Mol Microbiol, 1996 Jan, 19(1), 7 - 17 Involvement of sigma 54 in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter; Cases I et al.; The sigma 54-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida becomes activated by the prokaryotic enhancer-binding XyIR protein when cells encounter m-xylene in the medium . However, even in the presence of the aromatic inducer, Pu activity is silenced in vivo during rapid exponential growth of the cells in rich medium . Various elements known to be involved in the control of the transcriptional activity of the promoter were examined to ascertain the mechanism by which expression of Pu is limited during the exponential phase of growth . A truncated and fully constitutive XyIR derivative deleted of its signal-reception N-terminal domain was found to be subjected to the same exponential silencing as the wild-type XyIR when exposed to m-xylene . This indicated that the phenomenon is not due to a late activation of XyIR by the aromatic effector . A Pu variant in which the integration host factor (IHF)-binding site had been functionally replaced by a statically curved DNA segment showed the same induction pattern, thus ruling out variations in the intracellular levels of IHF changes during growth as the element responsible for the inactivity of Pu in rapidly growing cells . On the contrary, overproduction of the sigma 54 factor allowed Pu expression during exponential phase . As sigma 54 protein levels remained approximately constant during growth, the exponential silencing of Pu could be caused ultimately by changes in the activity of the factor itself . This effect may not be exclusive to Pu, but could be a general co-regulation mechanism in sigma 54-dependent promoters that connects transcription of a specific set of genes with the general physiological status of the cells. Gene, 1996, 173(1 Spec No), 59 - 65 Bacterial plasmid conjugation on semi-solid surfaces monitored with the green fluorescent protein (GFP) from Aequorea victoria as a marker; Christensen BB et al.; Horizontal transfer of the TOL plasmid was examined in Pseudomonas putida (Pp) KT2442 micro-colonies on semi-solid agar surfaces . Horizontal gene transfer is usually studied in large populations where all information is based on average estimates of the transfer events in the entire population . We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a plasmid marker, in combination with single-cell observations . This provided hitherto unknown details on the distribution of cells active in conjugation . In the present study, donor cells containing the gfp gene expressed from the bacteriophage T7 phi 10 promoter on the TOL plasmid, and recipient cells expressing the corresponding phage RNA polymerase allowed us to monitor the occurrence of ex-conjugants as green fluorescent cells upon illumination with blue light (470-490 nm) . Further, the recipients were labeled with the luxAB genes to distinguish micro-colonies of donor cells from recipient cells . We conclude that conjugal plasmid transfer in Pp KT2442 cells on semi-solid surfaces occurs mainly during a short period of time after the initial contact of donors and recipients, indicating that spread of the TOL plasmid is limited in static, but viable cultures. Gene, 1996, 173(1 Spec No), 53 - 8 A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments; Burlage RS et al.; The movement of bacteria through groundwater is a poorly understood process . Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow . The identification of transported bacteria is often a limiting factor in both laboratory and field transport experiments . Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harboring the pGFP plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1 . The Tn5GFP1 transposon incorporates the gene (gfp) encoding green fluorescent protein (GFP) and can be used to mutagenize Gram-bacteria . Fluorescent colonies were suspended in phosphate-buffered saline (PBS) at a concentration of approx . 10(9) bacteria/ml . A 10-cm glass column packed with quartz sand (diameter range 177-250 microns) was equilibrated with PBS prior to the forced flow introduction of the bacteria . Collected fractions were analyzed and the bacteria quantitated using a fluorescence spectrometer . Results demonstrate that the bacteria can be accurately tracked using their fluorescence, and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria . The data show a rapid breakthrough of the bacteria followed by a characteristic curve pattern . A lower limit of detection of 10(5) cells was estimated based on these experiments . The Tn5GFP1 transposon should become a valuable tool for labeling bacteria. Appl Biochem Biotechnol, 1996 Spring, 57-58, 803 - 15 Biodegradation of mixed wastes in continuously operated cyclic reactors; Wang KW et al.; The problem of simultaneous biodegradation of two dissimilar substrates in a continuously operated cyclic reactor was studied both at the theoretical and experimental levels using a simple model system . The system involved media containing mixtures of glucose and phenol as carbon sources . A pure culture of Pseudomonas putida (ATCC 17514) was employed . Independent kinetic experiments have revealed that glucose and phenol are involved in a crossinhibitory uncompetitive kinetic interaction . The dynamics of a cyclically operated reactor were analyzed using the principles of bifurcation theory for forced systems . Experimental results have confirmed the theoretical predictions . Implications of the results for the design of waste-treating facilities are discussed. Appl Environ Microbiol, 1996 Jan, 62(1), 230 - 6 Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39; Goyal AK et al.; Three strains of Comamonas testosteroni were isolated from river sediment for the ability to degrade phenanthrene; two of the strains also grew on naphthalene, and one strain also grew on anthracene . The homology of the genes for polycyclic aromatic hydrocarbon degradation in these strains to the classical genes (nah) for naphthalene degradation from Pseudomonas putida NCIB 9816-4 was determined . The three C . testosteroni strains showed no homology to the nah gene probe even under low-stringency conditions . The genes for naphthalene and phenanthrene degradation were cloned from one of the three C . testosteroni strains . Two cosmid clones expressing polycyclic aromatic hydrocarbon dioxygenase activity were identified from a library prepared with genomic DNA from C . testosteroni GZ39 . The genes coding for the first two enzymes in the catabolic pathway, phenanthrene dioxygenase and cis-phenanthrene dihydrodiol dehydrogenase, were localized to a 5.4-kb NcoI-PstI fragment by subcloning and gene expression experiments . Further subcloning and analysis revealed a novel organization of the genes, with the gene for cis-phenanthrene dihydrodiol dehydrogenase located between the genes for the individual phenanthrene dioxygenase components . A Southern blot with the cloned genes from C . testosteroni GZ39 confirmed that these genes are distinct from those found in P . putida NCIB 9816-4 . Southern blots also demonstrated that C . testosteroni GZ38A possesses genes for phenanthrene degradation that are similar to those cloned from C . testosteroni GZ39 . However, C . testosteroni GZ42 possesses genes for phenanthrene degradation that are not homologous to those cloned from C . testosteroni GZ39 . This suggests that there are at least two different sets of genes for the degradation of phenanthrene among the three C . testosteroni strains. Appl Environ Microbiol, 1996 Jan, 62(1), 214 - 20 Nondisruptive detection of activity of catabolic promoters of Pseudomonas putida with an antigenic surface reporter system; Cebolla A et al.; A simple procedure to detect the switching on and off of catabolic promoters of Pseudomonas putida, at the level of single cells based on the immunodetection of a reporter epitope expressed on the surface of bacterial cells, has been developed . To do this, the antigenic sequence Asp-Leu-Pro-Pro-Asn-Ser-Asp-Val-Val-Asp, from a coronavirus, was inserted genetically in the permissive site around amino acid position 153 of the LamB protein (maltose and lambda phage receptor) of Escherichia coli . When the hybrid lamB gene is transcribed, the epitope becomes presented on the surface of the bacterial cells in a configuration available to specific antibodies . To validate this notion in nonenteric bacteria, the expression and correct processing of LamB were confirmed by coupling the lamB gene to the salicylate-responsive Psa1 promoter of the NAH7 (naphthalene degradation) plasmid in Pseudomonas putida . Subsequently, a hybrid lamB gene carrying the sequence of the coronavirus antigen was placed downstream of the m-toluate-responsive Pm promoter of the TOL (toluene degradation) plasmid . Exposure of the epitope on the Pseudomonas cell surface was monitored through fluorescence of whole cells treated with a monoclonal antibody against the heterologous antigen . Fluorescence emission was dependent on the presence of m-toluate in the medium, thus permitting detection of the Pm promoter switching on by simple optical inspection of individual cells, even in situations when these are a very minor component of a complex bacterial community. J Bacteriol, 1996 Jan, 178(1), 111 - 20 Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster; Prieto MA et al.; We have determined and analyzed the nucleic acid sequence of a 14,855-bp region that contains the complete gene cluster encoding the 4-hydroxyphenylacetic acid (4-HPA) degradative pathway of Escherichia coli W (ATCC 11105) . This catabolic pathway is composed by 11 genes, i.e., 8 enzyme-encoding genes distributed in two putative operons, hpaBC (4-HPA hydroxylase operon) and hpaGEDFHI (meta-cleavage operon); 2 regulatory genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein related to the superfamily of transmembrane facilitators and appears to be cotranscribed with hpaA . Although comparisons with other aromatic catabolic pathways revealed interesting similarities, some of the genes did not present any similarity to their corresponding counterparts in other pathways, suggesting different evolutionary origins . The cluster is flanked by two genes homologous to the estA (carbon starvation protein) and tsr (serine chemoreceptor) genes of E . coli K-12 . A detailed genetic analysis of this region has provided a singular example of how E . coli becomes adapted to novel nutritional sources by the recruitment of a catabolic cassette . Furthermore, the presence of the pac gene in the proximity of the 4-HPA cluster suggests that the penicillin G acylase was a recent acquisition to improve the ability of E . coli W to metabolize a wider range of substrates, enhancing its catabolic versatility . Five repetitive extragenic palindromic sequences that might be involved in transcriptional regulation were found within the cluster . The complete 4-HPA cluster was cloned in plasmid and transposon cloning vectors that were used to engineer E . coli K-12 strains able to grow on 4-HPA . We report here also the in vitro design of new biodegradative capabilities through the construction of a transposable cassette containing the wide