-alcohol . In addition to the 1-(4-hydroxyphenyl)ethanol dehydrogenase activity of 4-EPMH, NAD(+)-linked dehydrogenase activity for both enantiomers of the alcohol was found in extracts of Ps . putida JD1.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6097 - 101
Molecular analysis of the hydrogenosomal ferredoxin of the anaerobic protist Trichomonas vaginalis; Johnson PJ et al.; We have determined the primary structure of the {2Fe-2S}ferredoxin of the anaerobic protist Trichomonas vaginalis . This protein, situated in the hydrogenosome, is composed of 93 amino acids . A comparison of T . vaginalis ferredoxin with greater than 80 other ferredoxins shows the closest similarity to {2Fe-2S}putidaredoxin of the aerobic bacterium Pseudomonas putida and a lesser one to mitochondrial {2Fe-2S}ferredoxins of vertebrates . This similarity is reflected in the overall primary structure and in the spacing of cysteine residues coordinating the iron-sulfur center . The primary structure, but not the environment of the iron-sulfur center, also shows similarity with {2Fe-2S}ferredoxins of photosynthetic organisms and halobacteria . We have cloned and analyzed the T . vaginalis ferredoxin gene . The gene is present in a single copy and devoid of introns . It gives rise to a transcript with unusually short 5' and 3' untranslated regions of 16 and 18 nucleotides, respectively . DNA sequence analysis of the gene predicts an additional 8 amino acids at the amino terminus which are absent from the purified protein . This amino-terminal region of the protein is characterized by properties typical of mitochondrial presequences.

Eur J Biochem, 1990 Jul 31, 191(2), 337 - 46
Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus; Hawkins CF et al.; A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli . Its nucleotide sequence was determined . Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains . The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon . It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex . All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences . The E1 alpha and E1 beta subunits of the B . stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida . In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.

J Biol Chem, 1990 Jul 25, 265(21), 12388 - 92
The downfield resonances in the 1H NMR spectra of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins; Cheng H et al.; Pseudomonas putida and Azotobacter vinelandii ferredoxins each contain one {4Fe-4S} cluster and one {3Fe-4S} cluster . Their polypeptide chains are nearly identical, differing by only 15 residues out of a total of 106 . T1 measurements and temperature dependence studies of the 1H NMR spectrum of each ferredoxin demonstrate that all six resolved downfield resonances are near an iron-sulfur center . The five most downfield resonances are shown to arise from protons on cysteinyl beta-carbons by incorporation of cysteine deuterated at the beta-carbon into cell protein . The sixth peak (10.5 ppm) is shown to be a non-cysteinyl proton . This peak resolves into two resonances of approximately equal intensity at temperatures below 15 degrees or above 25 degrees C . A nuclear Overhauser effect observed between the two downfield-most resonances of A . vinelandii ferredoxin indicates that they originate from a geminal pair of beta-cysteinyl protons . An Overhauser effect observed between the resonances at 22.3 and 15.7 ppm, in conjunction with other results, implies that the resonance at 22.3 ppm arises from a beta-proton on the 3Fe-center-bound Cys16, while the resonance at 15.7 ppm arises from Cys45 beta-proton, which is bound to the 4Fe center . The five most downfield resonances are pH-dependent . The sixth peak (10.5 ppm in P . putida ferredoxin) is pH-independent . Possible origins for the observed pH dependencies are discussed.

Appl Environ Microbiol, 1990 Jul, 56(7), 2268 - 70
Chromium reduction in Pseudomonas putida; Ishibashi Y et al.; Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000 . Chromate reductase activity was associated with soluble protein and not with the membrane fraction . The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-) . Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.

Plasmid, 1990 Jul, 24(1), 25 - 36
Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase; Kivisaar M et al.; Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation . Four types of PHE plasmids, pEST1024, pEST1026, pEST1028, and pEST1029, are characterized . They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes) . We cloned the genes determining phenol monooxygenase and catechol 1,2-dioxygenase from the Pseudomonas sp . parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA . The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P . putida PaW85 as the result of transposition of the 17-kb genetic element from the chromosome of PaW85 into the plasmid carrying cloned phe genes . The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site . In subcloning experiments we found two regions in the 17-kb DNA stretch that are involved in the expression of the cloned phe genes.

