J Bacteriol, 1998 Dec, 180(24), 6459 - 67 Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp . strain 61-3; Matsusaki H et al.; Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp . strain 61-3, which produces a blend of poly(3-hydroxybutyrate) {P(3HB)} homopolymer and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) {P(3HB-co-3HA}) consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level . In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), beta-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found . The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs . Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators . The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes . Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp . strain 61-3 elevated the levels of beta-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium . In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs . Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp . strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively. FEMS Microbiol Lett, 1998 Dec 1, 169(1), 37 - 43 Malonate decarboxylase of Pseudomonas putida is composed of five subunits; Chohnan S et al.; Two different forms of malonate decarboxylase were purified from Pseudomonas putida . The active form was composed of the five different subunits alpha (60 kDa), beta (33 kDa), gamma (28 kDa), delta (13 kDa), and epsilon (30 kDa) and the inactive form was composed of the four subunits lacking the epsilon subunit . The former catalyzed the decarboxylation of malonate to acetate, but the latter could not, although it retained both activities of acetyl-CoA:malonate CoA transferase and malonyl-CoA decarboxylase . The delta subunit of the active form was acylated by the incubation with {2-14C}malonyl-CoA, but the delta subunit of the inactive form was not labeled . From the above results and the N-terminal amino acid sequence analysis, it was concluded that the epsilon subunit was an essential subunit to function as malonyl-CoA:ACP transacylase, which was an indispensable component of the enzyme for the cyclic decarboxylation of malonate. Biochemistry, 1998 Nov 10, 37(45), 15965 - 73 Bacterial iron transport: 1H NMR determination of the three-dimensional structure of the gallium complex of pyoverdin G4R, the peptidic siderophore of Pseudomonas putida G4R; Atkinson RA et al.; Among the fluorescent Pseudomonas species, Pseudomonas putida is a rare case of a nitrogen-fixing bacterium that transforms nitrogen into ammonia . When grown under iron-deficient conditions, it produces two major pyoverdins: pyoverdin G4R and pyoverdin G4RA . Their primary structures have been established using FAB-MS and one- and two-dimensional 15N, 13C, and 1H NMR on both the unlabeled and 15N-labeled compounds {Salah El Din, A . L . M., et al . (1997) Tetrahedron 53, 12539-12552} . The two pyoverdins have a common chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline . The chromophore is bound to the linear heptapeptide L-Asp-L-Orn-D-beta-threo-OHAsp-L-Dab-Gly-L-Ser-L-cyclo-OHOrn . Circular dichroism spectra suggest that the absolute configuration of the metal complex is Delta . The three-dimensional structure in solution of pyoverdin G4R-Ga(III) was determined after interpretation of two-dimensional 1H NMR spectra recorded at 283 and 303 K . The complex is tightly defined with a compact structure with a Delta absolute configuration . The site of complexation of the metal ion is found to be located on the surface of the molecule, showing that the ion can be released without large conformational changes, while the polar groups of the peptide chain, which may be responsible for the recognition of the receptor, are placed on the opposite side of the overall shape . The three-dimensional structure of pyoverdin G4R-Ga(III) is compared with those of other pyoverdins, and the role of the structure in iron uptake is discussed. Appl Microbiol Biotechnol, 1998 Aug, 50(2), 233 - 40 Simultaneous degradation of chloro- and methyl-substituted aromatic compounds: competition between Pseudomonas strains using the ortho and meta pathway or the ortho pathway exclusively; Franck-Mokross AC et al.; Pseudomonas sp . D7-4 and Pseudomonas sp . B13 FR1(pFRC20P) degraded mixtures of chloro- and methyl-substituted benzoates exclusively via an extended ortho pathway, whereas in Pseudomonas putida WR 201 both ortho and meta fission were induced by mixtures of 3-chloro- and 3-methylbenzoate or even by 3-chloro-benzoate alone . The competition behaviour of these strains was compared in batch and in chemostat cultures. . Despite misrouting of metabolites, strain WR201 was competitive, in a lot of the competition experiments, with mixtures of these substrates . Only in a narrow range of the mixing ratio of chloro- and methylbenzoate was the presence of both the meta and ortho pathways a disadvantage for competitiveness . Outside these ranges other attributes, such as high growth rates or short lag periods, or a respective strain were even more essential for one strain to outcompete another. Appl Environ Microbiol, 1998 Dec, 64(12), 5049 - 52 Induction of the tod operon by trichloroethylene in Pseudomonas putida TVA8; Shingleton JT et al.; Bioluminescence, mRNA levels, and toluene degradation rates in Pseudomonas putida TVA8 were measured as a function of various concentrations of toluene and trichloroethylene (TCE) . TVA8 showed an increasing bioluminescence response to increasing TCE and toluene concentrations . Compared to uninduced TVA8 cultures, todC1 mRNA levels increased 11-fold for TCE-treated cultures and 13-fold for toluene-treated cultures . Compared to uninduced P . putida F1 cultures, todC1 mRNA levels increased 4.4-fold for TCE-induced cultures and 4.9-fold for toluene-induced cultures . Initial toluene degradation rates were linearly correlated with specific bioluminescence in TVA8 cultures. Appl Environ Microbiol, 1998 Dec, 64(12), 4904 - 11 Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes; Ronchel MC et al.; Active biological containment systems are based on the controlled expression of killing genes . These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides . The physiological effects that lead to cell death upon the induction of expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed . P . putida CMC4 and CMC12 carry in their chromosomes a fusion of the PA1-04/03 promoter to the Escherichia coli gef gene and the phiX174 lysis gene E, respectively . Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter . Under induced conditions, killing of P . putida CMC12 cells mediated by phiX174 lysis protein E was faster than that observed for P . putida CMC4, for which the Gef protein was the killing agent . In both cases, cell death occurred as a result of impaired respiration, altered membrane permeability, and the release of some cytoplasmic contents to the extracellular medium. Eur J Clin Microbiol Infect Dis, 1998 Sep, 17(9), 642 - 4 Neonatal Pseudomonas putida infection presenting as staphylococcal scalded skin syndrome; Ladhani S et al.; A case of neonatal Pseudomonas putida sepsis presenting as staphylococcal scalded skin syndrome is described . Staphylococcal scalded skin syndrome is a clinical term used for a spectrum of primarily neonatal blistering skin disorders caused by the exfoliative toxins of Staphylococcus aureus . The disease typically begins with general erythema and fever, followed by the formation of large fluid-filled bullae that coalesce and rupture on slightest pressure to leave extensive areas of denuded skin . The 9-day-old male infant described presented with a generalised non-tender, macular, erythematous rash that later developed into large, flaccid, clear fluid-filled bullae to leave extensive erythematous, weeping, and denuded areas covering over 90% of the total body surface . Despite aggressive antibiotic and symptomatic treatment, he died 11 days after admission . While Pseudomonas infections may present with vesico-bullous eruptions, this is believed to be the first case of neonatal Pseudomonas putida sepsis presenting as staphylococcal scalded skin syndrome. Extremophiles, 1998 Nov, 2(4), 395 - 400 Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance; Fukumori F et al.; A toluene-resistant variant of Pseudomonas putida KT2442, strain TOL, was isolated after liquid cultivation under xylene followed by toluene for 1 month in each condition . Almost all the populations of the variant strain formed small but readily visible colonies under toluene within 24 h at 30 degrees C . The toluene-resistant strain also showed an increase in resistance to some unrelated antibiotics . Several toluene-sensitive Tn5 mutants have been isolated from the toluene-resistant strain and showed various levels of sensitivity . Most of these mutations did not cause significant changes in antibiotic resistance; however, one of the mutants (TOL-4) was highly susceptible to both organic solvents and various antibiotics, especially beta-lactams . Sequencing analysis revealed that the mutation in TOL-4 had been introduced into a gene that may encode a transporter protein of an efflux system . This efflux system is very similar to one of the multidrug efflux systems of Pseudomonas aeruginosa . These observations indicate that a multidrug efflux system plays a major role in the organic solvent resistance of P . putida TOL . However, several other genes may also be involved. J Biotechnol, 1998 Oct 8, 64(2-3), 145 - 57 Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1; Valentin HE et al.; Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomonas oleovorans were purified and the N-terminal sequences of two major proteins migrating in sodium dodecyl sulfate polyacrylamide gels with a relative molecular mass of 18 and 43 kDa (GA1 and GA2, respectively) were analyzed . Radiolabeled degenerate probes deduced from these amino acid sequences were used to identify genomic DNA fragments from P . oleovorans and Pseudomonas putida encoding GA1 and GA2 . DNA sequence analysis of the fragments obtained from P . putida revealed that the genes encoding these proteins were adjacent to phaC2 and ORF3, the PHA synthase II gene and an open reading frame of unknown function, respectively, found at the P . oleovorans and P . aeruginosa PHA synthase gene locus . The open reading frames encoding GA1, GA2 and ORF3 or smaller fragments beginning at GA1 were inactivated by chromosomal insertion of the Tn5 kanamycin resistance gene block (neo) . When these mutants were grown on mineral salts agar media under nitrogen limitation, containing gluconate or decanoate as carbon sources, they appeared more translucent than the wild-type grown under similar conditions . Gas-chromatographic analysis of the cellular dry mass revealed that the mutant strains accumulated 30-50% less PHA than the P . putida wild type. J Biotechnol, 1998 Oct 8, 64(2-3), 137 - 44 Protein organization on the PHA inclusion cytoplasmic boundary; Stuart ES et al.; Polyhydroxyalkanoate (PHA) cellular inclusions consist of polyesters, phospholipids, and proteins . Both the polymerase and the depolymerase enzymes are active components of the structure . Recently, proteins associated with these inclusions have been described in a number of bacterial species . In order to further clarify the structure and function of these proteins in relation to polymer inclusions, ultrastructural studies of isolated polymer inclusions were initiated . The surface boundary characteristics of polymer inclusions, produced by several genera of bacteria, two different Pseudomonas putida deletion mutants and by Escherichia coli recombinants, were examined . The recombinant E . coli carried either the PHB biosynthesis operon (phaCAB) from Ralstonia eutropha alone, or both this operon and a gene encoding an inclusion surface protein of R . eutropha (phaP) . The