substrate range 4-HPA hydroxylase, in order to expand the ortho-cleavage pathway of Pseudomonas putida KT2442 and allow the new recombinant strain to use phenol as the only carbon source. Gene, 1995 Dec 29, 167(1-2), 93 - 8 Analysis of the rpoD gene encoding the principal sigma factor of Pseudomonas putida; Fujita M et al.; The gene (rpoD) encoding the principal sigma factor of Pseudomonas putida (Pp) was cloned and sequenced . The amino-acid sequence deduced from the nucleotide sequence of rpoD contained sequences with significant similarity to the conserved region of the principal sigma factors . In vivo transcriptional analyses revealed that the Pp rpoD is transcribed as a monocistronic mRNA of 2.1 kb and that the transcription of rpoD is under control of the heat-shock (HS) response . The transcription start point (tsp) of the gene was determined and found to be different depending on either normal growth (at 30 degrees C) or HS (at 42 degrees C) conditions . The possible promoter sequences for the principal (sigma 70) and the HS RNA polymerase of Pseudomonas were located in the upstream region of the tsp. J Biol Chem, 1995 Dec 29, 270(52), 31202 - 9 The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase; Kim J et al.; When p-cresol methylhydroxylase (PCMH) is expressed in its natural host Pseudomonas putida, or when the genes of the alpha and beta subunits of the enzyme are expressed together in the heterologous host Escherichia coli, flavin-adenine dinucleotide (FAD) is covalently attached to Tyr384 of the alpha subunit and the correct alpha 2 beta 2 form of the enzyme is assembled . The apoflavoprotein has been expressed in E . coli in the absence of the beta cytochrome c subunit and purified . While noncovalent FAD binding to apoflavoprotein in the absence of the cytochrome subunit could not be directly demonstrated, circumstantial evidence suggests that this indeed occurs . Covalent flavinylation requires one molecule each of FAD and cytochrome for each flavoprotein subunit . The flavinylation process leads to the 2-electron-reduced form of covalently bound FAD, and the resulting alpha 2 beta 2 enzyme is identical to wild-type PCMH . This work presents clear evidence that covalent flavinylation occurs by a self-catalytic mechanism; an external enzyme or chaperon is not required, nor is prior chemical activation of FAD or of the protein . This work is the first to define the basic chemistry of covalent flavinylation of an enzyme to produce the normal, active species, and confirms a long standing, postulated chemical mechanism of this process . It also demonstrates, for the first time, the absolute requirement for a partner subunit in the post-translational modification of a protein . It is proposed that the covalent FAD bond to Tyr384 and the phenolic portion of this Tyr are part of the essential electron transfer path from FAD to heme. Biochemistry, 1995 Dec 26, 34(51), 16533 - 42 Coordination of the Rieske-type {2Fe-2S} cluster of the terminal iron-sulfur protein of Pseudomonas putida benzene 1,2-dioxygenase, studied by one- and two-dimensional electron spin-echo envelope modulation spectroscopy; Shergill JK et al.; One- and two-dimensional (1D and 2D) electron spin-echo envelope modulation (ESEEM) spectroscopy has been used to investigate the ligand environment of the {2Fe-2S} cluster from the terminal dioxygenase (ISPBED) of the Pseudomonas putida benzene dioxygenase complex . The modulation frequencies observed in the 0.5-8.5 MHz region of the Fourier transforms of 1D and 2D ESEEM spectra measured across the electron paramagnetic resonance (EPR) absorbance envelope (from gz through to gx) are consistent with their assignment to two 14N nuclei . Using hyperfine sublevel correlation spectroscopy (HYSCORE), two sets of correlated double quantum transitions sharing the same hyperfine coupling were observed and were identified as being due to the same two 14N nuclei . On the basis of the isotropic hyperfine and quadrupolar couplings estimated for these 14N nuclei {N(1), Aiso = 3.6 MHz and e2qQ = 2.2-2.8 MHz; N(2), Aiso = 4.8 MHz and e2qQ = 2.2-2.4 MHz}, the ESEEM pattern of ISPBED is assigned to two histidine nitrogens which are directly coordinated to the reduced iron-sulfur cluster . Bonding parameters of the two {14N}histidine ligands were calculated from these hyperfine couplings . The primary covalent contributions to the hyperfine interaction arise from 14N-to-Fe2+ sigma bonds . For N(1), our analysis of the percentage of unpaired 2s and 2p electrons gave f2s approximately 1.3% and f2p approximately 0.2%, while values of f2s approximately 1.7% and f2p approximately 1.4% were estimated for N(2) . Comparison of these values with those determined from electron nuclear double resonance (ENDOR) data of the Rieske-type {2Fe-2S} center of Pseudomonas cepacia phthalate dioxygenase {Gurbiel, R . J., Batie, C . J., Sivaraja, M., True, A . E., Fee, J . A., Hoffman, B . M., & Ballou, D . P . (1989) Biochemistry 28, 4861-4871} indicates an apparent reduction in unpaired electron spin density residing on the two 14N ligands of ISPBED . Analysis of slices of the HYSCORE spectrum has provided evidence for another 14N nucleus (A approximately 1.1 MHz, e2qQ = 3.3 MHz), which we have attributed to a weakly coupled peptide nitrogen, similar to those observed for ferredoxin-type {2Fe-2S} clusters . This type of weak interaction has not been previously described by the detailed ENDOR and ESEEM studies of Rieske-type centers . The resolution of the spectra demonstrates the effectiveness of 2D ESEEM for the disentanglement of multiple hyperfine interactions to metalloprotein centers. Biochem J, 1995 Dec 15, 312 ( Pt 3), 671 - 8 Bacterial morphinone reductase is related to Old Yellow Enzyme; French CE et al.; Morphinone reductase, produced by Pseudomonas putida M10, catalyses the NADH-dependent saturation of the carbon-carbon double bond of morphinone and codeinone, and is believed to be involved in the metabolism of morphine and codeine . The structural gene encoding morphinone reductase, designated morB, was cloned from Ps . putida M10 genomic DNA by the use of degenerate oligonucleotide probes based on elements of the amino acid sequence of the purified enzyme . Sequence analysis and structural characteristics indicated that morphinone reductase is related to the flavoprotein alpha/beta-barrel oxidoreductases, and is particularly similar to Old Yellow Enzyme of Saccharomyces spp . and the related oestrogen-binding protein of Candida albicans . Expressed sequence tags from several plant species show high homology to these enzymes, suggesting the presence of a family of enzymes conserved in plants and fungi . Although related bacterial proteins are known, morphinone reductase appears to be more similar to the eukaryotic proteins . Morphinone reductase was overexpressed in Escherichia coli, and has potential applications for the industrial preparation of semisynthetic opiates. J Mol Biol, 1995 Dec 15, 254(5), 918 - 41 The refined X-ray structure of muconate lactonizing enzyme from Pseudomonas putida PRS2000 at 1.85 A resolution; Helin S et al.; We report here the refined X-ray crystal structure of muconate lactonizing enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 A with an R-factor of 16.8% . An enzyme from the beta-ketoadipate pathway, MLE catalyses the conversion of cis,cis-muconate to muconolactone . It is a homo-octamer, one monomer consisting of 373 amino acid residues . MLE has two large domains and a C-terminal subdomain: an alpha + beta domain, an alpha beta-barrel domain and a C-terminal meandering subdomain . The alpha beta-barrel domain is highly irregular . Its structure is (beta/alpha)7 beta, with the structural role of the last alpha-helix being replaced by both the C-terminal subdomain and part of the N-terminal domain . The fifth, seventh and eighth barrel strands are unusual because they have left-handed twist about their axes . The strand crossing angles also vary enormously, from +9 degrees to -69 degrees; the first and last strands, which close the barrel, cross at an angle of -69 degrees, making extensive strand-strand hydrogen bonding impossible . The first barrel strand is also unusual because it starts in the N-terminal domain and forms hydrogen bonds to the C-terminal subdomain beta-sheet as well as to its neighbouring strands in the barrel . It thus cements the whole protein together . As in other alpha beta-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel beta-strands . The active site cleft contains an essential manganese ion, is lined with charged and other polar residues, and contains many of the crystallographic water molecules . The manganese ion is octahedrally co-ordinated to three side-chain carboxylate groups and three water molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding interactions . Additionally, two water molecules are buried in the centre of the barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic and hydrophilic packing interactions with much of the barrel interior . The barrel interior is thus also unusual because it is so hydrophilic; the dominating force appears to be the need to solvate the metal ion effectively . This might account for the irregularity of the barrel . The catalytic mechanism has been investigated by docking both substrate and product in the active site with the C-COO- of muconolactone superimposed on the corresponding atoms of cis,cis-muconate . In agreement with earlier kinetic and spectroscopic results, the manganese ion does not interact directly with substrate or product.