J Bacteriol, 1990 Jul, 172(7), 3707 - 10
Mutations leading to constitutive expression from the TOL plasmid meta-cleavage pathway operon are located at the C-terminal end of the positive regulator protein XylS; Zhou LM et al.; The XylS protein is the positive activator of the TOL plasmid meta-cleavage pathway operon for the metabolism of alkylbenzoates in Pseudomonas putida . The regulator stimulates transcription from the TOL meta pathway operon promoter (Pm) when activated by benzoate effectors or in the absence of effectors when overproduced . xylS mutant alleles that encode regulators which constitutively mediate expression from Pm were isolated and characterized . The mutant proteins all exhibit single amino acid substitutions adjacent to putative alpha-helix-turn-alpha-helix domains at their C-terminal ends . The XylS mutant proteins can still be partially activated by the usual and unusual benzoate effectors for the wild-type regulator and when activated stimulate higher levels of transcription from Pm.

Appl Environ Microbiol, 1990 Jul, 56(7), 2036 - 45
Chlorinated biphenyl mineralization by individual populations and consortia of freshwater bacteria; Pettigrew CA et al.; Comparative studies were performed to investigate the contribution of microbial consortia, individual microbial populations, and specific plasmids to chlorinated biphenyl biodegradation among microbial communities from a polychlorinated biphenyl-contaminated freshwater environment . A bacterial consortium, designated LPS10, was shown to mineralize 4-chlorobiphenyl (4CB) and dehalogenate 4,4'-dichlorobiphenyl . The LPS10 consortium involved three isolates: Pseudomonas testosteroni (LPS10A), which mediated the breakdown of 4CB and 4,4'-dichlorobiphenyl to 4-chlorobenzoic acid; an isolate tentatively identified as an Arthrobacter sp . (LPS10B), which mediated 4-chlorobenzoic acid degradation; and Pseudomonas putida bv . A (LPS10C), whose role in the consortium has not been determined . None of these isolates contained detectable plasmids or sequences homologous to the 4CB-degradative plasmid pSS50 . A freshwater isolate, designated LBS1C1, was found to harbor a 41-megadalton plasmid that was related to the 35-megadalton plasmid pSS50, and this isolate was shown to mineralize 4CB . In chemostat enrichments with biphenyl and 4CB as primary carbon sources, the LPS10 consortium was found to outcomplete bacterial populations harboring plasmids homologous to pSS50 . These results demonstrate that an understanding of the biodegradative capacity of individual bacterial populations as well as interacting populations of bacteria must be considered in order to gain a better understanding of polychlorinated biphenyl biodegradation in the environment.

Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 1049 - 54
Purification and characterization of salicylate hydroxylase from Pseudomonas putida PpG7; You IS et al.; The salicylate hydroxylase from P . putida PpG7 was purified and characterized . The enzyme appears to be monomeric, and it showed one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 45 kDa . The sequence of the first 25 amino acids of salicylate hydroxylase (PpG7) was determined . Also, the total amino acid composition of salicylate hydroxylase (PpG7) was obtained and compared with that of the known salicylate hydroxylase from P . putida.

J Bacteriol, 1990 Jun, 172(6), 3335 - 45
Genes for two herbicide-inducible cytochromes P-450 from Streptomyces griseolus; Omer CA et al.; Streptomyces griseolus ATCC 11796 contains two inducible, herbicide-metabolizing cytochromes P-450 previously designated P-450SU1 and P-450SU2 (P-450CVA1 and P-450CVB1, respectively, using nomenclature of Nebert et al . {D . W . Nebert, M . Adesnik, M . J . Coon, R . W . Estabrook, F . J . Gonzalez, F . P . Guengerich, I . C . Gunsalus, E . F . Johnson, B . Kemper, W . Levin, I . R . Phillips, R . Sato, and M . R . Waterman, DNA 6:1-11, 1987}) . Using antibodies directed against cytochrome P-450SU1, its N-terminal amino acid sequence, and amino acid composition, we cloned the suaC gene encoding cytochrome P-450SU1 . Similar information about the cytochrome P-450SU2 protein confirmed that a gene cloned by cross-hybridization to the suaC gene was the subC gene encoding cytochrome P-450SU2 . The suaC and subC genes were expressed in Escherichia coli, DNA for both genes was sequenced, and the deduced amino acid sequences were compared with that of the well-characterized cytochrome P-450CAM from Pseudomonas putida . Both cytochromes P-450SU1 and P-450SU2 contain several regions of strong similarity with the amino acid sequence of P-450CAM, primarily in regions of the protein responsible for attachment and coordination of the heme prosthetic group.