results support two suggestions: (i) specific genes in the PHA gene cluster code for the proteins forming the surface boundary arrays which characterize the polymer inclusion; and (ii) transfer of such a gene would result in subcellular compartmentalization of accumulating polymer . Although the proteins appear to serve a similar function among different genera, nevertheless, the different surface proteins are encoded by a variety of non-homologous genetic sequences. Biodegradation, 1998, 9(2), 119 - 32 Isopropylbenzene catabolic pathway in Pseudomonas putida RE204: nucleotide sequence analysis of the ipb operon and neighboring DNA from pRE4; Eaton RW et al.; Pseudomonas putida RE204 employs a set of plasmid-specified enzymes in the catabolism of isopropylbenzene (cumene) and related alkylbenzenes . A 21,768 bp segment of the plasmid pRE4, whose sequence is discussed here, includes the ipb (isopropylbenzene catabolic) operon as well as associated genetic elements . The ipb operon, ipbAaAbAcAdBCEGFHD, encodes enzymes catalyzing the conversion of isopropylbenzene to isobutyrate, pyruvate, and acetyl-coenzyme A as well as an outer membrane protein (IpbH) of uncertain function . These gene products are 75 to 91% identical to those encoded by other isopropylbenzene catabolic operons and are somewhat less similar to analogous proteins of related pathways for the catabolism of mono-substituted benzenes . Upstream of ipbAa, ipbR encodes a positive regulatory protein which has about 56% identity to XylS regulatory proteins of TOL (xylene/toluate) catabolic plasmids . This similarity and that of the DNA sequence in the proposed ipb operator-promoter region (ipbOP) to the same region of the xyl meta operon (xylOmPm) suggest that, although the IpbR and XylS regulatory proteins recognize very different inducers, their interactions with DNA to activate gene expression are similar . Upstream of ipbR is an 1196 bp insertion sequence, IS1543, related to IS52 and IS1406 . Separating ipbR from ipbAa are 3 additional tightly clustered IS elements . These are IS1544, related to IS1543, IS52, and other members of the IS5 family; IS1545, related to IS1240; and IS1546, related to IS1491 . Encompassing the ipb catabolic genes and the other genetic elements and separated from each other by 18,492 bp, are two identical, directly repeated 1007 bp DNA segments . Homologous recombination between these segments appears to be responsible for the occasional deletion of the intervening DNA from pRE4. Mol Gen Genet, 1998 Oct, 259(6), 674 - 8 The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn5501 and Tn5502, are cryptic transposons of the Tn3 family; Lauf U et al.; Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1 . Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons) . The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily . The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons . Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies . Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501 . While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors . This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment . It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat) . Transposition of phenol degradation genes could not be detected . Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon. DNA Cell Biol, 1998 Oct, 17(10), 915 - 20 Cloning and nucleotide sequence of amidase gene from Pseudomonas putida; Wu S et al.; Amidases are a class of enzymes which convert amides to acids and have potential value in the development of commercial bioprocesses for the production of useful chemicals . A gene encoding an amidase in Pseudomonas putida 5B has been cloned, sequenced, and overexpressed in Escherichia coli . An additional open reading frame (P38K) encoding a putative protein of 38 kDa was found immediately upstream of the amidase gene . This work continues our characterization of a P . putida operon, which now appears to include P38K, amidase, and a stereo-specific nitrile hydratase . This characterization underlies continuing efforts in biocatalyst development. Eur J Biochem, 1998 Oct 1, 257(1), 92 - 100 Quantitative structure/activity relationship for the rate of conversion of C4-substituted catechols by catechol-1,2-dioxygenase from Pseudomonas putida (arvilla) C1; Ridder L et al.; The influence of various C4/C5 substituents in catechol (1,2-dihydroxybenzene) derivatives on the overall rate of conversion by catechol-1,2-dioxygenase from Pseudomonas putida (arvilla) C1 was investigated . Using catechol, 4-methylcatechol, 4-fluorocatechol, 4-chlorocatechol, 4-bromocatechol, 4,5-difluorocatechol and 4-chloro-5-fluorocatechol, it could be demonstrated that substituents at the C4 and/or C5 position decrease the rate of conversion, from 62% (4-methylcatechol) down to 0.7% (4-chloro-5-fluorocatechol) of the activity with non-substituted catechol . The inhibition was reversible upon addition of excess catechol for all substrates tested . This indicates that the lower activities are neither due to irreversible inactivation of the enzyme nor to product inhibition . Based on the reaction mechanism proposed in the literature {Que, L . & Ho, R . Y . N . (1996) Chem . Rev . 96, 2606-2624}, the nucleophilic reactivity of the catecholate was expected to be an essential characteristic for its conversion by catechol-1,2-dioxygenase . Therefore, the rates of conversion were compared with calculated energies of the highest occupied molecular orbital (E(HOMO)) of the substrates . A clear quantitative relationship (R>0.97) between the ln kcat and the calculated electronic parameter E(HOMO) was obtained . This indicates that the rate-limiting step of the reaction cycle is dependent on the nucleophilic reactivity of the substrate and not sterically hindered by the relatively large bromine or methyl substituents used in the present study . Possible steps in the reaction mechanism determining the overall rate at 20 degrees C are discussed. J Bacteriol, 1998 Nov, 180(21), 5505 - 14 Int-B13, an unusual site-specific recombinase of the bacteriophage P4 integrase family, is responsible for chromosomal insertion of the 105-kilobase clc element of Pseudomonas sp . Strain B13; Ravatn R et al.; Pseudomonas sp . strain B13 carries the clcRABDE genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb clc element . The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (glyV) as the integration site . We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration . The integrase gene (int-B13) was located near the right end of the clc element . It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa) . The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation . The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases . The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1 . Similar to the bacteriophages phiR73 and P4, the clc element integrates into the 3' end of the target tRNA gene . This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence specificity of the integration . In Pseudomonas sp . strain B13, a circular form of the clc element, which carries an 18-bp DNA sequence identical to the 3'-end portion of glyV as part of its attachment site (attP), could be detected . Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstructed at the right end of the element . The integration process could be demonstrated in RecA-deficient Escherichia coli with two recombinant plasmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1. Protein Expr Purif, 1998 Nov, 14(2), 267 - 74 High expression, purification, and properties of recombinant homocysteine alpha, gamma-lyase; Han Q et al.; Homocysteine alpha,gamma-lyase from the anaerobic protozoan parasite Trichomonas vaginalis has been cloned from genomic DNA using PCR methods and expressed in Escherichia coli with a vector containing the T7 promoter . The recombinant homocysteine alpha,gamma-lyase (rHCYase) is expressed as the major protein in the host E . coli cells . The enzyme was purified to approximately 90% purity using heat treatment at 50 degreesC, precipitation steps with polyethyleneimine, polyethylene glycol 8000, and high sodium chloride, DEAE-Sepharose FF chromatography, and phenyl-Sepharose 6 FF chromatography . The final yield was greater than 50%, which encompassed an approximate 18-fold purification . The enzyme is a homotetramer with a monomer molecular weight of 43K and contains pyridoxal phosphate . The Trichomonas rHCYase is selective for homocysteine with respect to very low cysteinase activity in contrast to the alpha,gamma-lyase from Pseudomonas putida, which has very high cysteinase activity with respect to homocysteine . The T . vaginalis and P . putida alpha,gamma-lyases readily separate on a phenyl-Sepharose 6 FF column with the T . vaginalis enzyme eluting first . rHCYase is stable up to 50 degreesC and active over a pH range of 6-8 . These properties of high recombinant expression in E . coli, a simple and effective high-yield purification procedure and high relative specificity for homocysteine with respect to cysteine, make rHCYase a promising candidate to use for the diagnosis of hyperhomocystenemia, which has been demonstrated to be a major risk factor for the onset and mortality of cardiovascular disease of all types . Can J Microbiol, 1998 Jul, 44(7), 687 - 91 Cloning of the Nocardia corallina polyhydroxyalkanoate synthase gene and production of poly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate); Hall B et al.; The polyhydroxyalkanoate (PHA) synthase gene (phaCNc) from Nocardia corallina was identified in a lambda library on a 6-kb BamHI fragment . A 2.8-kb XhoII subfragment was found to contain the intact PHA synthase . This 2.8-kb fragment was subjected to DNA sequencing and was found to contain the coding region for the PHA synthase and a small downstream open reading frame of unknown function . On the basis of DNA sequence, phaCNc is closest in homology to the PHA synthases (phaCPaI and phaCPaII) of Pseudomonas aeruginosa (approximately 41% identity and 55% similarity) . The 2.8-kb XhoII fragment containing phaCNc was subcloned into broad host range mobilizable plasmids and transferred into Escherichia coli, Klebsiella aerogenes (both containing a plasmid bearing phaA and phaB from Ralstonia eutropha), and PHA-negative strains of R . eutropha and Pseudomonas putida . The recombinant strains were grown on various carbon sources and the resulting polymers were analyzed . In these strains, the PHA synthase from N . corallina was able to mediate the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) containing high levels of 3-hydroxyhexanoate when grown on hexanoate and larger even-chain fatty acids and poly(3-hydroxyvalerate-co-3-hydroxyheptanoate) containing high levels of 3-hydroxyheptanoate when grown on heptanoate or larger odd-chain fatty acids. Int J Biol Macromol, 1998 Oct, 23(3), 165 - 73 Differential scanning calorimetric study of poly(3-hydroxyoctanoate) inclusions in bacterial cells; Song JJ et al.; Medium chain length polyhydroxyalkanoates, MCL-PHAs, produced by bacteria as inclusion bodies or granules were analyzed in situ by differential scanning calorimetry (DSC) without isolation from the cells . The kinetic DSC study of PHA granules, which contained mostly 3-hydroxyoctanoate units (PHO), in Pseudomonas putida BM01 cells showed that the polymer within the granules existed in an amorphous state, but it crystallized after dehydration of the cells under freeze-drying condition (below -50 degrees C) followed by annealing at ambient temperature . In this manner, PHO within the cells readily crystallized to the maximum degree of crystallinity within 24 h at room temperature, which was much faster than for the same polymer isolated by solvent extraction . This observation suggests that the polymer within the cellular granules may be well organized . The DSC endothermic melting peak areas for the room-temperature annealed polymers within the cells were directly proportional to the amount of polymer in the cell, and the results from this type of quantitative analysis were essentially identical to those obtained by gas chromatographic and gravimetric analysis of the polymers . X-Ray diffraction analysis of the polymer in the freeze-dried, whole cells and of the isolated, fully crystallized polymer showed that the two types of PHO samples had similar crystal structures, but the polymer in the granules exhibited better side-chain packing and higher crystallinity. Biochemistry, 1998 Oct 13, 37(41), 14358 - 68 Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida; Gulick AM et al.; The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0% . The protein crystallizes in the space group I222 with one subunit in the asymmetric unit . The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A . The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000 . The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues . The structure was determined without a bound substrate or substrate analogue . Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423 . On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily {Babbitt, P . C., Hasson, M . S., Wedekind, J . E., Palmer, D . R . J., Barrett, W . C., Reed, G . J., Rayment, I., Ringe, D., Kenyon, G . L., and Gerlt, J . A . (1996) Biochemistry 35, 16489-16501} . This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase . Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR . The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed. Biochemistry, 1998 Oct 13, 37(41), 14350 - 7 Evolution of enzymatic activities in the enolase superfamily: partitioning of reactive intermediates by (D)-glucarate dehydratase from Pseudomonas putida; Palmer DR et al.; Glucarate dehydratase (GlucD) from Pseudomonas putida catalyzes the dehydration of both (D)-glucarate and (L)-idarate to 3-deoxy-(L)-threo-2-hexulosarate as well as their epimerization . (D)-{6-13C}Glucarate and (L)-{6-13C}idarate have been synthesized for use in continuous assay of the reactions catalyzed by GlucD by both 13C and 1H NMR spectroscopies, thereby allowing the simultaneous measure of both the dehydration and epimerization reactions . Substrate and solvent isotope effects for the dehydration reactions have been quantitated . The mechanism of the GlucD-catalyzed reaction is discussed in the context of that previously established for the homologous mandelate racemase from P . putida, also a member of the enolase superfamily whose members catalyze reactions initiated by abstraction of a proton alpha to a carboxylate group. Res Microbiol, 1997 Feb, 148(2), 153 - 61 Isolation of a soil psychrotrophic toluene-degrading Pseudomonas strain: influence of temperature on the growth characteristics on different substrates; Chablain PA et al.; Two psychrotrophic toluene-degrading Pseudomonas putida strains were isolated at low temperature from a toluene-polluted soil, thereby demonstrating that toluene degradation at low temperature occurred in nature, a finding of possible interest for soil bioremediation procedures . In one of these strains, two aromatic compounds (toluene and benzoate) were degraded, most likely through different pathways . To study the effect of the growth temperature on the metabolism of these substrates, we studied the evolution of the maximal growth rates with respect to both temperature and substrate . It was shown that not only cardinal temperatures but also temperature characteristics deduced from the Arrhenius plot of maximal growth rates differed when the different substrates were used as sole carbon and energy source. J Bacteriol, 1998 Oct, 180(20), 5357 - 68 A chaperone in the HSP70 family controls production of extracellular fibrils in Myxococcus xanthus; Weimer RM et al.; Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development . The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK) . The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue . Unlike sglK, grpS is not required for social motility or development . Wild-type M . xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein . Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils . The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida . Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M . xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways . M . xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria. J Bacteriol, 1998 Oct, 180(20), 5306 - 12 Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida; Kallastu A et al.; A new insertion sequence (IS element), IS1411, was identified downstream of the phenol degradation genes pheBA that originated from plasmid DNA of Pseudomonas sp . strain EST1001 . According to sequence analysis, IS1411 belongs to a new family of IS elements that has recently been named the ISL3 family (J . Mahillon and M . Chandler, Microbiol . Mol . Biol . Rev . 62:725-774, 1998) . IS1411 generates 8-bp duplication of the target DNA and carries 24-bp inverted repeats (IRs), highly homologous to the IRs of other IS elements belonging to this family . IS1411 was discovered as a result of insertional activation of promoterless pheBA genes in Pseudomonas putida due to the presence of outward-directed promoters at the left end of IS1411 . Both promoters located on the IS element have sequences that are similar to the consensus sequence of Escherichia coli sigma70 . IS1411 can produce IS circles, and the circle formation is enhanced when two copies of the element are present in the same plasmid. Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 681 - 3 Crystallization and preliminary X-ray studies of Pseudomonas putida histidine ammonium-lyase; Teo B et al.; Histidine ammonium-lyase from P . putida was expressed in Escherichia coli, purified to homogeneity, and crystallized by the vapour-diffusion method using polyethylene glycol 3350 as the precipitant . The crystals, which diffract to at least 2.5 A resolution, exhibit the symmetry of space group P212121, with unit-cell parameters a = 89.7, b = 138.2 and c = 164.8 A . The asymmetric unit contains a tetramer, and the crystals have a Vm value of 2.41 A3 Da-1. Appl Environ Microbiol, 1998 Oct, 64(10), 3633 - 40 Plasmids responsible for horizontal transfer of naphthalene catabolism genes between bacteria at a coal tar-contaminated site are homologous to pDTG1 from pseudomonas putida NCIB 9816-4 Stuart-Keil KG, Hohnstock AM, Drees KP, Herrick JB, Madsen EL. The presence of a highly conserved nahAc allele among phylogenetically diverse bacteria carrying naphthalene-catabolic plasmids provided evidence for in situ horizontal gene transfer at a coal tar-contaminated site (J . B . Herrick, K . G . Stuart-Keil, W . C . Ghiorse, and E . L . Madsen, Appl . Environ . Microbiol . 63:2330-2337, 1997) . The objective of the present study was to identify and characterize the different-sized naphthalene-catabolic plasmids in order to determine the probable mechanism of horizontal transfer of the nahAc gene in situ . Filter matings between naphthalene-degrading bacterial isolates and their cured progeny revealed that the naphthalene-catabolic plasmids were self-transmissible . Limited interstrain transfer was also found . Analysis of the restriction fragment length polymorphism (RFLP) patterns indicated that catabolic plasmids from 12 site-derived isolates were closely related to each other and to the naphthalene-catabolic plasmid (pDTG1) of Pseudomonas putida NCIB 9816-4, which was isolated decades ago in Bangor, Wales . The similarity among all site-derived naphthalene-catabolic plasmids and pDTG1 was confirmed by using the entire pDTG1 plasmid as a probe in Southern hybridizations . Two distinct but similar naphthalene-catabolic plasmids were retrieved directly from the microbial community indigenous to the contaminated site in a filter mating by using a cured, rifampin-resistant site-derived isolate as the recipient . RFLP patterns and Southern hybridization showed that both of these newly retrieved plasmids, like the isolate-derived plasmids, were closely related to pDTG1 . These data indicate that a pDTG1-like plasmid is the mobile genetic element responsible for transferring naphthalene-catabolic genes among bacteria in situ . The pervasiveness and persistence of this naphthalene-catabolic plasmid suggest that it may have played a role in the adaptation of this microbial community to the coal tar contamination at our study site. Appl Environ Microbiol, 1998 Oct, 64(10), 4040 - 6 Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains; Sanchez-Romero JM et al.; Resistance to the toxic compound potassium tellurite (Telr) has been employed as a selection marker built into a set of transposon vectors and broad-host-range plasmids tailored for genetic manipulations of Pseudomonas strains potentially destined for environmental release . In this study, the activated Telr determinants encoded by the cryptic telAB genes of plasmid RK2 were produced, along with the associated kilA gene, as DNA cassettes compatible with cognate vectors . In one case, the Telr determinants were assembled between the I and O ends of a suicide delivery vector for mini-Tn5 transposons . In another case, the kilA and telAB genes were combined with a minimal replicon derived from a variant of Pseudomonas plasmid pPS10, which is able to replicate in a variety of gram-negative hosts and is endowed with a modular collection of cloning and expression assets . Either in the plasmid or in the transposon vector, the Telr marker was combined with a 12-kb DNA segment of plasmid pWW0 of Pseudomonas putida mt-2 encoding the upper TOL pathway enzymes . This allowed construction of antibiotic resistance-free but selectable P . putida strains with the ability to grow on toluene as the sole carbon source through an ortho-cleavage catabolic pathway. Appl Environ Microbiol, 1998 Oct, 64(10), 3556 - 62 The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1; de Vrind JP et al.; A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase . It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation . After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified . Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation . The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway . Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production . These strains were chosen for detailed analysis . Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon . They were cytochrome oxidase negative and did not contain c-type cytochromes . Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain . These results indicate that a functional ccm operon is required for Mn2+ oxidation in P . putida GB-1 . A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed. Appl Environ Microbiol, 1998 Oct, 64(10), 3549 - 55 c-type cytochromes and manganese oxidation in Pseudomonas putida MnB1; Caspi R et al.; Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides . We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants . The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan . The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes . Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation. Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1035 - 8 Crystallization and preliminary X-ray diffraction studies of the oxygenating subunit of 3,6-diketocamphane monooxygenase from Pseudomonas putida; McGhie EJ et al.; The oxygenating constituent of the 3,6-diketocamphane monooxygenase isozyme from Pseudomonas putida NCIMB 10007 has been crystallized under two different conditions . Crystals were initially grown from polyethylene glycol (PEG) 8000 and sodium acetate using the vapour-phase diffusion method . The crystals were of orthorhombic P212121 space group, with cell dimensions a = 55.8, b = 94.5 and c = 163.7 A and diffracted to 2.8 A resolution . More recently, improved crystals, which diffracted beyond 2 A, have been grown from ammonium sulfate . These crystals also belong to the orthorhombic P212121 space group, with cell dimensions of a = 54.6, b = 93.2 and c = 154 . 