(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1995 Dec 8, 254(4), 608 - 22 Dissection of the core and auxiliary sequences in the vegetative replication origin of promiscuous plasmid RK2; Shah DS et al.; The vegetative replication origin (oriV) of promiscuous IncP plasmid RK2 can function in many Gram-negative bacterial species when supplied with the plasmid-encoded replication protein TrfA and host-encoded replication proteins including DnaA . Nine TrfA binding sites (iterons) are known, and also two DnaA binding sites, box 1, between TrfA iterons 4 and 5, and box 2, downstream of repeat 9 . The deletion analysis presented here shows that the core oriV requires DnaA box 1 for function in Escherichia coli and Pseudomonas putida . This DnA box is not essential in Pseudomonas aeruginosa, although its deletion does reduce plasmid copy number in this species . A putative IHF binding site is located upstream of DnaA box 1, but IHF deficiency in E . coli seems not to alter replication efficiency or copy number control . Cloned oriV can interfere with maintenance of an independent RK2 replicon . Analysis of replication inhibition functions associated with oriV showed that a short putative orf between TrfA iterons 1 and 2 is not necessary for replication inhibition, the presence of repeats 5 to 9 in target and inhibitor plasmid are not sufficient for efficient inhibition and inhibition does not correlate directly with the number of direct repeats present . Rather, the results showed that the isolated repeats 1 and 2 to 4, potentiate replication inhibition disproportionately to their effect on the number of TrfA binding sites . The results are consistent with the idea that repeats 1 to 4, arranged as a single copy and as an irregular group of three, potentiate the ability of the oriV region to form complexes which inhibit replication . We suggest that TrfA bound at these iterons may be more susceptible to forming pairs between oriV sequences on different plasmids. Mol Microbiol, 1995 Dec, 18(5), 851 - 7 Role of sigma S in transcription from the positively controlled Pm promoter of the TOL plasmid of Pseudomonas putida; Marques S et al.; Transcription from the TOL plasmid Pm promoter is dependent on the XylS regulator activated by benzoate effectors . We analysed transcription from Pm in several backgrounds with differing Escherichia coli alpha and sigma subunits of RNA polymerase . In different RpoA backgrounds, transcription from Pm was as high as in the wild-type background throughout the growth curve . In the sigma S-deficient background provided by E . coli RH90, high levels of transcription from Pm (XylS/3-methylbenzoate dependent) were observed in the early logarithmic growth phase but not in the late logarithmic phase or early stationary phase . This contrasted with the results obtained in the isogenic sigma S-proficient background, in which high levels of transcription were observed throughout the growth curve . XylS/3-methylbenzoate-dependent transcription from Pm in the late logarithmic growth phase in the RH90 background was restored by cloned rpoS . The transcription initiation point of Pm was the same regardless of the growth phase and the sigma S background . The requirement of sigma S for stimulation of transcription from Pm in the late logarithmic and early stationary phase was overcome by using certain mutant Pm promoters, e.g . Pm5 (C-47-->G, A-44-->G), and the mutant regulator XylSG44S . It is suggested that the transcription from Pm involves the use of two sigma factors: sigma 70 during the early logarithmic phase and sigma S thereafter. FEMS Microbiol Lett, 1995 Dec 1, 134(1), 51 - 6 Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction; Porter J et al.; Use of the polymerase chain reaction, coupled with flow cytometry, to detect a plasmid encoded xylE gene sequence in intact cells of Escherichia coli and Pseudomonas putida was investigated . Optimal incorporation of fluorescently labelled dUTP into a full length PCR product required substitution at a level of 2:3 dUTP:dTTP . Formaldehyde fixed cells of both species were counted before and after thermal cycling . Sufficient numbers of cells remained intact for subsequent detection using microscopy and flow cytometry but light scatter properties were altered . Intact cell suspensions of both species containing plasmid pLV1013 were subjected to thermal cycling with fluorescent dUTP in the reaction mix . Subsequent analysis by flow cytometry allowed detection of a fluorescent PCR product associated with cells . Control samples (without the plasmid) showed only background fluorescence . This method demonstrates the potential for applying DNA amplification methods for sensitive detection of specific sequences localized inside intact bacterial cells. J Bacteriol, 1995 Dec, 177(24), 7033 - 40 Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida beta-ketoadipate pathway; Nichols NN et al.; Pseudomonas putida PRS2000 degrades the aromatic acids benzoate and 4-hydroxybenzoate via two parallel sequences of reactions that converge at beta-ketoadipate, a derivative of which is cleaved to form tricarboxylic acid cycle intermediates . Structural genes (pca genes) required for the complete degradation of 4-hydroxybenzoate via the protocatechuate branch of the beta-ketoadipate pathway have been characterized, and a specific transport system for 4-hydroxybenzoate has recently been described . To better understand how P . putida coordinates the processes of 4-hydroxybenzoate transport and metabolism to achieve complete degradation, the regulation of pcaK, the 4-hydroxybenzoate transport gene, and that of pcaF, a gene required for both benzoate and 4-hydroxybenzoate degradation, were compared . Primer extension analysis and lacZ fusions showed that pcaK and pcaF, which are adjacent on the chromosome, are transcribed independently . PcaR, a transcriptional activator of several genes of the beta-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate beta-ketoadipate induces both genes . In addition to these expected regulatory elements, expression of pcaK, but not pcaF, is repressed by benzoate . This previously unrecognized layer of regulatory control in the beta-ketoadipate pathway appears to extend to the first two steps of 4-hydroxybenzoate degradation, since levels of 4-hydroxybenzoate hydroxylase and protocatechuate 3,4-dioxygenase activities were also depressed when cells were grown on a mixture of 4-hydroxybenzoate and benzoate . The apparent consequence of benzoate repression is that cells degrade benzoate in preference to 4-hydroxybenzoate . These findings indicate that 4-hydroxybenzoate transport is an integral feature of the beta-ketoadipate pathway in P . putida and that transport plays a role in establishing the preferential degradation of benzoate over 4-hydroxybenzoate . These results also demonstrate that there is communication between the two branches of the beta-ketoadipate pathway. J Bacteriol, 1995 Dec, 177(23), 6983 - 8 Formation of indigo and related compounds from indolecarboxylic acids by aromatic acid-degrading bacteria: chromogenic reactions for cloning genes encoding dioxygenases that act on aromatic acids; Eaton RW et al.; The p-cumate-degrading strain Pseudomonas putida F1 and the m- and p-toluate-degrading strain P . putida mt-2 transform indole-2-carboxylate and indole-3-carboxylate to colored products identified here as indigo, indirubin, and isatin . A mechanism by which these products could be formed spontaneously following dioxygenase-catalyzed dihydroxylation of the indolecarboxylates is proposed . Indolecarboxylates were employed as chromogenic substrates for identifying recombinant bacteria carrying genes encoding p-cumate dioxygenase and toluate dioxygenase . Dioxygenase gene-carrying bacteria could be readily distinguished as dark green-blue colonies among other colorless recombinant Escherichia coli colonies on selective agar plates containing either indole-2-carboxylate or indole-3-carboxylate. Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 1024 - 33 Production of a form-specific, inhibitory antibody against rat cytochrome P450 2B1 using a synthetic peptide antigen against a putative substrate binding site; Charnecki J et al.; Rat cytochrome P450 2B1 antipeptide antibodies were produced by immunizing rabbits with a synthetic peptide antigen . The anti-CYP2B1 IgG obtained did not cross-react with CYP2B2, which has 97% identity in primary sequence of CYP2B1 . This result demonstrates that a difference of 2 amino acid residues among 12 is sufficient to produce a form-specific antibody . The CYP2B1 antipeptide IgG inhibited pentoxyresorufin O-dealkylase activity of microsomes obtained from phenobarbital-treated rats in a dose-dependent manner, whereas it did not inhibit ethoxyresorufin O-deethylase activity of microsomes obtained from 3-methylcholanthrene-treated rats . These results suggest that the selected amino acid sequence, which coincides with one of the substrate binding sites of Pseudomonas putida CYP101A (P450cam) and one of the putative substrate binding sites of CYP2B2, is located on the surface of the CYP2B1 molecule, as opposed to inside the molecule or in the lipid bilayer of microsomes. Arch Microbiol, 1995 Nov, 164(5), 353 - 7 Purification and characterization of dihydroorotase from Pseudomonas putida; Ogawa J et al.; Dihydroorotase was purified to homogeneity from Pseudomonas putida . The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa . The enzyme only hydrolyzed dihydro-L-orotate and its methyl ester, and the reactions were reversible . The apparent Km and Vmax values for dihydro-L-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 mumol min-1 mg-1, respectively; and those for N-carbamoyl-DL-aspartate (at pH 6.0) were 2.2 mM and 68 mumol min-1 mg-1, respectively . The enzyme was inhibited by metal ion chelators and activated by Zn2+ . However, excessive Zn2+ was inhibitory . The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited by N-carbamoylamino acids such as N-carbamoylglycine, with a Ki value of 2.7 mM . The enzyme was also inhibited non-competitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with a Ki value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis. Eur J Biochem, 1995 Nov 1, 233(3), 828 - 36 Purification of active E1 alpha 2 beta 2 of Pseudomonas putida branched-chain-oxoacid dehydrogenase; Hester K et al.; Active E1 component of Pseudomonas putida branched-chain-oxoacid dehydrogenase was purified from P . putida strains carrying pJRS84 which contains bkdR (encoding the transcriptional activator) and bkdA1 and bkdA2 (encoding the alpha and beta subunits) . Expression was inducible, however, 45-, 39- and 37-kDa proteins were produced instead of the expected 45-kDa and 37-kDa proteins . The 45-kDa protein was identified as E1 alpha and the 37-kDa and 39-kDa proteins were identified as separate translational products of bkdA2 by their N-terminal sequences . The N-terminal amino acid of the 39-kDa protein was leucine instead of methionine . The 45-, 39- and 37-kDa proteins were also produced in wild-type P.putida . Translation of bkdA1 and bkdA2 from an Escherichia coli expression plasmid produced only 45-kDa and 39-kDa proteins, with N-terminal methionine on the 39-kDa protein . The insertion of guanine residues 5' to the first ATG of bkdA2 did not affect expression of E1 beta in P . putida including the N-terminal leucine which appears to eliminate the possibility of ribosome jumping . The Z-average molecular mass of the E1 component was determined by sedimentation equilibrium to be 172 +/- 9 kDa compared to a calculated value of 166 kDa for the heterotetramer and a Stokes radius of 5.1 nm . E1 alpha Ser313, which is homologous to the phosphorylated residue of rat liver E1 alpha, was converted to alanine resulting in about a twofold increase in Km, but no change in Kcat . S315A and S319A mutations had no effect on Km or Kcat indicating that these residues do not play a major part in catalysis of E1 alpha beta 2. Biochim Biophys Acta, 1995 Oct 25, 1252(2), 177 - 9 The 2Fe2S centres of the 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 studied by EPR spectroscopy; Rosche B et al.; The 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 comprises two components with four redox active sites necessary for activity . We present an EPR characterization of the iron-sulfur centres in the purified reductase and oxygenase component of this novel enzyme system . The oxygenase component was identified as a Rieske {2Fe2S} protein on the basis of its characteristic EPR spectrum with gz,y,x = 2.01, 1.91, 1.76 and gav = 1.893 . The reductase component, an iron-sulfur flavoprotein, contained a {2Fe2S} cluster with gz,y,x = 2.03, 1.94, 1.89 and the average g-value (gav) of 1.953, typical of a ferredoxin-type centre . In redox titrations at pH 7, the midpoint potentials were determined to be -180 mV +/- 30 mV and -100 mV +/- 10 mV for the reductase and oxygenase component, respectively . A detailed comparison to other multicomponent enzyme systems is presented pointing out the EPR and redox properties of the FeS centres involved. Mol Gen Genet, 1995 Oct 25, 248(6), 735 - 43 Multiple outer membrane receptors for uptake of ferric pseudobactins in Pseudomonas putida WCS358; Koster M et al.; Under iron limitation Pseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA . In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads . Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA . Eleven amplification products within the expected size range were obtained . Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors . Two complete receptor genes were isolated from a genomic library of P . putida WCS358 . Both protein products are involved in the transport of a limited number of specific ferric pseudobactins . These results indicate that the ability of P . putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins. Gene, 1995 Oct 16, 164(1), 117 - 22 Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl/chlorobiphenyl degradation by Rhodococcus sp . M5; Wang Y et al.; The nucleotide (nt) sequence of the bpdC1C2BADE genes which encode the first three enzymes in the biphenyl (BP) degradation pathway of Gram+ Rhodococcus sp . M5 (formerly Arthrobacter M5) was determined . Except for the ferredoxin component (BpdB) of the initial BP dioxygenase, the predicted amino acid (aa) sequences of the remaining proteins are found to be more closely related to the counterpart proteins (TodC1C2BADE) present in the toluene-degrader, Pseudomonas putida F1, than those of three BP-degrading pseudomonads . The cloned bpd genes were verified by their expression in the Escherichia coli T7 RNA polymerase/promoter system . In E . coli, BpdA was able to complement TodC1C2B in indigo biosynthesis, although the M5 native or cloned BP dioxygenase does not carry out this reaction. J Bacteriol, 1995 Oct, 177(20), 5799 - 805 Desaturation and oxygenation of 1,2-dihydronaphthalene by toluene and naphthalene dioxygenase; Torok DS et al.; Bacterial strains expressing toluene and naphthalene dioxygenase were used to examine the sequence of reactions involved in the oxidation of 1,2-dihydronaphthalene . Toluene dioxygenase of Pseudomonas putida F39/D oxidizes 1,2-dihydronaphthalene to (+)-cis-(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene, (+)-(1R)-hydroxy-1,2-dihydronaphthalene, and (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene . In contrast, naphthalene dioxygenase of Pseudomonas sp . strain NCIB 9816/11 oxidizes 1,2-dihydronaphthalene to the opposite enantiomer, (-)-cis-(1R,2S)-dihydroxy-1,2,3,4-tetrahydronaphthalene and the identical (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene . Recombinant Escherichia coli strains expressing the structural genes for toluene and naphthalene dioxygenases confirmed the involvement of these enzymes in the reactions catalyzed by strains F39/D and NCIB 9816/11 . 1-Hydroxy-1,2-dihydronaphthalene was not formed by strains expressing naphthalene dioxygenase . These results coupled with time course studies and deuterium labelling experiments indicate that, in addition to direct dioxygenation of the olefin, both enzymes have the ability to desaturate (dehydrogenate) 1,2-dihydronaphthalene to naphthalene, which serves as a substrate for cis dihydroxylation. Microbiology, 1995 Oct, 141 ( Pt 10), 2713 - 8 The use of two-dimensional gradient plates to investigate the range of conditions under which conjugal plasmid transfer occurs; Venables WA et al.; Gel-stabilized two-dimensional gradient plates were used to study the effects of pH, salt concentration and temperature on the conjugal transfer of plasmid RP4 between strains of Escherichia coli and Pseudomonas putida . The combinations of pH and salt concentration that permitted conjugation were mapped as a two-dimensional growth area occupied by transconjugants following conjugation . This conjugation domain was less extensive than the areas that supported growth of the parental strains, and showed evidence for the interactive effects of pH and salt concentration in determination of conditions that permitted conjugation . The size and shape of the conjugation domain was influenced by time, temperature, the identities of the donor and recipient bacteria, and the combination of donor and recipient bacteria. Appl Environ Microbiol, 1995 Oct, 61(10), 3645 - 9 Transformations of morphine alkaloids by Pseudomonas putida M10; Long MT et al.; The oxidation of morphine by washed-cell incubations of Pseudomonas putida M10 gave rise to a large number of transformation products including hydromorphone (dihydromorphinone), 14 beta-hydroxymorphine, 14 beta-hydroxymorphinone, and dihydromorphine . Similarly, in incubations with oxymorphone (14 beta-hydroxydihydromorphinone) as substrate, the major transformation product was identified as oxymorphol (14 beta-hydroxydihydromorphine) . The identities of all these biological products were confirmed by mass spectrometry and 1H nuclear magnetic resonance spectroscopy . This is the first report describing structural evidence for the biological synthesis of 14 beta-hydroxymorphine and 14 beta-hydroxymorphinone . These products have applications as intermediates in the synthesis of semisynthetic opiate drugs. Gene, 1995 Sep 22, 163(1), 81 - 5 Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor; Watanabe I et al.; Pravastatin, a drug for treating hypercholesterolemia, is produced by hydroxylation of ML-236B-Na in Streptomyces carbophilus (Sc) catalyzed by the cytochrome P-450sca (CytP-450sca) monooxygenase system . The gene (cytP-450sca-2) encoding CytP-450sca-2 was cloned from Sc . The gene had an open reading frame of 1233 bp, encoding a 410-amino-acid protein . The partial sequencing of the purified CytP-450sca-2 revealed that the N-terminal Met had been removed . CytP-450sca-2 contained the heme-binding HR2 region characteristic of all CytP-450, as well as the putative oxygen-binding site proposed in CytP-450cam from Pseudomonas putida . ML-236B.Na enhanced transcription of cytP-450sca-2, suggesting that substrate induction in Sc is transcriptionally regulated . S . lividans (Sl) transfected with cytP-450sca-2 converted ML-236B.Na to pravastatin, indicating the cloned gene to be functional in Sl. Z Naturforsch {C}, 1995 Sep-Oct, 50(9-10), 622 - 9 Structure and characterization of isopyoverdin from Pseudomonas putida BTP1 and its relation to the biogenetic pathway leading to pyoverdins; Jacques P et al.; Pyoverdin type siderophores produced by six fluorescent Pseudomonas strains isolated from different rhizospheres were purified and characterized . The purified ferri-pyoverdins were tested for their ability to promote the growth of other strains grown under iron deficiency conditions . Only the one obtained from Pseudomonas putida BTP1 did not act as a growth promoter . The structure of the BTP1 siderophore was elucidated by spectroscopic methods and degradation studies . It turned out that it contains a chromophore which differs from the one typical for pyoverdins insofar as it carries the carboxyl group in 3- rather than in 1-position ((3S)-5-amino-1,2-dihydro-8,9-dihydroxy-3H-pyrimido{1,2a}quinoline-3- carboxylic acid) . The amino group of the chromophore is substituted with the 5-carboxyl group of L-glutamic acid and its carboxyl group with the N-terminus of the peptide L-Asp-L-Ala-L-Asp-D-N5-Ac-N5-OH-Orn-L-Ser-L-c-N5-OH-Orn . This isopyoverdin fits into the biogenetic scheme which postulates ferribactins as the precursors of pyoverdins. J Bacteriol, 1995 Sep, 177(18), 5374 - 8 Growth phase-dependent induction of stationary-phase promoters of Escherichia coli in different gram-negative bacteria; Miksch G et al.; RSF1010-derived plasmids carrying a fusion of a promoterless lacZ gene with the sigma s-dependent growth phase-regulated promoters of Escherichia coli, bolAp1 and fic, were constructed . The plasmids were mobilized into the gram-negative bacterial species Acetobacter methanolicus, Xanthomonas campestris, Pseudomonas putida, and Rhizobium meliloti . The beta-galactosidase activities of bacterial cultures were determined during exponential and stationary growth phases . Transcriptional activation of the fic promoter in the different bacteria was growth phase dependent as in E . coli and was initiated generally during the transition to stationary phase . The induction of the bolA promoter was also growth phase dependent in the bacteria tested . While the expression in E . coli and R . meliloti was initiated during the transition from exponential to stationary phase, the induction in A . methanolicus, P . putida, and X . campestris started some hours after stationary growth phase was reached . In all the species tested, DNA fragments hybridizing with the rpoS gene of E . coli were detected . The results show that in different gram-negative bacteria, stationary-phase-specific sigma factors which are structurally and functionally homologous to sigma s and are able to recognize the promoter sequences of both bolA and fic exist. Can J Microbiol, 1995 Sep, 41(9), 776 - 84 Low temperature growth, freezing survival, and production of antifreeze protein by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2; Sun X et al.; The plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was originally isolated from the rhizosphere of plants growing in the Canadian High Arctic . Here we report that this bacterium was able to grow and promote root elongation of both spring and winter canola at 5 degrees C, a temperature at which only a relatively small number of bacteria are able to proliferate and function . In addition, the bacterium survived exposure to freezing temperatures, i.e., -20 and -50 degrees C . In an effort to determine the mechanistic basis for this behaviour, it was discovered that following growth at 5 degrees C, P . putida GR12-2 synthesized and secreted to the growth medium a protein with antifreeze activity . Analysis of the spent growth medium, following concentration by ultrafiltration, by SDS-polyacrylamide gel electrophoresis revealed the presence of one major protein with a molecular mass of approximately 32-34 kDa and a number of minor proteins . However, at this point it is not known which of these proteins contains the antifreeze activity. Biodegradation, 1995 Sep, 6(3), 247 - 55 The nucleotide sequence of a transposable haloalkanoic acid dehalogenase regulatory gene (dehRI) from Pseudomonas putida strain PP3 and its relationship with sigma 54-dependent activators; Topping AW et al.; The mobile genetic element, DEH found in Pseudomonas putida PP3 carries a 2-haloalkanoic acid dehalogenase structural gene, dehI, and its associated regulatory gene, dehRI . The nucleotide sequence of dehRI was determined . The gene had an open reading frame putatively encoding for a 64 kDa protein containing 571 amino acid residues . The protein was similar to previously published sequences of several other sigma 54-dependent activator proteins . Amino acid sequence analysis showed that the deduced DehRI protein clustered with the NifA nitrogenase regulatory activator family, and was most