Biochim Biophys Acta, 1990 May 8, 1038(3), 300 - 5
L-pipecolic acid metabolism in human liver: L-alpha-aminoadipate delta-semialdehyde oxidoreductase; Chang YF et al.; A soluble enzyme that catalyzes the oxidation of L-alpha-aminoadipate delta-semialdehyde to L-alpha-aminoadipic acid in the presence of NAD+ has been isolated and characterized from human liver . This enzyme L-alpha-aminoadipic delta-semialdehyde oxidoreductase has been found to be localized in the cytosol using subcellular fractionation and marker enzyme assays . The reaction product of this enzyme has been identified as L-alpha-aminoadipic acid by use of an amino acid analyzer and thin layer chromatography . The enzymatic reaction was irreversible and has a pH optimum of 8 . The enzyme was stimulated by Mg2+, Cu2+ and Mn2+, and has a requirement of free -SH groups . The Km and Vmax values for its substrate L-alpha-aminoadipate delta-semialdehyde were shown to be 181 microM and 71.4 pmol.min-1.mg-1, respectively, and for its coenzyme NAD+ to be 454 microM and 142.9 pmol.min-1.mg-1, respectively . The characteristics of the oxidoreductase obtained from the human liver and Pseudomonas putida were compared.

J Gen Microbiol, 1990 May, 136 ( Pt 5), 881 - 6
Purification and characterization of D-2-haloacid dehalogenase from Pseudomonas putida strain AJ1/23; Smith JM et al.; A D-2-haloacid dehalogenase was isolated and purified to homogeneity from Pseudomonas putida strain AJ1/23 . The enzyme catalysed the stereospecific dehalogenation of the D-isomer of 2-chloropropionate . Using a new ion-chromatograph assay, the enzyme was found to catalyse the dehalogenation of short-chain 2-halocarboxylic acids . Maximum enzyme activity occurred at pH 9.5 and 50 degrees C and the enzyme was insensitive to most -SH reagents . The enzyme has an Mr of about 135,000 and appears to be composed of four subunits of identical Mr.

Appl Environ Microbiol, 1990 May, 56(5), 1501 - 3
Chemotaxis of Pseudomonas putida toward chlorinated benzoates; Harwood CS et al.; The chlorinated aromatic acids 3-chlorobenzoate and 4-chlorobenzoate are chemoattractants for Pseudomonas putida PRS2000 . These compounds are detected by a chromosomally encoded chemotactic response to benzoate which is inducible by beta-ketoadipate, an intermediate of benzoate catabolism . Plasmid pAC27, encoding enzymes for 3-chlorobenzoate degradation, does not appear to carry genes for chemotaxis toward chlorinated compounds.

J Bacteriol, 1990 May, 172(5), 2224 - 9
Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida; Consevage MW et al.; The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli . The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis . Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence . This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits . Comparison of the P . putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level . Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.

J Gen Microbiol, 1990 May, 136 ( Pt 5), 905 - 12
Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance; Gleave AP et al.; In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPG1000 (5.0 kb) which carries both a selectable marker (chloramphenicol resistance from Staphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonas putida expressed from a cloned promoter of Bacillus phage SPO2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion . To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPG1000 insertion and insertion/deletion derivatives . We have identified the replication region as well as separate regions required for segregational and structural stability . The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.

Appl Microbiol Biotechnol, 1990 May, 33(2), 217 - 20
Mechanism of formaldehyde biodegradation by Pseudomonas putida; Adroer N et al.; Formaldehyde biodegradation by a strain of Pseudomonas putida has been studied . The results indicate that this biodegradation is initiated by a dismutation reaction, yielding as products formic acid and methanol . The degradation of methanol and formic acid begins after exhaustion of formaldehyde in the medium, and presents a diauxic pattern: first formic acid is consumed followed by methanol . Moreover, cell viability, which is affected by the amount of added formaldehyde, has been determined.

Gene, 1990 Apr 30, 89(1), 37 - 46
A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for gram-negative bacteria; Labes M et al.; A series of broad-host-range expression and lac fusion vectors, based on RSF1010 derivatives, was constructed . The expression vectors contain various promoters (pNm, plac, ptac and pS1) for expression of foreign genes . The efficiency of the promoters was determined in Escherichia coli, Rhizobium meliloti, Rhizobium leguminosarum and Pseudomonas putida by beta-galactosidase activity measurements . Of the promoters assayed in E . coli, the most effective is the tac promoter, whereas in soil bacteria the appropriate promoter for overexpression of foreign genes is the NmR promoter . The GmR gene, serving as a selectable marker for the plasmids, was efficiently expressed in R . meliloti as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thus, pGm was also used to construct an expression vector . The translational fusion vectors allow the identification and characterization of promoter-carrying cloned fragments on the translational level, whereas the transcriptional fusion vectors can be used to identify and to study promoters on cloned fragments . All lac fusion vectors contain the E . coli lacZ gene or the complete lac operon facilitating quantification of expression.