1 A . A full native data set to 2.5 A resolution has been collected from the ammonium sulfate grown crystals. Mol Gen Genet, 1998 Jul, 259(1), 72 - 8 The rpoS gene regulates OP2, an operon for the lower pathway of xylene catabolism on the TOL plasmid, and the stress response in Pseudomonas putida mt-2; Miura K et al.; Operon OP2 on the Pseudomonas putida TOL plasmid encodes enzymes for m-toluate catabolism; transcription of this operon is activated by XylS in the presence of m-toluate . Because transcriptional activation of OP2 specifically occurs in the stationary phase of growth both in P . putida and in Escherichia coli, we suspected that its transcription is dependent on RpoS (sigmaS) . Therefore, we constructed a rpoS disruption strain of P . putida mt-2, and assayed OP2 expression and other phenotypes . OP2 transcription was dependent on rpoS, indicating that the stationary-phase specific activation of OP2 is due to positive regulation by RpoS in P . putida mt-2 . The rpoS mutant exhibited reduced viability during the stationary phase and was sensitive to high salt concentrations and H2O2 . P . putida mt-2 has two catalase isozymes, KatA and KatB . Expression of the katB gene was specific to the stationary phase and entirely dependent on the rpoS gene, while the katA gene expressed during log phase partially required rpoS . There were no significant changes in tolerance to high temperature or UV light in the rpoS mutant . No difference was observed between the E . coli rpoS mutants and their parents in their capacity to activate OP2, suggesting that the mechanism of OP2 activation in the stationary phase of growth differs between P . putida and E . coli. J Biol Chem, 1998 Sep 11, 273(37), 24044 - 51 A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis . The PHAG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme a transferase; Rehm BH et al.; To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route . The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201 . S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon . Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source . No complementation of the rhlA mutant P . aeruginosa UO299-harboring plasmid pBHR81, expressing phaG gene under lac promoter control, was obtained . Heterologous expression of phaG in Pseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source . Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P . oleovorans-harboring plasmid pBHR81 . It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10396 - 401 Evolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase; Hasson MS et al.; Muconate lactonizing enzyme (MLE), a component of the beta-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the "enolase superfamily") that catalyze the abstraction of the alpha-proton of a carboxylic acid in the context of different overall reactions . New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-A resolution . The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits . The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn2+-bound structure . Thus, absence of the metal ion does not perturb the structure of the active site . The structures of enolase, mandelate racemase, and MLE were superimposed . A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family . Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary. EMBO J, 1998 Sep 1, 17(17), 5120 - 8 Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD; Bertoni G et al.; The sequence elements determining the binding of the sigma54-containing RNA polymerase (sigma54-RNAP) to the Pu promoter of Pseudomonas putida have been examined . Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs . Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the alpha subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in sigma70 promoters . Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending . These results support the view that binding of sigma54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g . IHF) other than the sole intrinsic affinity of sigma54-RNAP for the -12/-24 site. J Bacteriol, 1998 Sep, 180(17), 4360 - 9 Chromosomal integration, tandem amplification, and deamplification in Pseudomonas putida F1 of a 105-kilobase genetic element containing the chlorocatechol degradative genes from Pseudomonas sp . Strain B13; Ravatn R et al.; Analysis of chlorobenzene-degrading transconjugants of Pseudomonas putida F1 which had acquired the genes for chlorocatechol degradation (clc) from Pseudomonas sp . strain B13 revealed that the clc gene cluster was present on a 105-kb amplifiable genetic element (named the clc element) . In one such transconjugant, P . putida RR22, a total of seven or eight chromosomal copies of the entire genetic element were present when the strain was cultivated on chlorobenzene . Chromosomal integrations of the 105-kb clc element occurred in two different loci, and the target sites were located within the 3' end of glycine tRNA structural genes . Tandem amplification of the clc element was preferentially detected in one locus on the F1 chromosome . After prolonged growth on nonselective medium, transconjugant strain RR22 gradually diverged into subpopulations with lower copy numbers of the clc element . Two nonadjacent copies of the clc element in different loci always remained after deamplification, but strains with only two copies could no longer use chlorobenzene as a sole substrate . This result suggests that the presence of multiple copies of the clc gene cluster was a prerequisite for the growth of P . putida RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence of chlorobenzene. J Bacteriol, 1998 Sep, 180(17), 4332 - 8 Mechanism for biotransformation of nonylphenol polyethoxylates to Xenoestrogens in Pseudomonas putida; John DM et al.; A strain of Pseudomonas putida isolated from activated sewage grew aerobically on the xenoestrogen precursor, nonylphenol polyethoxylate (NPEOx, where x is the number of ethoxylate units) as sole carbon source . Comparative growth yields on NPEOav6, NPEOav9, and NPEOav20 (mixtures with average ethoxylate numbers as indicated) were consistent with utilization of all but two ethoxylate units, and the final accumulating metabolite was identified by gas chromatography-mass spectroscopy as nonylphenol diethoxylate (NPEO2) . There was no growth on nonylphenol or polyethylene glycols, and there was no evidence for production of carboxylic acid analogs of NPEOx . Biodegradation kinetics measured by high-pressure liquid chromatography (HPLC) for each component in NPEOx mixtures showed that biodegradation proceeded via successive exoscission of the ethoxylate chain and not by direct scission between the second and third ethoxylate residues . The NPEOx-degrading activity was inducible by substrate, and cell extracts of NPEOav9-induced cells were also active on the pure alcohol ethoxylate, dodecyl octaethoxylate (AEO8), producing sequentially, under either aerobic or anaerobic conditions, AEO7, AEO6, AEO5, etc., thus demonstrating that the pathway involved removal of single ethoxylate units . HPLC analysis of 2,4-dinitrophenylhydrazone derivatives revealed acetaldehyde (ethanal) as the sole aldehydic product from either NPEOav9 or AEO8 under either aerobic or anaerobic conditions . We propose a mechanism for biotransformation which involves an oxygen-independent hydroxyl shift from the terminal to the penultimate carbon of the terminal ethoxylate unit of NPEOx and dissociation of the resulting hemiacetal to release acetaldehyde and the next-lower homolog, NPEOx-1, which then undergoes further cycles of the same reaction until x = 2. Plant Cell, 1998 Aug, 10(8), 1251 - 66 The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9 Hammond-Kosack KE, Tang S, Harrison K, Jones JD. The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9 . We investigated whether the genomic Cf-9 gene functions in potato and tobacco . Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection . Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco . A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent . However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed . Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings . Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9 . Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed. Mikrobiologiia, 1998 May-Jun, 67(3), 349 - 55 {Features of the production of CryIIIA delta-endotoxin from Bacillus thuringiensis in a recombinant strain of Pseudomonas putida under control of Pm and xylS regulatory elements}; Koretskaia NG et al.; The addition of 3-methyl benzoate to the culture of the recombinant strain Pseudomonas putida IPM-36 (bearing the cryIIIA gene of B . thuringiensis subsp, tenebrionis under the control of the Pm promoter and the regulator gene xylS) slowed down the growth rate of the recombinant strain and increased, under non-selective conditions, the number of plasmid-free cells . Intense synthesis of the Coleoptera-specific delta-endotoxin encoded by the cryIIIA gene began 6-8 h after the addition of the inducer 3-methyl benzoate, no matter whether it was added in the early or late logarithmic phase . Maximal production of endotoxin (0.5-0.6 g/l) was observed when the inducer was added in the early logarithmic phase (3 h of growth) . Overproduction of delta-endotoxin impaired cell division, so that filamentous cells became predominant in the culture . delta-Endotoxin accumulated in overproducing cells as irregular crystalloid inclusions. Can J Microbiol, 1998 May, 44(5), 482 - 6 Amplification of putative chlorocatechol dioxygenase gene fragments from alpha- and beta-Proteobacteria; Leander M et al.; Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes . These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and cholorobenzoate-degrading strains, including species of Sphingomonas . Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain . The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems. J Bacteriol, 1998 Aug, 180(15), 3816 - 22 Physical and genetic map of the obligate intracellular bacterium Coxiella burnetii; Willems H et al.; Pulsed-field gel electrophoresis and PCR techniques have been used to construct a NotI macrorestriction map of the obligate intracellular bacterium Coxiella burnetii Nine Mile . The size of the chromosome has been determined to be 2,103 kb comprising 29 NotI restriction fragments . The average resolution is 72.5 kb, or about 3 . 5% of the genome . Experimental data support the presence of a linear chromosome . Published genes were localized on the physical map by Southern hybridization . One gene, recognized as transposable element, was found to be present in at least nine sites evenly distributed over the whole chromosome . There is only one copy of a 16S rRNA gene . The putative oriC has been located on a 27.5-kb NotI fragment . Gene organization upstream the oriC is almost identical to that of Pseudomonas putida and Bacillus subtilis, whereas gene organization downstream the oriC seems to be unique among bacteria . The physical map will be helpful in investigations of the great heterogeneity in restriction fragment length polymorphism patterns of different isolates and the great variation in genome size . The genetic map will help to determine whether gene order in different isolates is conserved. Macromolecules, 1998 Jul 28, 31(15), 4760 - 3 Bacterial Poly(3-hydroxyalkanoates) Bearing Carbon-Carbon Triple Bonds; Kim DY et al.; Production of poly(3-hydroxyalkanoates), PHAs, by Pseudomonas oleovorans (P . oleovorans) and Pseudomonas putida (P . putida) grown with mixtures of nonanoic acid, NA, and 10-undecynoic acid, 10-UND( identical with), were investigated . Both microorganisms produced PHAs containing carbon-carbon triple bonds in fractions from 0 to 100%, depending on the composition of the carbon substrate mixture . The amounts of unsaturated repeating units in PHAs produced by P . oleovorans were higher than those in PHAs produced by P . putida grown with the same carbon substrates . The repeating units containing carbon-carbon triple bonds were 3-hydroxy-8-nonynoate, 3HN(&tbd1;), and 3-hydroxy-10-undecynoate, 3HUD(&tbd1;) . 