closely related, with 47.7% similarity, to a 'NifA-like' deduced partial sequence from a plasmid-encoded ORF in Pseudomonas sp . strain NS671, associated with L-amino acid production . The domain structure of DehRI was analysed by alignment with other NifA-like and NtrC-like sequences and showed a highly conserved central region of approximately 230 amino acids, and a potential DNA-binding domain . No homology was detected between the deduced DehRI and other sigma 54-dependent activator sequences at the N-terminus, a result which was consistent with that region being the domain which recognised inducer. Biodegradation, 1995 Sep, 6(3), 213 - 6 Use of haloacetate dehalogenase genes as selection markers for Escherichia coli and Pseudomonas vectors; Kawasaki H et al.; The haloacetate dehalogenase gene, dehH2, cloned from Moraxella sp . strain B could be used a selection marker gene for vectors in Escherichia coli and Pseudomonas putida . Haloacetates, especially iodoacetate, inhibit the growth of some microorganisms . The dehH2 gene introduced into the cells conferred iodoacetate resistance on them . Therefore, E . coli and P . putida transformed with vectors marked with dehH2 could be easily selected on plates containing iodoacetate. Lett Appl Microbiol, 1995 Sep, 21(3), 167 - 72 Quantification of the effect of substrate concentration on the conjugal transfer rate of the TOL plasmid in short-term batch mating experiments; Smets BF et al.; Batch mating experiments with Pseudomonas putida PAW 1 (TOL) as a donor and Pseudomonas aeruginosa PAO 1162 as a recipient strain were performed to quantify the effect of the substrate concentration in the mating medium on the observed plasmid transfer rate coefficient . The impact of the substrate concentration in the mating medium was highly correlated with the growth history of the donor strain . When the donor strain was harvested in exponential growth phase, no impact was observed; when the donor strain was taken from the stationary phase, however, a strong impact of the substrate concentration was measured: a 10-fold reduction in the substrate concentration decreased the observed plasmid transfer rate by 55%. Mol Gen Genet, 1995 Aug 30, 248(4), 487 - 90 Roa307, a protein encoded on Coxiella burnetii plasmid QpH1, shows homology to proteins encoded in the replication origin region of bacterial chromosomes; Lin Z et al.; TnphoA mutagenesis identified an open reading frame, roa307, immediately upstream of the partition locus qsopAB on the Coxiella burnetii plasmid QpH1 . The protein sequence deduced from roa307 displayed homology to Orf290 of Pseudomonas putida, Orf283 and Orf282 (SpoOJ) of Bacillus subtilis-hypothetical products of genes in the chromosomal replication origin region . Expression of roa307 was demonstrated by PhoA activity of an Roa307-PhoA fusion. Arch Biochem Biophys, 1995 Aug 20, 321(2), 353 - 62 Cloning, DNA sequencing, and amino acid sequencing of catechol 1,2-dioxygenases (pyrocatechase) from Pseudomonas putida mt-2 and Pseudomonas arvilla C-1; Nakai C et al.; Catechol 1,2-dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis-muconic acid and is encoded by a catA gene . We have cloned a catA gene from Pseudomonas putida mt-2 using a PCR product of amino acid sequence-based primers as a probe . The amino acid sequence deduced from the 930 nucleotides was in complete agreement with the chemically determined sequence of the protein . Crude extracts of Escherichia coli cells carrying the catA gene downstream from the lac promoter showed the enzyme activity . By using the same probe, we also cloned and sequenced the catA beta gene for catechol 1,2-dioxygenase isozyme beta beta from Pseudomonas arvilla C-1, which has three isozymes, alpha alpha, alpha beta, and beta beta (C . Nakai, H . Horiike, S . Kuramitsu, H . Kagamiyama, and M . Nozaki, 1990, J . Biol . Chem . 265, 660-665) . There was very high homology between isozyme beta beta of the C-1 strain and the enzyme of the mt-2 strain in both the amino acid (98%) and the DNA sequences (97%) . A preference for the use of codons terminating in C and G was found in the coding region of both the enzymes, which contributed to the high G + C content (65-66%) of the genes . A comparison of the DNA sequences of various catA genes from other sources revealed their common ancestry, whereas a comparison of the amino acid sequences of the enzymes revealed clear reflection of substrate specificity . Tyrosyl and histidyl residues for proposed ligands of ferric ion are conserved in all catechol 1,2-dioxygenases. J Bacteriol, 1995 Aug, 177(16), 4713 - 20 Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida; Leddy MB et al.; Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway . After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU . Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s) . Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose . Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function . Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor . Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes . Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O . Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants . Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes . No variants formed in identical ethylbenzene-exposed controls . The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pathway. Lett Appl Microbiol, 1995 Aug, 21(2), 109 - 14 Assessment of rapid bioassays for detecting cyanobacterial toxicity; Lahti K et al.; Simple and easy-to-use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato- and neurotoxins . The hepatotoxins and a neurotoxin, anatoxin-a, were extracted from laboratory-grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays . The toxin concentration of cyanobacterial extracts was determined with HPLC . The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3-17 mg l-1 . The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin-a . The fractionated extract of anatoxin-a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards . Filtered cyanobacterial cultures with anatoxin-a, on the other hand, caused mortality of Artemia larvae at concentrations of 2-14 mg l-1 . With the solid phase fractionation of cyanobacterial samples, no non-specific toxicity due to compounds other than hepato- and neurotoxins was observed . In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin-a . The growth of Ps . putida was enhanced, rather than inhibited by cyanobacterial toxin fractions. Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7277 - 81 Integration host factor suppresses promiscuous activation of the sigma 54-dependent promoter Pu of Pseudomonas putida; Perez-Martin J et al.; In the presence of m-xylene, the Pu promoter of the TOL plasmid of Pseudomonas putida is activated by the prokaryotic enhancer-binding protein XylR . The intervening DNA segment between the upstream activating sequences (UASs) and those for RNA polymerase binding contains an integration host factor (IHF) attachment site that is required for full transcriptional activity . In the absence of IHF, the Pu promoter can be cross-activated by other members of the sigma 54-dependent family of regulatory proteins . Such illegitimate activation does not require the binding of the heterologous regulators to DNA and it is suppressed by bent DNA structures, either static or protein induced, between the promoter core elements (UAS and RNA polymerase recognition sequence) . The role of IHF in some sigma 54 promoters is, therefore, not only a structural aid for assembling a correct promoter geometry but also that of an active suppressor (restrictor) of promiscuous activation by heterologous regulators for increased promoter specificity. Biokhimiia, 1995 Aug, 60(8), 1251 - 60 {Modification of the ortho-route in Pseudomonas putida strain 87: purification and properties of dienlactone hydrolase}; Solianikova IP et al.; Dienelactone hydrolase (DLH) was purified to electrophoretic homogeneity from the biomass of the Pseudomonas putida strain 87 grown on 3-chlorobenzoate . The specific activity of the purified enzyme is 50.5 U./mg of protein . The enzyme is a monomer with a molecular mass of 22 kDa, has a pH optimum at 7.6 and is active within a broad temperature range (20-45 degrees C) . The enzyme activity is inhibited by pCMB (which requires up to two equivalents of the reagent) but not by EDTA . The Vmax values of DLH for trans-dienelactone and 2-chlorodienelactone are practically identical (247.9 and 247 u./mg, respectively) and exceeded two fold that for cis-dienelactone . However, the Km for 2-chlorodienelactone is two times as high as that for trans-dienelactone (6.9 and 14.2 microM, respectively) . A comparison of physico-chemical and kinetic properties of P . putida 87 DLH with those of the enzymes responsible for the decomposition of chlorinated and fluorinated compounds revealed that the enzyme can be assigned to the third group, i.e., the DLH-modified ortho-cleavage path way characteristic of gram-negative bacteria. Appl Environ Microbiol, 1995 Aug, 61(8), 2990 - 4 Construction and behavior of biologically contained bacteria for environmental applications in bioremediation; Ronchel MC et al.; The survival of microorganisms can be predicted through the use of active biological containment systems . We have constructed contained Pseudomonas putida strains that degrade alkylbenzoates . The modified strain carries a fusion of the Plac promoter to the gef gene, which encodes a killing protein . Expression from Plac is controlled through a regulatory cascade, so that Plac is switched on or off by the absence or presence of alkylbenzoates, respectively . Similar uncontained strains were also constructed and tested as a control . Contained and uncontained strains were genetically stable, and their survival and functionality in soil microcosms were as expected . Both contained and uncontained strains survived well in soils supplemented with alkylaromatics, whereas survival of the contained strain in soil microcosms without methylbenzoates was markedly reduced, in contrast to the control strain, which survived in these soils in the absence of alkylbenzoates . The TOL plasmid was transferred in soils between Pseudomonas strains but was not able to mobilize the elements of the containment system. Appl Environ Microbiol, 1995 Aug, 61(8), 2859 - 62 Role of competition for inorganic nutrients in the biodegradation of mixtures of substrates; Steffensen WS et al.; A study was conducted to determine whether competition for inorganic nutrients affects the biodegradation of mixtures of substrates . Little benzylamine was mineralized by Pseudomonas putida in solutions with no added P, but the substrate was degraded if the medium contained 100 nM P . The enhancement by P addition did not occur if the medium also contained caprolactam and a caprolactam-utilizing strain of Pseudomonas aeruginosa . The suppression by the second bacterium was overcome by a higher P concentration . The rate of caprolactam utilization by P . aeruginosa was reduced if benzylamine and P . putida were also present in media with 100 nM P, but the suppression was absent if the solution contained a higher P concentration . Glutamate increased and inorganic N plus P decreased the length of the acclimation phase prior to benzylamine mineralization in lake water . We suggest that the effect of one biodegradable substrate on the metabolism of a second often results from a competition for inorganic nutrients. J Biol Chem, 1995 Jul 28, 270(30), 17836 - 42 2-Oxo-1,2-dihydroquinoline 8-monooxygenase, a two-component enzyme system from Pseudomonas putida 86; Rosche B et al.; 2-Oxo-1,2-dihydroquinoline 8-monooxygenase, which catalyzes the NADH-dependent oxygenation of 2-oxo-1,2-dihydroquinoline to 8-hydroxy-2-oxo-1,2-dihydroquinoline, is the second enzyme in the quinoline degradation pathway of Pseudomonas putida 86 . This enzyme system consists of two inducible protein components, which were purified, characterized, and identified as reductase and oxygenase . The yellow reductase is a monomeric iron-sulfur flavoprotein (M(r), 38,000), containing flavin adenine dinucleotide and plant-type ferredoxin {2Fe-2S} . It transferred electrons from NADH to the oxygenase or to some artificial electron acceptors . The red-brown oxygenase (M(r), 330,000) consists of six identical subunits (M(r), 55,000) and was identified as an iron-sulfur protein, possessing about six Rieske-type {2Fe-2S} clusters and additional iron . It was reduced by NADH plus catalytic amounts of reductase . For monooxygenase activity, reductase, oxygenase, NADH, molecular oxygen, and substrate were required . The activity was considerably enhanced by the addition of polyethylene glycol and Fe2+ . 