J Biol Chem, 1990 Apr 25, 265(12), 7084 - 90
Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida . A specific enzyme for the catabolism of phenylacetic acid; Martinez-Blanco H et al.; A new enzyme, phenylacetyl-CoA ligase (AMP-forming) (PA-CoA ligase, EC 6.2.1-) involved in the catabolism of phenylacetic acid (PAA) in Pseudomonas putida is described and characterized . PA-CoA ligase was specifically induced by PAA when P . putida was grown in a chemically defined medium in which phenylacetic acid was the sole carbon source . Hydroxyl, methyl-phenylacetyl derivatives, and other PAA close structural molecules did not induce the synthesis of this enzyme and neither did acetic, butyric, succinic, nor fatty acids (greater than C5 atoms carbon length) . PA-CoA ligase requires ATP, CoA, PAA, and MgCl2 for its activity . The maximal rate of catalysis was achieved in 50 mM HCl/Tris buffer, pH 8.2, at 30 degrees C and under these conditions, the Km calculated for ATP, CoA, and PAA were 9.7, 1.0, and 16.5 mM, respectively . The enzyme is inhibited by some divalent cations (Cu2+, Zn2+, and Hg2+) and by the sulfhydryl reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-chloromercuribenzoate . PA-CoA ligase was purified to homogeneity (513-fold) . It runs as a single polypeptide in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a molecular mass of 48 +/- 1 kDa . PA-CoA ligase does not use as substrate either 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, or 3,4-dihydroxyphenylacetic acids and shows a substrate specificity different from other acyl-CoA-activating enzymes . The enzyme is detected in P . putida from the early logarithmic phase of growth and is repressed by glucose, suggesting that PA-CoA ligase is a specific enzyme involved in the utilization of PAA as energy source.

J Biol Chem, 1990 Apr 15, 265(11), 6066 - 73
Putidaredoxin reductase and putidaredoxin . Cloning, sequence determination, and heterologous expression of the proteins; Peterson JA et al.; The oxidation of camphor by cytochrome P-450cam requires the participation of a flavoprotein, putidaredoxin reductase, and an iron-sulfur protein, putidaredoxin, to mediate the transfer of electrons from NADH to P-450 for oxygen activation . A 2.2-kilobase pair BamHI-StuI fragment from whole cell DNA of camphor-grown Pseudomonas putida has been cloned and sequenced . Translation of the sequence revealed two open reading frames that could code for putidaredoxin reductase and putidaredoxin . In the case of putidaredoxin, the translated sequence matched the published sequence (Tanaka, M., Haniu, M., Yasunobu, K . T., Dus, K., and Gunsalus, I . C . (1974) J . Biol . Chem . 249, 3689-3701) with the exception of one amino acid . Codon usage in these proteins, like the proteins of other Pseudomonads, is strongly biased to G + C in the third nucleotide . A potential transcription termination site was found 3' to the putidaredoxin coding region . The "FAD-binding" amino acid consensus sequence, present in other flavoproteins, was found in putidaredoxin reductase beginning at residue 11 and a second occurrence of this sequence was found beginning with amino acid 156 . The second sequence could represent the NAD-binding site . The regions encoding putidaredoxin reductase and putidaredoxin were subcloned and independently expressed in Escherichia coli at the level of 0.4 and 4.8 mg of enzymatically active protein/g wet weight of cells, respectively . Site-directed mutagenesis was used to change the rare start codon, GTG, of putidaredoxin reductase to ATG which resulted in an 18-fold increase in the level of expression of this protein to 7.4 mg/g wet weight of cells . The construction of these two clones, which express these important proteins, will facilitate studies of their interaction with each other and with P-450cam.

Genetika, 1990 Apr, 26(4), 770 - 2
{New plasmids of herbicide 2,4-dichlorophenoxyacetic acid biodegradation}; Ausmees NR et al.; Three herbicide 2,4-D metabolizing bacterial strains were isolated from three independent soil samples of Estonia . The strains, although belonging to various species, contain 2,4-D degradative plasmids with identical restriction patterns . pEST4001 is a 78 kb conjugative plasmid . All Pseudomonas putida PaW340 2,4-D+ transconjugants obtained a 70 kb plasmid pEST4011 - a deletion derivative of the pEST4001 . The restriction patterns of the plasmids mentioned above are considerably different from those of the other 2,4-D plasmids pJP4 and pRC10 reported previously.

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