3HN(&tbd1;) was the major repeating unit formed from 10-UND(&tbd1;) . The relative amounts of 3HN(&tbd1;) and 3HUD(&tbd1;) in PHAs produced by P . putida were slightly different from those in PHAs produced by P . oleovorans . The number average molecular weights of PHAs produced in this study were approximately 50 000, and polydispersity indices were approximately 2.5 as determined by gel permeation chromatography . The molecular weight distribution and the relative amounts of 3HN(&tbd1;) and 3HUD(&tbd1;) were not affected by either growth time or the composition of the carbon substrate . PHAs bearing triple bonds were soft and differential scanning calorimetry thermograms of these polymers showed very small melting endotherms at approximately 60 degreesC . The glass transition temperatures were in the range of -33 to -21 degreesC. Biochemistry, 1998 Jul 21, 37(29), 10345 - 53 Active monomeric and dimeric forms of Pseudomonas putida glyoxalase I: evidence for 3D domain swapping; Saint-Jean AP et al.; 3D domain swapping of proteins involves the interconversion of a monomer containing a single domain-domain interface and a 2-fold symmetrical dimer containing two equivalent intermolecular interfaces . Human glyoxalase I has the structure of a domain-swapped dimer {Cameron, A . D., Olin, B., Ridderstrom, M., Mannervik, B., and Jones, T . A . (1997) EMBO J . 16, 3386-3395} but Pseudomonas putida glyoxalase I has been reported to be monomeric {Rhee, H.-I., Murata, K., and Kimura, A . (1986) Biochem . Biophys . Res . Commun . 141, 993-999} . We show here that recombinant P . putida glyoxalase I is an active dimer (kcat approximately 500 +/- 100 s-1; KM approximately 0.4 +/- 0.2 mM) with two zinc ions per dimer . The zinc is required for structure and function . However, treatment of the dimer with glutathione yields an active monomer (kcat approximately 115 +/- 40 s-1; KM approximately 1.4 +/- 0.4 mM) containing a single zinc ion . The monomer is metastable and slowly reverts to the active dimer in the absence of glutathione . Thus, glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms . It is unique among domain-swapped proteins in that the active site and an essential metal binding site are apparently disassembled and reassembled by the process of domain swapping . Furthermore, it is the only example to date in which 3D domain swapping can be regulated by a small organic ligand. Biochemistry, 1998 Jul 14, 37(28), 9918 - 30 The crystal structure of benzoylformate decarboxylase at 1.6 A resolution: diversity of catalytic residues in thiamin diphosphate-dependent enzymes; Hasson MS et al.; The crystal structure of the thiamin diphosphate (ThDP)-dependent enzyme benzoylformate decarboxylase (BFD), the third enzyme in the mandelate pathway of Pseudomonas putida, has been solved by multiple isomorphous replacement at 1.6 A resolution and refined to an R-factor of 15.0% (free R = 18.6%) . The structure of BFD has been compared to that of other ThDP-dependent enzymes, including pyruvate decarboxylase . The overall architecture of BFD resembles that of the other family members, and cofactor- and metal-binding residues are well conserved . Surprisingly, there is no conservation of active-site residues not directly bound to the cofactor . The position of functional groups in the active site may be conserved, however . Three classes of metal ions have been identified in the BFD crystal structure: Ca2+ bound to the cofactor in each subunit, Mg2+ on a 2-fold axis of the tetramer, and Ca2+ at a crystal contact . The structure includes a non-proline cis-peptide bond and an unusually long and regular polyproline type II helix that mediates the main contact between tetramers in the crystal . The high-quality electron-density map allowed the correction of errors totaling more than 10% of the amino acid sequence, which had been predicted from the reported sequence of the mdlC gene . Analysis of the BFD structure suggests that requirements for activation of the cofactor, the nature of the reaction intermediates, and architectural considerations relating to the protein fold have been dominant forces in the evolution of ThDP-dependent enzymes. J Gastroenterol, 1998 Jun, 33(3), 412 - 8 Fulminant hepatitis complicated by small intestine infection and massive hemorrhage; Tamura H et al.; A 34-year-old man diagnosed with fulminant hepatitis, caused by hepatitis B virus, and acute renal failure was referred to our hospital . After admission to the intensive care unit, the liver and renal failure were ameliorated . Melena requiring transfusion occurred during the course of his illness . Endoscopic examination demonstrated pseudomembranes, erosions, ulcers, and hemorrhage in the duodenum, the upper jejunum, and the terminal ileum, suggesting widespread lesions throughout the small intestine . Pseudomonas putida, Xanthomonas maltophilia, and Candida glabrata were cultured from ileal fluid . Candida glabrata was also detected in sputum, feces, and on an intravenous catheter tip . The patient was treated with amphotericin B and miconazole . The melena was ameliorated, but inflammation of the small intestine persisted . Although we had difficulty in treating the enteritis, the patient survived, and 1 year later colonoscopic examination demonstrated no abnormalities . The small intestine is a difficult site to examine, but endoscopic examination of this site is important when massive hemorrhage develops. J Bacteriol, 1998 Jul, 180(14), 3692 - 6 Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73; Kim K et al.; To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants . None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene . When wild-type cells were treated with toluene (1% {vol/vol}) for 5 min, about 2% of the cells could form colonies . In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10(-6)) . In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies . We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined . From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows . Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon . The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases . Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance. Cancer Res, 1998 Jun 15, 58(12), 2583 - 7 Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil, and safety of recombinant methioninase; Yoshioka T et al.; The elevated exogenous-methionine dependency of tumors for growth has been observed in all major cancer cell types . We have previously cloned a methioninase (rMETase) from Pseudomonas putida to deplete methionine . Growth inhibition followed by apoptotic cell death was induced by treatment of tumor cells with rMETase in vitro . A single i.p . injection of 300 units of rMETase can lower the serum methionine level in the mice from 70 microM to less than 1 microM within 2 h and maintain this depleted level for 8 h . Repeated dosing of rMETase of tumor-bearing mice could be administered without acute immune-hypersensitivity . rMETase treatment demonstrated growth inhibitory activity against human tumors in nude mice, including those which were multiple drug-resistant . No body weight loss or hematotoxicity, except a slight anemia, was found throughout the therapy . The combined treatment of the Lewis lung carcinoma with a fixed rMETase dose and increasing doses of 5-fluorouracil (5-FU) resulted in a dose-dependent enhanced antitumor efficacy for survival as well as tumor growth inhibition . Thus, methionine depletion by rMETase potentiates the antitumor efficacy of 5-FU . The data presented in this report thus indicate that rMETase is active alone, is synergistic in combination with 5-FU, and has negligible toxicity suggesting a novel clinical approach for effective cancer therapy. Appl Environ Microbiol, 1998 Jul, 64(7), 2730 - 5 A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethybenzene, and xylene (BTEX) sensing; Applegate BM et al.; A tod-luxCDABE fusion was constructed and introduced into the chromosome of Pseudomonas putida F1, yielding the strain TVA8 . This strain was used to examine the induction of the tod operon when exposed to benzene, toluene, ethylbenzene, and xylene (BTEX) compounds and aqueous solutions of JP-4 jet fuel constituents . Since this system contained the complete lux cassette (luxCDABE), bacterial bioluminescence in response to putative chemical inducers of the tod operon was measured on-line in whole cells without added aldehyde substrate . There was an increasing response to toluene concentrations from 30 micrograms/liter to 50 mg/liter, which began to saturate at higher concentrations . The detection limit was 30 micrograms/liter . There was a significant light response to benzene, m- and p-xylenes, phenol, and water-soluble JP-4 jet fuel components, but there was no bioluminescence response upon exposure to o-xylene . The transposon insertion was stable and had no negative effect on cell growth. Appl Environ Microbiol, 1998 Jul 1, 64(7), 2670 - 5 In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities; Dahlberg C et al.; Gene transfer of the conjugative plasmid pBF1 from Pseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C . Dahlberg et al., Mol . Biol . Evol . 15:385-390, 1998) . pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacIq) . The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp . Transfer to recipient strains lacking the repressor results in expression of gfp . The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level . By using this method, transfer of pBF1::gfp and expression of the gfp gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater . Second, we measured transfer of pBF1 from P . putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria . Plasmid transfer was detected on surfaces and in bulk seawater . Seawater bacteria with different morphologies were shown to receive the plasmid . Gene transfer frequencies of 2.3 x 10(-6) to 2.2 x 10(-4) transconjugants per recipient were recorded after 3 days of incubation. Appl Environ Microbiol, 1998 Jul, 64(7), 2560 - 5 Spatial and temporal variation of phenanthrene-degrading bacteria in intertidal sediments; Berardesco G et al.; Phenanthrene-degrading bacteria were isolated from a 1-m2 intertidal sediment site in Boston Harbor . Samples were taken six times over 2 years . A total of 432 bacteria were isolated and characterized by biochemical testing . When clustered on the basis of phenotypic characteristics, the isolates could be separated into 68 groups at a similarity level of approximately 70% . Several groups (a total of 200 isolates) corresponded to well-characterized species belonging the genera Vibrio and Pseudomonas . Only 51 of the 437 isolates (< 11.7% of the total) hybridized to a DNA probe that encodes the upper pathway of naphthalene and phenanthrene degradation in Pseudomonas putida NCIB 9816 . A cluster analysis indicated that the species composition of the phenanthrene-degrading community changed significantly from sampling date to sampling date . At one sampling time, 12 6-mm-diameter core subsamples were taken within the 1-m2 site to determine the spatial variability of the degrading communities . An analysis of molecular variance, performed with the phenotypic characteristics, indicated that only 6% of the variation occurred among the 12 subsamples, suggesting that the subsamples were almost identical in composition . We concluded that the communities of phenanthrene-degrading bacteria in the sediments are very diverse, that the community structure undergoes significant change with time but does not vary significantly on a spatial scale of centimeters, and that the predominant genes that encode phenanthrene degradation in the communities are not well-characterized. Appl Environ Microbiol, 1998 Jul 1, 64(7), 2463 - 72 Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil; Kuske CR et al.; Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil . A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use . Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization . Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community . Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B . subtilis subsp . niger) endospores or Fusarium moniliforme conidia . The hot-detergent-bead mill procedure was simplified and miniaturized . By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B . globigii spores . In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture) . The detection limit of P . putida cells and B . globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR. Appl Environ Microbiol, 1998 Jul, 64(7), 2454 - 62 Construction and use of an ipb DNA module to generate Pseudomonas strains with constitutive trichloroethene and isopropylbenzene oxidation activity; Berendes F et al.; Pseudomonas sp . strain JR1 exhibits trichloroethene (TCE) oxidation activity with isopropylbenzene (IPB) as the inducer substrate . We previously reported the genes encoding the first three enzymes of the IPB-degradative pathway (ipbA1, ipbA2, ipbA3, ipbA4, ipbB, and ipbC) and identified the initial IPB dioxygenase (IpbA1 A2A3A4) as responsible for TCE cooxidation (U . Pflugmacher, B . Averhoff, and G . Gottschalk, Appl . Environ . Microbiol . 62:3967-3977, 1996) . Primer extension analyses revealed multiple transcriptional start points located upstream of the translational initiation codon of ipbA1 . The transcription from these start sites was found to be IPB dependent . Thirty-one base pairs upstream of the first transcriptional start point tandemly repeated DNA sequences overlapping the -35 region of a putative sigma 70 promoter were found . These repeats exhibit significant sequence similarity to the operator-promoter region of the xyl meta operon in Pseudomonas putida, which is required for the binding of XylS, a regulatory protein of the XylS (also called AraC) family . These similarities suggest that the transcription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family . The construction of an ipb DNA module devoid of this ipb operator-promoter region and the stable insertion of this DNA module into the genomes of different Pseudomonas strains resulted in pseudomonads with constitutive IPB and TCE oxidation activities . Constitutive TCE oxidation of two such Pseudomonas hybrid strains, JR1A::ipb and CBS-3::ipb, was found to be stable for more than 120 generations in antibiotic-free medium . Evaluation of constitutive TCE degradation rates revealed that continuous cultivation of strain JR1A::ipb resulted in a significant increase in rates of TCE degradation. J Ind Microbiol Biotechnol, 1998 Feb, 20(2), 90 - 4 Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments; Khan AA et al.; A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction . A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism . For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample . Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100 degrees C . After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min . Supernatant containing nucleic acid was purified with a PCR purification kit . The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences . The detection limit was 10(2) cells per gram of soil . This method is rapid and obviates the need for lengthy DNA purification from soil samples. J Bacteriol, 1998 Jul, 180(13), 3421 - 31 Cloning, sequencing, and phenotypic characterization of the rpoS gene from Pseudomonas putida KT2440; Ramos-Gonzalez MI et al.; A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strain E . coli ZK918 . The rpoS gene of P . putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E . coli and stimulated expression of the RpoS-controlled promoter, bolAp1 . The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria . Like in other gram-negative bacteria, a homolog of the nlpD gene was found upstream to the rpoS gene . A transcriptional fusion of the promoter of the P . putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele of rpoS for gene replacement of the wild-type copy in the chromosome of P . putida . The resultant rpoS mutant of P.putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol . Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P . putida C1R1 . The RpoS protein of P . putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation. J Bacteriol, 1998 Jul, 180(13), 3323 - 9 Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E; Ramos JL et al.; The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes . The solvent-tolerant P . putida DOT-T1E grows in the presence of high concentrations (e.g., 1% {vol/vol}) of toluene and octanol . Growth of P . putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P . putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon . One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-'phoA mutagenesis of wild-type P . putida DOT-T1E cells . The mutant, called P . putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number . Solvent exclusion was tested with 1,2,4-{14C}trichlorobenzene . The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different . In contrast, the mutant was unable to remove 1,2,4-{14C}trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase . The amount of 14C-labeled substrate in whole cells increased in competition assays in which toluene-and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor . This finding suggests that the exclusion system works specifically with certain aromatic substrates . The mutation in P . putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type. Infect Immun, 1998 Jul, 66(7), 3050 - 8 Inhibition of human peripheral blood mononuclear cell proliferation by Streptococcus pyogenes cell extract is associated with arginine deiminase activity; Degnan BA et al.; Streptococcus pyogenes (group A Streptococcus) cell extracts (CE) have a remarkably powerful and dose-dependent inhibitory effect on antigen, superantigen, or mitogen-stimulated human peripheral blood mononuclear cell (PBMC) proliferation in vitro . Purification of the inhibitory component present in S . pyogenes type M5 (Manfredo strain) CE by anion-exchange chromatography followed by gel filtration chromatography showed that the inhibitor had an approximate native molecular mass of 100 kDa . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitory fractions followed by silver staining gave a single band with an approximate molecular mass of 47 kDa, indicating that the inhibitor is composed of two identical subunits . NH2-terminal sequencing of the protein revealed that it was identical to the previously characterized streptococcal acid glycoprotein (SAGP); this protein possesses between 31.5 and 39.0% amino acid identity with arginine deiminase (AD) from Mycoplasma hominis, Mycoplasma arginini, Pseudomonas putida, and Pseudomonas aeruginosa . AD enzyme activity was present in unfractionated CE prepared from a range of streptococcal strains, and partially purified inhibitory fractions of Manfredo CE also had high levels of activity . The inhibitory effect of Manfredo CE was overcome by the addition of L-arginine to proliferation assays in which human PBMC were stimulated with phytohemagglutinin . We conclude that SAGP, or its homolog, possesses AD activity and that the potent inhibition of proliferation of human T cells by streptococcal CE is due to activity of this enzyme. Biotechnology (N Y), 1995 Jul, 13(7), 674 - 6 Biological production of semisynthetic opiates using genetically engineered bacteria; French CE et al.; Semisynthetic derivatives of morphine and related alkaloids are in widespread clinical use . Due to the complexity of these molecules, however, chemical transformations are difficult to achieve in high yields . We recently identified the powerful analgesic hydromorphone as an intermediate in the metabolism of morphine by Pseudomonas putida M10 . Here we describe the construction of recombinant strains of Escherichia coli that express morphine dehydrogenase and morphinone reductase . These strains are capable of efficiently transforming the naturally occurring alkaloids morphine and codeine to hydromorphone and the antitussive hydrocodone, respectively . Our results demonstrate the potential for recombinant DNA technology to provide biological routes for the synthesis of known and novel semisynthetic opiate drugs. Lett Appl Microbiol, 1998 Apr, 26(4), 265 - 9 Segregational and structural instability of recombinant plasmid carrying genes for naphthalene degrading pathway; Samanta SK et al.; The stability of recombinant plasmid carrying genes for naphthalene mineralization was determined . A strain of Pseudomonas putida capable of mineralizing naphthalene (Nap+) via salicylate (Sal+) was isolated, and all regulatory and structural genes for the whole pathway were found to be encoded on a 25 kb EcoRI fragment of an approximately 83 kb plasmid present in this strain . The 25 kb EcoRI fragment was cloned into a tetracycline-resistant (TcR) cloning vector pLAFR3 and the recombinant plasmid, pRKJ3 (Nap+, Sal+, TcR), thus obtained was transferred into the plasmid-free strain Pseudomonas putida KT2442 in order to test the stability of the plasmid . Plasmid pRKJ3 was found to be segregationally and/or structurally unstable, depending on the growth conditions . Two types of novel derivative strains having the phenotypes Nap-, Sal+, TcR and Nap-, Sal-, TcR with specific deletions of approximately 2 kb and 18 kb, respectively, were obtained. Biochemistry, 1998 Jun 9, 37(23), 8325 - 30 Crystal structure and enzyme mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni; Cho HS et al.; Bacterial Delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been intensively studied as a prototype for understanding an enzyme-catalyzed allylic rearrangement involving intramolecular proton transfer . Asp38 serves as a general base to abstract the proton from the steroid C4-H, which is a much stronger base than the carboxyl group of this residue . This unfavorable proton transfer requires 11 kcal/mol of energy which has to be provided by favorable interactions between catalytic residues and substrate in the course of the catalytic reaction . How this energy is provided at the active site of KSI has been a controversial issue, and inevitably the enzyme mechanism is not settled . To resolve these issues, we have determined the crystal structure of this enzyme at 2.3 A resolution . The crystal structure revealed that the active site environment of P . testosteroni KSI is nearly identical to that of Pseudomonas putida KSI, whose structure in complex with a reaction intermediate analogue we have determined recently . Comparison of the two structures clearly indicates that the two KSIs should share the same enzyme mechanism involving the stabilization of the dienolate intermediate by the two direct hydrogen bonds to the dienolate oxyanion, one from Tyr14 OH and the other from Asp99 COOH . Mutational analysis of the two residues and other biochemical data strongly suggest that the hydrogen bond of Tyr14 provides the more significant contribution than that of Asp99 to the requisite 11 kcal/mol of energy for the catalytic power of KSI. Biosci Biotechnol Biochem, 1998 Apr, 62(4), 747 - 52 Purification, characterization, and gene analysis of catechol 2,3-dioxygenase from the aniline-assimilating bacterium Pseudomonas species AW-2; Murakami S et al.; Catechol 2,3-dioxygenase (C23D; EC 1.13.1.2) was purified to homogeneity from a cell extract of Pseudomonas sp . AW-2 grown on aniline, and the purified C23D was characterized . The molecular mass estimated by gel filtration was 110 kDa . The enzyme dissociated into four identical subunits each with the molecular mass of 33 kDa . The enzyme had high activity for 3-methylcatechol as well as catechol, and differed from the enzyme from Pseudomonas putida mt-2, which carries the TOL plasmid, in optimal pH for catechol, extradiol cleavage activities for 3-methylcatechol and 4-methylcatechol, and immunochemical properties . The amino acid sequence deduced from a C23D gene, alnE, from Pseudomonas sp . AW-2 was 85.7% identical to that of 3-methylcatechol 2,3-dioxygenase from toluidine-assimilating Pseudomonas putida UCC22 . AlnE was 44.1% identical to the C23D encoded by xylE in P . putida mt-2 . Because XylE has low activity for 3-methylcatechol, these results suggest that the differences in substrate specificity for 3-methylcatechol among the C23Ds reflected their sequence similarity. J Bacteriol, 1998 Jun, 180(11), 2889 - 94 Activation and repression of transcription at the double tandem divergent promoters for the xylR and xylS genes of the TOL plasmid of Pseudomonas putida; Marques S et al.; The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism . Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive sigma70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive sigma70-dependent promoter, and by the regulatable sigma54 class Ps1 promoter . In Ps1 the XylR targets (upstream activator sequences {UASs}) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions -2 to -30 (-2/-30 region) and the -137/-156 region, the latter overlapping the Pr promoters . When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters . In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active . In this background and in the presence of an effector, XylR increases autorepression . In a sigma54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector . However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable . This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible . In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing sigma54 modulates expression from the downstream Ps2 sigma70-dependent promoter . In a P . putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed . This finding suggests that IHF acts in vivo as a repressor of the sigma54-dependent Ps1 promoter . In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible . Thus, binding of RNA polymerase containing sigma54 at the upstream promoter may modulate expression from the Ps2 promoter. J Bacteriol, 1998 Jun, 180(11), 2822 - 9 Expression of the transposase gene tnpA of Tn4652 is positively affected by integration host factor; Horak R et al.; Tn4652 is a derivative of the toluene degradation transposon Tn4651 that belongs to the Tn3 family of transposons (M . Tsuda and T . Iino, Mol . Gen . Genet . 210:270-276, 1987) . We have sequenced the transposase gene tnpA of transposon Tn4652 and mapped its promoter to the right end of the element . The deduced amino acid sequence of tnpA revealed 96.2% identity with the putative transposase of Tn5041 . Homology with other Tn3 family transposases was only moderate (about 20 to 24% identity), suggesting that Tn4652 and Tn5041 are distantly related members of the Tn3 family . Functional analysis of the tnpA promoter revealed that it is active in Pseudomonas putida but silent in Escherichia coli, indicating that some P . putida-specific factor is required for the transcription from this promoter . Additionally, tnpA promoter activity was shown to be modulated by integration host factor (IHF) . The presence of an IHF-binding site upstream of the tnpA promoter enhanced the promoter activity . The positive role of IHF was also confirmed by the finding that the enhancing effect of IHF was not detected in the P . putida ihfA-deficient strain A8759 . Moreover, the Tn4652 terminal sequences had a negative effect on transcription from the tnpA promoter in the ihfA-defective strain . This finding suggests that IHF not only enhances transcription from the tnpA promoter but also alleviates the negative effect of terminal sequences of Tn4652 on the promoter activity . Also, an in vitro binding assay demonstrated that both ends of Tn4652 bind IHF from a cell lysate of E . coli. Microbiology, 1998 May, 144 ( Pt 5), 1387 - 96 Implications of the xylQ gene of TOL plasmid pWW102 for the evolution of aromatic catabolic pathways; Aemprapa S et al.; Pseudomonas putida strain O2C2 is able to grow on toluene, m-xylene and p-xylene through benzoate and the corresponding methylbenzoates (toluates) . The catabolic genes are encoded on a large TOL plasmid, pWW102, of > 220 kb . The complete catabolic genes were cloned on four large overlapping restriction fragments covering a total of 28 kb of the plasmid, which was carefully mapped by restriction enzyme analysis . The presence of the xyl genes on the cloned DNA was confirmed by assay of representative enzymes of both operons . Virtually all the genes were located on the cloned DNA by hybridization of Southern blots with gene-specific probes from related pathways of other catabolic plasmids . Within the limitations of available restriction sites, the analysis showed that the genes are in two blocks . The major block carries the meta pathway operon xylXYZLTEGFJQKIH with the two regulatory genes xylSR immediately downstream . The upper pathway operon xylUWCMAB(N) is about 2-3 kb downstream of the regulatory genes and transcribed in the same direction as the meta pathway operon . Within each operon the gene order appears to be identical to that found in other TOL plasmids, but the relative location of the operons most closely resembles that found on plasmid pWW53, although there is no evidence of any xyl duplications on pWW102 . The nucleotide sequence of the xylQ gene for the acetaldehyde dehydrogenase (acylating; ADA), together with the 3'-end of the upstream xylJ (for 2-oxopent-4-enoate hydratase) and the 5'-end of the downstream xylK (for 4-hydroxy-2-oxovalerate aldolase), was determined . The xylQ gene was ligated into expression vector pTrc99a and high levels of XylQ protein were detected by enzyme assay and by SDS-PAGE . All three genes xylJQK showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas meta pathways, the highest being with the naphthalene catabolic genes nahLOM from the plasmid of Pseudomonas sp . NCIB 9816 . The implications of the sequence homologies to the evolution of these pathways are discussed. Biochemistry, 1998 May 19, 37(20), 7598 - 607 Reductive and oxidative half-reactions of morphinone reductase from Pseudomonas putida M10: a kinetic and thermodynamic analysis; Craig DH et al.; The reaction of morphinone reductase (MR) with the physiological reductant NADH and the oxidizing substrate codeinone has been studied by multiple and single wavelength stopped-flow spectroscopy . Reduction of the enzyme with NADH proceeds in two kinetically resolvable steps . In the first step, the oxidized enzyme forms a charge-transfer intermediate with NADH . The charge-transfer complex is characterized by an increase in absorbance at long wavelength (540 to 650 nm), and its rate of formation is dependent on substrate concentration and is controlled by a second-order rate constant of 4 . 8 x 10(5) M-1 s-1 at pH 7.0 and 5 degrees C . In the second step, the enzyme-bound flavin is reduced to the dihydroflavin form . The rate of flavin reduction (23.4 s-1 at pH 7.0 and 5 degrees C) is independent of substrate concentration and is observed as a monophasic decrease in absorbance at 462 nm . The oxidative half-reaction proceeds in three kinetically resolvable steps . The first is due to the formation of a reduced enzyme-codeinone charge-transfer complex and is observed at long wavelength (about 650 nm) . The rate of charge-transfer complex formation is dependent on codeinone concentration and is controlled by a second-order rate constant of 11.5 x 10(3) M-1 s-1 at pH 7.0 and 5 degrees C . The second step represents flavin reoxidation and is observed at 462 (absorption increase) and 650 nm (absorption decrease) and progresses with a rate (about 45 s-1) which is independent of codeinone concentration . The third step is observed as a further small increase in absorbance at 462 nm and proceeds with a rate of about 2.5 s-1 . This step most likely represents hydrocodone release from the oxidized enzyme . Analysis of the temperature dependence of the reductive half-reaction has enabled calculation of the entropic and enthalpic contributions for charge-transfer formation, charge-transfer decay (yielding free enzyme and substrate), and electron transfer to the enzyme-bound FMN, and the construction of a partial energy profile for the reaction catalyzed by MR . The reaction scheme and redox properties of MR are compared with those described previously for the closely related flavoprotein, old yellow enzyme . Although common features are identified, there are notable differences in the kinetic and redox properties of the two enzymes. Appl Environ Microbiol, 1998 Jun, 64(6), 2072 - 8 Construction of an efficient biologically contained pseudomonas putida strain and its survival in outdoor assays Molina L, Ramos C, Ronchel MC, Molin S, Ramos JL. Active biological containment systems consist of two components, a killing element designed to induce cell death and a control element which modulates the expression of the killing function . We constructed a mini-Tn5 transposon bearing a fusion of the Plac promoter to the gef killing gene and a fusion of the Pm promoter to the lacI gene plus the positive regulator of the Pm promoter, the xylS gene . This mini-Tn5 transposon was transferred to the chromosome of Pseudomonas putida CMC4, and in culture this strain survived in the presence of 3-methylbenzoate (an XylS effector) and committed suicide in the absence of this aromatic compound . The rate of killing escape was on the order of 10(-8) per cell and per generation . This contained strain and an uncontained control strain were used in outdoor tests performed in the spring-summer and autumn-winter periods to determine their survival in planted and unplanted soils with and without 3-methylbenzoate . In unplanted soils the numbers of both the contained strain and the uncontained strain per gram of soil tended to decrease, but the numbers of the contained strain decreased faster in soils without 3-methylbenzoate . The decrease in the number of CFU per gram of soil was faster in the spring-summer period than in the autumn-winter period . In planted soils survival in the rhizosphere and survival in bulk soil were studied . In the rhizosphere the uncontained control strain tended to become established at levels on the order of 10(5) to 10(6) CFU/g of soil regardless of the presence of 3-methylbenzoate . In the bulk soil the numbers of bacterial cells were 2 to 3 orders of magnitude lower . In planted soils the contained strain tended to disappear, but this tendency was more pronounced in the absence of 3-methylbenzoate and occurred faster in the summer assay than in the winter assay . We found no evidence of dispersal of the test strains outside the experimental plots. Appl Environ Microbiol, 1998 Jun, 64(6), 2240 - 6 New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria; Andersen JB et al.; Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition . To expand the use of Gfp as a reporter protein, new variants have been constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp . This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli and Pseudomonas putida . The new Gfp variants should be useful for in situ studies of temporal gene expression. Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6419 - 24 Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: the phenylacetyl-CoA catabolon; Olivera ER et al.; Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U . This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a beta-oxidation-like system, and (vi) two regulatory genes . This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA). J Med Chem, 1998 May 7, 41(10), 1671 - 8 Modular fluorescent-labeled siderophore analogues; Nudelman R et al.; Biomimetic analogues 1 of the microbial siderophore (iron carrier) ferrichrome were labeled via piperazine with various fluorescent markers at a site not interfering with iron binding or receptor recognition (compounds 10-12) . These iron carriers were built from a tetrahedral carbon symmetrically extended with three strands, each containing an amino acid (G = glycyl, A = alanyl, L = leucyl and P = phenylalanyl) and terminated by a hydroxamic acid, which together define an octahedral iron-binding domain . A fourth exogenous strand provided the site for connecting various fluorescent markers via a short bifunctional linker . Iron(III) titrations, along with fluorescence spectroscopy, generated quenching of fluorescence emission of some of the probes used . The quenching process fits the Perrin model which reinforces the intramolecular quenching process, postulated previously.1 All tested compounds, regardless of their probe size, polarity, or the linker binding them to the siderophore analogue, promote growth of Pseudomonas putida with the same efficacy as the nonlabeled analogues 1, with the added benefit of signaling microbial activity by fluorescence emission . All G derivatives of compounds 10-12 were found to parallel the behavior of natural ferrichrome, whereas A derivatives mediated only a modest iron(III) uptake by P . putida . Incubation of various Pseudomonas strains with iron(III)-loaded G derivatives resulted in the build-up of the labels' fluorescence in the culture medium to a much larger extent than from the corresponding A derivatives . The fluorescence buildup corresponds to iron utilization by the cells and the release of the fluorescent labeled desferrisiderophore from the cell to the media . The fact that the microbial activity of these compounds is not altered by attachment of various fluorescent markers via a bifunctional linker proposes their application as diagnostic tools for detecting and identifying pathogenic microorganisms. J Biol Chem, 1998 Apr 17, 273(16), 9622 - 9 A novel -2Fe-2S- ferredoxin from Pseudomonas putida mt2 promotes the reductive reactivation of catechol 2,3-dioxygenase; Hugo N et al.; Catechol 2,3-dioxygenase (XylE) is a component of the TOL plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring . Purified XylE is oxygen-sensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, XylT, encoded by the xylT gene located just upstream of xylE . In this study, we have purified to homogeneity the XylT product from a recombinant Escherichia coli strain containing a hyperexpressible xylT gene and characterized it as a novel {2Fe-2S} ferredoxin . It is the first example of a soluble ferredoxin with a net positive charge at neutral pH . The EPR signal of the iron sulfur cluster has rhombic symmetry as is the case for plant-type ferredoxins, but the XylT absorbance spectrum resembles more closely that of adrenodoxin . The midpoint redox potential was determined to be -373 +/- 6 mV, at pH 8 . 5 . XylT was unusually unstable for a {2Fe-2S} ferredoxin, with half-lives of 69 min at 25 degrees C in air and 70 min at 37 degrees C in argon . With photochemically reduced 5-deazaflavin for the controlled generation of reductant, it was demonstrated that XylT mediates the rapid reactivation of purified inactive catechol 2,3-dioxygenase in vitro . Inactivation of XylE by 4-methylcatechol resulted in oxidation of the active site iron to a high spin ferric state that was detectable by EPR . Spectroscopic evidence presented here demonstrates that XylT reactivates XylE through reduction of the iron atom in the active site of the enzyme . It is the first instance of a ferredoxin-mediated reactivation of an enzyme . The level of expression of XylT in Pseudomonas putida mt2 cells is low and the calculated XylT/XylE molar ratio is consistent with the proposal that XylE reactivation involves catalytic nonstoichiometric amounts of XylT. Protein Eng, 1997 Dec, 10(12), 1357 - 61 The dimerization of Pseudomonas putida cytochrome P450cam: practical consequences and engineering of a monomeric enzyme; Nickerson DP et al.; Cytochrome P450cam dimerizes via the formation of an intermolecular disulfide bond, complicating the storage and handling of the enzyme, particularly at higher concentrations . The dimeric enzyme is 14% less active than the monomer and forms at a slow but significant rate even at 4 degrees C {k = 1.09 x 10(-3) mM(-1) h(-1)} . To eliminate any ambiguity introduced by dimer formation and to simplify handling and storage of the enzyme, site-directed mutagenesis was used to identify C334 as the single cysteine residue responsible for the formation of the disulfide linkage and to engineer a monomeric enzyme by substituting an alanine in its place . The C334A mutant is identical with the wild-type P450cam monomer in terms of optical spectra, camphor binding and turnover activity, but shows no evidence of dimerization and aggregation even at millimolar concentrations . Preliminary 1H NMR investigations also indicate a significant improvement in the quality of spectra obtained with this mutant . (C334A)P450cam is therefore proposed as an alternative to the wild-type enzyme-a base mutant otherwise identical with the wild-type but with improved handling characteristics. J Antibiot (Tokyo), 1998 Mar, 51(3), 341 - 52 A 7.6kb DNA region from Streptomyces kasugaensis M338-M1 includes some genes responsible for kasugamycin biosynthesis; Ikeno S et al.; A 7.6kb PstI-KpnI DNA fragment including a sequence highly similar to kasugamycin acetyltransferase gene (kac) was isolated from Streptomyces kasugaensis M338-M1 and sequenced . Nine open reading frames (ORFs), designated as ORF A, B, C, D, E, F, G, H and I, were recognized in this region, although ORF A was incomplete . ORF G runs in the opposite direction to the others . The amino acid sequence deduced from ORF H showed 98% similarity to that of the kasugamycin acetyltransferase from S . kasugaensis MB273-C4, another kasugamycin (KSM) producer . Transformation of E . coli JM109 with ORF H made the strain highly resistant to KSM . The deduced amino acid sequences of the ORF A, C and D products were similar, respectively, to glucosyltransferase I from E . coli (26%), beta-alanine: pyruvate transaminase from Pseudomonas putida (32%) and dTDP-D-glucose 4,6-dehydratase (StrE) from Streptomyces griseus (37%) . The strE-like ORF (ORF D) seems to be the gene responsible for formation of the 6-deoxy structure of the kasugamine moiety . ORF A and ORF C are also likely to have roles in KSM biosynthesis . Taken together, our analyses strongly suggest that this DNA region includes at least a part of the gene cluster of KSM biosynthesis. J Formos Med Assoc, 1998 Apr, 97(4), 286 - 8 Hypothermia predisposing to Pseudomonas putida sepsis in a child with panhypopituitarism; Chiu CH et al.; A 14-year-old boy presented with a 1-week history of hypothermia and obtundation . His medical history included surgical resection of craniopharyngioma with postoperative visual impairment and panhypopituitarism . The patient's rectal temperature remained persistently lower than 35 degrees C during the first 3 days of hospitalization . His blood pressure was 90/56 mmHg on admission . The peripheral blood leukocyte count was 2.7 x 10(10)/L with 18% neutrophils, 19% band forms, 44% metamyelocytes, 3% myelocytes, and 16% lymphocytes . The C-reactive protein concentration was 133.9 mg/L . Two separate blood cultures both yielded Pseudomonas putida . The patient was treated with amikacin and ceftazidime along with aggressive fluid therapy . Replacement therapy directed at his hormonal deficiencies was initiated as soon as his hemodynamic status was stabilized . The patient responded well to therapy with a gradual rise in body temperature and improvement in general activity . A growth experiment carried out on the P . putida isolate showed that the bacteria grew more rapidly at 30 degrees C than at 37 degrees C . The clinical course of the patient, as well as the results of the laboratory study, suggest that hypothermia may predispose human infection with P . putida. Microbiology, 1998 Apr, 144 ( Pt 4), 965 - 73 Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358; Venturi V et al.; Transposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferulic and coumaric acids as sole sources of carbon and energy . Genetic studies confirmed previous biochemical data showing that ferulic acid is degraded via vanillic acid, and coumaric acid via hydroxybenzoic acid . The genes involved in these enzymic steps were cloned and characterized . Two proteins designated Fca (26.5 kDa) and Vdh (50.3 kDa) were identified as responsible for the conversion of ferulic acid to vanillic acid; the proteins are encoded by the fca and vdh genes which are organized in an operon structure in the chromosome . The Vdh protein is 69% identical at the amino acid level to the Vdh protein recently identified in Pseudomonas sp . strain HR199 and converts vanillin to vanillic acid . Homology studies revealed that the Vdh proteins exhibited significant identity to aldehyde dehydrogenases from different organisms whereas Fca belonged to the enoyl-CoA hydratase family of proteins . Two proteins, designated VanA (39.9 kDa) and VanB (34.3 kDa), encoded by two genes, vanA and vanB, are organized in an operon in the chromosome . They were found to be responsible for the demethylation of vanillic acid to protocatechuic acid . The VanA proteins showed no homology to any other known protein, while VanB belonged to the ferredoxin family of proteins . This two-component enzyme system demethylated another phenolic monomer, veratric acid, thus indicating broad specificity . Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid. J Bacteriol, 1998 May, 180(9), 2367 - 72 Transcriptional repression mediated by LysR-type regulator CatR bound at multiple binding sites; Chugani SA et al.; The catBCA operon of Pseudomonas putida encodes enzymes involved in the catabolism of benzoate . Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate . Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of the inducer . We report the presence of an additional binding site for CatR downstream of the catBCA promoter within the catB structural gene . This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame . Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity . CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites . Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site . We measured beta-galactosidase activity of catB-lacZ transcriptional fusions in vivo . Our results suggest a probable cis-acting repressor function for the internal binding site . Site-directed mutagenesis of the IBS verified this finding . The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA. Appl Environ Microbiol, 1998 May, 64(5), 1902 - 9 Effect of bacterial distribution and activity on conjugal gene transfer on the phylloplane of the bush bean (Phaseolus vulgaris); Normander B et al.; Conjugal plasmid transfer was examined on the phylloplane of bean (Phaseolus vulgaris) and related to the spatial distribution pattern and metabolic activity of the bacteria . The donor (Pseudomonas putida KT2442) harbored a derivativ