2-Oxo-1,2-dihydroquinoline 8-monooxygenase revealed a high substrate specificity toward 2-oxo-1,2-dihydroquinoline, since none of 25 other tested compounds was converted . Based on its physical, chemical, and catalytic properties, we presume 2-oxo-1,2-dihydroquinoline 8-monooxygenase to belong to the class IB multicomponent non-heme iron oxygenases. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6424 - 8 Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa; Ochsner UA et al.; The opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, including exotoxin A, elastase, alkaline protease, alginate, phospholipases, and extracellular rhamnolipids . The previously characterized rhlABR gene cluster encodes a regulatory protein (RhlR) and a rhamnosyltransferase (RhlAB), both of which are required for rhamnolipid synthesis . Another gene, rhII, has now been identified downstream of the rhlABR gene cluster . The putative RhlI protein shares significant sequence similarity with bacterial autoinducer synthetases of the LuxI type . A P . aeruginosa rhlI mutant strain carrying a disrupted rhlI gene was unable to produce rhamnolipids and lacked rhamnosyltransferase activity . Rhamnolipid synthesis was restored by introducing a wild-type rhlI gene into such strains or, alternatively, by adding either the cell-free spent supernatant from a P . aeruginosa wild-type strain or synthetic N-acylhomoserine lactones . Half-maximal induction of rhamnolipid synthesis in the rhlI mutant strain required 0.5 microM N-butyrylhomoserine lactone or 10 microM N-(3-oxohexanoyl)homoserine lactone . The P . aeruginosa rhlA promoter was active in the heterologous host Pseudomonas putida when both the rhlR and rhlI genes were present or when the rhlR gene alone was supplied together with synthetic N-acylhomoserine lactones . The RhlR-RhlI regulatory system was found to be essential for the production of elastase as well, and cross-communication between the RhlR-RhlI rhamnolipid regulatory system and the LasR-LasI elastase regulatory system was demonstrated. Biosci Biotechnol Biochem, 1995 Jul, 59(7), 1204 - 7 Nucleotide sequence of the gene for a thermostable esterase from Pseudomonas putida MR-2068; Ozaki E et al.; The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109 . An 8-kb inserted DNA directed synthesis of an esterase in E . coli . The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA . The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828 bp coding for a protein of 276 amino acid residues . The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase . A potential Shine-Dalgarno sequence is followed by the open reading frame . The esterase activity of the recombinant E . coli was more than 200 times higher than that of parental strain, P . putida MR-2068. FEMS Microbiol Rev, 1995 Jul, 16(4), 295 - 307 Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins; Braun V; Iron in the form of ferric siderophore complexes and vitamin B12 are transported through the outer membrane of Gram-negative bacteria by a mechanism which consumes energy . There is no known energy source in the outer membrane or in the adjacent periplasmic space so that energy is provided by the electrochemical potential across the cytoplasmic membrane . Energy flows from the cytoplasmic into the outer membrane via a complex consisting of the TonB, ExbB and ExbD proteins which are anchored in the cytoplasmic membrane . It is proposed that the TonB--ExbB--ExbD complex opens--via an energized conformation of the TonB protein--channels in the outer membrane, formed by proteins which serves as highly specific binding sites for the various ferric siderophores and vitamin B12 . In addition, outer membrane receptors together with the TonB--ExbB--ExbD complex are directly involved in induction of the transcription of ferric citrate and pseudobactin transport genes of Escherichia coli and Pseudomonas putida, respectively. Appl Environ Microbiol, 1995 Jul, 61(7), 2754 - 8 Simultaneous chromium reduction and phenol degradation in a coculture of Escherichia coli ATCC 33456 and Pseudomonas putida DMP-1; Shen H et al.; In a defined coculture of a Cr(VI) reducer, Escherichia coli ATCC 33456, and a phenol degrader, Pseudomonas putida DMP-1, simultaneous reduction of Cr(VI) and degradation of phenol was observed . When Cr(VI) was present in the coculture, quantitative transformation of Cr(VI) into Cr(III) proceeded with simultaneous degradation of phenol . Cr(VI) reduction was correlated to phenol degradation in the coculture as demonstrated by a regression analysis of the cumulative Cr(VI) reduction and the cumulative phenol degradation . Both the rate and extent of Cr(VI) reduction and phenol degradation were significantly influenced by the population composition of the coculture . Although Cr(VI) reduction occurred as a result of E . coli metabolism, the rate of phenol degradation by P . putida may become a rate-limiting factor for Cr(VI) reduction at a low population ratio of P . putida to E . coli . Phenol degradation by P . putida was very susceptible to the presence of Cr(VI), whereas Cr(VI) reduction by E . coli was significantly influenced by phenol only when phenol was present at high concentrations (> 9 mM). Appl Environ Microbiol, 1995 Jul, 61(7), 2727 - 31 Cyclodextrin-enhanced degradation of toluene and p-toluic acid by Pseudomonas putida; Schwartz A et al.; Degradation of an immiscible aromatic solvent, toluene, and a water-soluble aromatic compound, p-toluic acid, by a Pseudomonas putida strain in the presence of beta-cyclodextrin (beta-CD) was investigated . The ability of CDs to interact with hydrophobic organics and form inclusion compounds was exploited in this study to remove or alleviate the toxicities of substrates and consequently to enable or enhance degradation . Liquid toluene was found to be highly toxic to P . putida . However, this phase toxicity was removed when crystalline beta-CD-complexed toluene was provided as the substrate . The latter was fully degraded at a concentration of up to 10 g/liter . Degradation of toluene vapors was enhanced in the presence of beta-CD as a result of reduced molecular toxicity and facilitated absorption of the gaseous substrate . Similarly, beta-CD alleviated the inhibitory effect of p-toluic acid on P . putida . This protective effect of CD was remarkably more prominent when the microbial culture was shock loaded with an otherwise toxic dose of p-toluic acid (1.8 g/liter). J Bacteriol, 1995 Jul, 177(14), 3911 - 6 Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons; Ramos JL et al.; Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source . The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log P(ow) (octanol/water partition coefficient) was higher than 2.3 . Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted . Mg2+ ions in the culture medium improved solvent tolerance . Electron micrographs showed that cells growing on high concentrations of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity . Polarographic studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolyze toluene . Although the strain also thrived in high concentrations of m- and p-xylene, these hydrocarbons could not be used as the sole C source for growth . The catabolic potential of the isolate was expanded to include m- and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km. J Bacteriol, 1995 Jul, 177(13), 3758 - 63 The sigma 54-dependent promoter Ps of the TOL plasmid of Pseudomonas putida requires HU for transcriptional activation in vivo by XylR; Perez-Martin J et al.; In the presence of toluene and xylenes, the sigma 54-dependent Ps promoter of the TOL (toluene biodegradation) plasmid pWW0 of Pseudomonas putida is activated at a distance by the XylR protein, of the NtrC family of transcriptional regulators . Since contacts between XylR bound to upstream activating sites and the RNA polymerase require the looping out of the intervening DNA segment, the intrinsic curvature, the bendability of the corresponding sequence, and the spatial effects of protein-induced DNA bending have an influence on promoter activity . Unlike other sigma 54-dependent promoters, Ps does not require the structural aid of the integration host factor to assemble a specific promoter geometry required for transcriptional initiation . In vivo analysis of transcriptional activity in various genetic backgrounds suggests, instead, that the looping out of intervening DNA sequences in Ps would result from the exacerbation of a preexisting static bend within the region, assisted by the histone-like protein HU. Biochem Biophys Res Commun, 1995 Jun 15, 211(2), 382 - 8 Cloning of salicylate hydroxylase gene and catechol 2,3-dioxygenase gene and sequencing of an intergenic sequence between the two genes of Pseudomonas putida KF715; Lee J et al.; The salicylate hydroxylase can convert the salicylate to catechol, and catechol 2,3-dioxygenase catalizes the conversion of catechol to 2-hydroxymuconic semialdehyde . A salicylate hydroxylase gene and a catechol 2,3-dioxygenase have been cloned from chromosomal DNA of P . putida KF715 . The two genes have different promoters . An open reading frame with 339 nucleotides preceded by a putative ribosome-binding sequence (GGAGG) was identified in the intergenic sequence between salicylate hydroxylase gene and catechol 2,3-dioxygenase gene of P . putida KF715 and its sequence analyzed . This open reading frame can encode a polypeptide of molecular weight 13 kDa containing 112 amino acids, whose sequence exhibited 87% homology with that of ferredoxin encoded in NAH7 of P . putida PpG7 and significant homology with those of redox components in phenol hydroxylase, benzoate 1,2-dioxygenase, toluate 1,2-dioxygenase, xylene monooxygenase, and toluene 4-monooxygenase. J Biol Chem, 1995 Jun 2, 270(22), 12957 - 60 Evidence for a (triosephosphate isomerase-like) "catalytic loop" near the active site of glyoxalase I; Lan Y et al.; The conformational mobility of glyoxalase I (Glx I) during catalysis has been probed using stable analogs of the enediol intermediate that forms along the reaction pathway: GSC(O)N(OH)R, where GS = glutathionyl and R = CH3 (1), C6H5 (2), C6H4Cl (3), or C6H4Br (4) . For human erythrocyte Glx I, catalysis is unlikely to be coupled to major changes in protein secondary structure, as the circular dichroism spectrum of the enzyme (190-260 nm) is insensitive to saturating concentrations of either enediol analog or S-D-lactoylglutathione, the product of the Glx I reaction . However, a small conformational change is indicated by the fact that binding of enediol analog to the active site decreases intrinsic protein fluorescence by 11%, and protects the enzyme from proteolytic cleavage by Pronase E at the C-side of Ala-92 and Leu-93 . In contrast, binding of S-D-lactoylglutathione does not affect protein fluorescence, and increases the rate of proteolytic cleavage by 1.5-fold . These observations are consistent with a model of catalysis in which a flexible peptide loop folds over and stabilizes the enediol intermediate bound to the active site . Indeed, a highly conserved sequence of amino acid residues is found near the proteolytic cleavage sites, for human Glx I (100-111) and Pseudomonas putida Glx I (93-105), that shows significant sequence homology to the "catalytic loop" of chicken muscle triosephosphate isomerase (TIM) (165-176) . The active site base (Glu-165) of TIM, which catalyzes the proton transfer reaction during isomerization, corresponds in position to Glu-93 of P . putida Glx I . Consistent with a functional role for Glu-93, a mutant enzyme in which Glu-93 is replaced by Asp shows no detectable catalytic activity. Environ Health Perspect, 1995 Jun, 103 Suppl 5, 45 - 8 Recruitment of co-metabolic enzymes for environmental detoxification of organohalides; Wackett LP; Polyhalogenated compounds are often environmentally persistent and toxic to mammals . Microorganisms that metabolize these compounds can detoxify contaminated environments . Different biochemical mechanisms are used to metabolize polyhalogenated compounds, but few naturally occurring bacteria have this capability . A recombinant bacterium was constructed to metabolize polyhalogenated compounds to nonhalogenated end products . Seven genes were expressed in Pseudomonas putida G786 to biosynthesize cytochrome P450CAM and toluene dioxygenase . Cytochrome P450CAM catalyzed reductive dechlorinated reactions and toluene dioxygenase catalyzed oxidative dechlorination . With pentachloroethane, reductive dechlorination yielded trichloroethylene, which was further oxidized to formate and glyoxylate . The sequential action of cytochrome P450CAM and toluene dioxygenase with polyhalogenated compounds constitutes a novel engineered metabolic pathway. Environ Health Perspect, 1995 Jun, 103 Suppl 5, 107 - 11 Natural selection of PAH-degrading bacterial guilds at coal-tar disposal sites; Ghiorse WC et al.; Microbial activity patterns at buried coal-tar disposal sites have been under investigation for several years to determine the response of naturally occurring microflora to polycyclic aromatic hydrocarbons (PAHs) at the sites . At one site in upstate New York, data have shown enrichment of PAH-degrading bacteria in subsurface contaminated zones but not in uncontaminated zones . Similar work at a midwestern site showed that the same trends existed in a heterogeneous disposal site except that a borehole outside the plume showed some PAH-mineralization activity . Polymerase chain reaction amplification of DNA extracted from sediment samples from the New York site indicated the presence of naphthalene metabolism genes nahAc and nahR, similar to those found on the NAH7 plasmid of Pseudomonas putida G7 . Significant sequence polymorphism was observed in amplified nahAc products, indicating that divergent homologs of nahAc were present in the native community . Protozoan numbers were elevated in sediment samples displaying relatively high PAH-degrading activity, suggesting that a food chain was established based on PAH-degrading bacteria . Removal of the coal-tar source at the site occurred in 1991 . In 1992, sampling of three key borehole stations revealed that mixing and backfilling operations had introduced soil microorganisms into the source area and introduced 14C-PAH-mineralization activity into the previously inactive pristine area . Thus removal of the source of the contaminants and restoration at the site have altered the microbial activity patterns outside the contaminant plume as well as in the source area. Appl Environ Microbiol, 1995 Jun, 61(6), 2211 - 7 Combination of the tod and the tol pathways in redesigning a metabolic route of Pseudomonas putida for the mineralization of a benzene, toluene, and p-xylene mixture; Lee JY et al.; Construction of a hybrid strain which is capable of mineralizing components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida . Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture . Consequently, a hybrid strain was constructed by cloning todC1C2BA genes encoding toluene dioxygenase on RSF1010 and introducing the resulting plasmid into P . putida mt-2 . The hybrid strain of P . putida TB105 was found to mineralize a benzene, toluene, and p-xylene mixture without accumulation of any metabolic intermediate. Appl Environ Microbiol, 1995 Jun, 61(6), 2079 - 85 Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp . strain RHA1; Masai E et al.; Rhodococcus sp . strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days . We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence . On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation . The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB . This gene order differs from that of other PCB degraders reported previously . The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp . strains KF707 and KKS102 (30 to 65%) . In Escherichia coli, bphA gene activity was not observed even when expression vectors were used . The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S . Irie, S . Doi, T . Yorifuji, M . Takagi, and K . Yano, J . Bacteriol . 169:5174-5179, 1987) . The expected products of the cloned bph genes, except bphA3, were observed in E . coli in an in vitro transcription-translation system . Insertion mutations of bphA1 and bphC of Rhodococcus sp . strain RHA1 were constructed by gene replacement with cloned gene fragments.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1995 Jun, 177(11), 3312 - 5 Involvement of IHF protein in expression of the Ps promoter of the Pseudomonas putida TOL plasmid; Holtel A et al.; Regulation of the xyl gene operons of the Pseudomonas putida TOL plasmid is mediated by the products of the downstream clustered and divergently oriented xylR and xylS regulatory genes . The xylR-xylS intergenic region contains the xylR and xylS promoters Pr and Ps, respectively . A binding site for the XylR activator protein is located upstream of Ps and overlapping Pr . DNase I footprint experiments showed that one of these sites, which overlaps the recognition site for XylR activator, as well as an AT-rich region comprising the Ps promoter consensus were protected by integration host factor (IHF) . IHF was found to act negatively in the in vivo activation of the Ps promoter, since the activity of a Ps promoter::lacZ fusion was elevated in an Escherichia coli mutant lacking IHF . In contrast, no alteration in the synthesis of XylR protein in the E . coli IHF-deficient mutant was detected. J Bacteriol, 1995 Jun, 177(11), 3120 - 7 Purification and characterization of a cam repressor (CamR) for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid; Aramaki H et al.; The cytochrome P-450cam hydroxylase operon of Pseudomonas putida PpG1 (ATCC 17543) encodes proteins responsible for early steps of the degradation of D-camphor . Transcription of this operon is negatively controlled by the cam repressor (CamR), and the expression of camR is autoregulated . CamR was purified from Escherichia coli harboring an overproducing plasmid . The repressor forms a homodimer with a molecular mass of 40 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and gel filtration . CamR protected a specific DNA region from attack by DNase I . This region contains a palindromic operator of the cytochrome P-450cam hydroxylase operon and of the camR gene . Protection was inhibited by the addition of 60 microM D-camphor and also by certain camphor analogs and degradation products, including D-3-bromocamphor, adamantane, 2-adamantanone, 5-exo-hydroxycamphor, and 2,5-diketocamphane . These analogs and degradation products induced cytochrome P-450cam hydroxylase operon expression in vivo. Biochemistry, 1995 May 23, 34(20), 6649 - 59 X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase . Implications for the extradiol cleavage mechanism; Shu L et al.; The extradiol-cleaving catechol 2,3-dioxygenase (2,3-CTD) isolated from Pseudomonas putida mt-2 and its catechol and ternary E.S.NO complexes are characterized by X-ray absorption spectroscopy (XAS) . The intensities of the 1s-->3d transitions in the pre-edge spectra of the uncomplexed enzyme and its substrate complex show that the Fe(II) center is five-coordinate in both complexes, in agreement with earlier magnetic circular dichroism studies {Mabrouk, P . A., Orville, A . M., Lipscomb, J . D., & Solomon, E . I . (1991) J . Am . Chem . Soc . 113, 4053-4061} . Analysis of the EXAFS region of uncomplexed 2,3-CTD shows five N/O ligand atoms 2.09 A from the active site Fe(II) . In the 2,3-CTD.catechol complex, one N/O atom is located at 1.93 A and four N/O type ligands are at 2.10 A . By comparison with {FeII-(6TLA)(DBCH)}(ClO4), the first well-characterized mononuclear Fe(II).catechol model complex, the 1.93 A scatterer is proposed to be the oxygen from the deprotonated hydroxyl group of the coordinated catecholate monoanion . Nitric oxide binds to the Fe(II) center in the enzyme.catechol complex without displacing the existing ligands, resulting in the formation of a six-coordinate complex, as indicated by the addition of a new N/O type scatterer at 1.74 A . Bond valence sum (BVS) analysis of the bond lengths derived from the EXAFS fits gives values that correspond to the iron oxidation states established for these complexes, thus lending credence to the coordination environment deduced for the iron center in those complexes . The present study provides the first evidence for a monoanionic substrate binding mode in an extradiol dioxygenase, which is distinct from the dianionic binding mode proposed for intradiol dioxygenases . We speculate that this difference in binding mode may have important ramifications for the site of aromatic ring cleavage in the subsequent oxygen insertion reactions. Gene, 1995 May 19, 157(1-2), 55 - 7 Cloning of the ppu21IM gene using a in vivo selection method; Vaisvila R et al.; A genetic system enabling the in vivo selection of genes encoding the DNA-modifying enzymes was developed . A gene library is transformed into a strain harboring the restriction-modification (R-M) system which a recognition sequence is a subset of the target sequence of the DNA methyltransferase (MTase) to be cloned . If the residing MTase is temperature sensitive, the inability of transformants to grow at 42 degrees C provides a simple and convenient procedure for the isolation of new MTase-encoding genes . The feasibility of this procedure has been demonstrated by the isolation of the ppu21IM gene from a Pseudomonas putida RFL21 gene library. Biochim Biophys Acta, 1995 May 17, 1262(1), 73 - 8 Nucleotide sequence of the Staphylococcus aureus RNA polymerase rpoB gene and comparison of its predicted amino acid sequence with those of other bacteria; Aboshkiwa M et al.; The complete nucleotide sequence of the rpoB gene which encodes the beta subunit of S . aureus RNA polymerase has been determined . The RpoB protein, consists of 1182 amino acids and has a novel initiation codon UUG which initiates protein synthesis with methionine . There is a very strong Shine-Dalgarno complementarity and the -10 and -35 promoter hexameric sequences are TAATAT and CCGTTT, respectively . A rho-dependent termination site, CAATCAA, is present which overlaps the -35 promoter sequence of the adjacent rpoC gene a feature which may have a role in the co-ordinate expression of the two genes . A strong homology and conserved regions were found to exist over the predicted amino acid sequences coding for S . aureus rpoB and the equivalent proteins in Escherichia coli, Pseudomonas putida, Salmonella typhimurium, Chlamydia trachomatis, cyanobacterium Anabaena sp . strain PCC 7120. J Biochem (Tokyo), 1995 May, 117(5), 1120 - 5 Structural analysis of the L-methionine gamma-lyase gene from Pseudomonas putida; Inoue H et al.; The gene encoding L-methionine gamma-lyase from Pseudomonas putida was cloned and the primary structure of the enzyme was deduced from its nucleotide sequence . The L-methionine gamma-lyase gene was expressed in Escherichia coli . The amino acid sequences of BrCN-digested peptides agreed with the corresponding parts of the L-methionine gamma-lyase sequence determined from the gene structure . The polypeptide is composed of 398 amino acid residues with a calculated molecular weight of 42,626, corresponding to the subunit of the homotetrameric enzyme . The deduced amino acid sequence of L-methionine gamma-lyase only showed extensive homology with other well known alpha,gamma-elimination and/or gamma-replacement pyridoxal 5'-phosphate-dependent enzymes, such as cystathionine gamma-lyase, cystathionine gamma-synthase, and O-acetylhomoserine O-acetylserine sulfhydrylase, that participate in the biosynthesis of sulfur amino acids . However, the deduced essential cysteine residue of L-methionine gamma-lyase was not conserved in these enzymes . We confirmed the presence of a part of an open reading frame in the 3'-flanking region of the L-methionine gamma-lyase gene, which showed high homology with the N-terminal region of pyruvate dehydrogenase (lipoamide) from E . coli, suggesting that it participates in the degradative pathway for L-methionine together with L-methionine gamma-lyase. J Bacteriol, 1995 May, 177(9), 2602 - 5 Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B; Kim SW et al.; In order to assess the roles of specific amino acid residues in the delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B during catalysis, we replaced aspartic acid 40 with asparagine (D40N) and tyrosine 16 with phenylalanine (Y16F) in the enzyme by site-directed mutagenesis . Both purified mutant enzymes resulted in profound decreases in catalytic activities, 10(3.3)-fold in the Y16F mutant and 10(6.2)-fold in the D40N mutant . Aspartic acid 40 and tyrosine 16 of the enzyme are the corresponding amino acids in the active site of the homologous enzyme from Comamonas testosteroni . Our results indicate that active-site residues of the two homologous enzymes are similar . This is opposite to the previous identification of a cysteine in an active site-directed photoinactivation study of the enzyme. J Bacteriol, 1995 May, 177(9), 2442 - 50 Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols; Toyama H et al.; A bacterial strain that can utilize several kinds of alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5 . Three distinct dye-linked alcohol dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different alcohols . ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P . putida (H . Gorisch and M . Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point . The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively . Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein dehydrogenases . Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I . The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers . Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another . Immunoblot analysis showed that ADH I was detected in cells grown on each alcohol tested, but ethanol was the most effective inducer . ADH IIB was formed in the cells grown on alcohols of medium chain length and also on 1,3-butanediol . Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former alcohol was more effective . These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes . Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions. Protein Sci, 1995 May, 4(5), 955 - 9 Purification and crystallization of benzoylformate decarboxylase; Hasson MS et al.; A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme . The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification . The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry . The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits . Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5 . The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation . Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222 . The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A . An average Vm value for the crystals is consistent with one subunit per asymmetric unit . The subunits of the tetramer must be arranged with tetrahedral 222 symmetry. Mol Gen Genet, 1995 Apr 20, 247(2), 240 - 6 Localization and organization of phenol degradation genes of Pseudomonas putida strain H; Herrmann H et al.; The genetic organization of the DNA region encoding the phenol degradation pathway of Pseudomonas putida H has been investigated . This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy . The first step in this process is the conversion of phenol into catechol . Catechol is then further metabolized via the meta-cleavage pathway into TCA cycle intermediates . Genes encoding these enzymes are clustered on the plasmid pPGH1 . A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation . The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon . This codes for a multicomponent phenol hydroxylase and meta-cleavage pathway enzymes . Catabolic genes are subject to positive control by the gene product(s) of a second locus. Gene, 1995 Apr 14, 156(1), 11 - 8 The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria; Asturias JA et al.; The Gram+ bacterium Rhodococcus globerulus P6 (RgP6) catabolizes a range of polychlorinated biphenyl (PCB) congeners, thus being of interest in bioelimination processes for PCB . The first step in the pathway, a dioxygenase attack of one of the biphenyl (BP) rings, is catalyzed by biphenyl dioxygenase (BDO) . In this study, the nucleotide (nt) sequences of the four clustered cistrons, bphA1A2A3A4, encoding the subunits of BDO and forming part of the bph operon of RgP6 for BP degradation, were determined . A conserved motif proposed to bind a Rieske-type {2Fe-2S} cluster was identified in the deduced amino acid (aa) sequence of both the a subunit of the terminal oxygenase (BphA1) and ferredoxin (BphA3) . The ferredoxin reductase subunit (BphA4) contains conserved sites for FAD and NADH binding . Deduced aa sequences of the BDO subunits shared homologies to multicomponent aromatic ring-hydroxylating dioxygenases from Gram- microorganisms . Stronger identity was found to toluene dioxygenase (TDO) of Pseudomonas putida F1 than to other BDO . Aa sequence comparisons suggest that BP degradation genes of RgP6 may have originated in Gram- microorganisms, probably Pseudomonas, and subsequently transferred to this Gram+ bacterium. Can J Microbiol, 1995 Apr-May, 41(4-5), 354 - 65 Characterization of the pTFI91-family replicon of Thiobacillus ferrooxidans plasmids; Chakravarty L et al.; Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication . Four strains yielded an identical 9.8-kb plasmid, pTFI91 . Restriction mapping and Southern blot hybridization analysis were used to confirm this finding . Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91 . DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons . A comparison of the pTFI91 origin with those of T . ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences . To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5 alpha cells but no transformants were identified . To test the replication of pTFI91 independent of DNA polymerase I in E . coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected . Electrotransformation of T . ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants . The results suggested that the pTFI91 plasmid replicon does not function either in E . coli or in P . putida . Since pTFI91 contains the same origin of replication as other plasmids in several other T . ferrooxidans strains, this replicon may be commonly distributed in T . ferrooxidans. J Bacteriol, 1995 Apr, 177(7), 1850 - 9 A carbon starvation survival gene of Pseudomonas putida is regulated by sigma 54; Kim Y et al.; By using mini-Tn5 transposon mutagenesis, two mutants of Pseudomonas putida ATCC 12633 were isolated which showed a marked increase in their sensitivity to carbon starvation; these mutants are presumably affected in the Pex type of proteins that P . putida induces upon carbon starvation (M . Givskov, L . Eberl, and S . Molin, J . Bacteriol . 176:4816-4824, 1994) . The affected genes in our mutants were induced about threefold upon carbon starvation . The promoter region of the starvation gene in the mutant MK107 possessed a strong sigma 54-type-promoter sequence, and deletion analysis suggested that this was the major promoter regulating expression; this was confirmed by transcript mapping in rpoN+ and rpoN mutant backgrounds . The deletion analysis implicated a sequence upstream of the sigma 54 promoter, as well as a region downstream of the transcription start site, in the functioning of the promoter . Two sigma 70-type Pribnow boxes were also detected in the promoter region, but their transcriptional activity in the wild type was very weak . However, in a sigma 54-deficient background, these promoters became stronger . The mechanism and possible physiological role of this phenomenon and the possibility that the sequence upstream of the sigma 54 promoter may have a role in carbon sensing are discussed. J Bacteriol, 1995 Apr, 177(7), 1751 - 9 Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme; Schurr MJ et al.; The nucleotide sequences of the genes encoding the enzyme aspartate transcarbamoylase (ATCase) from Pseudomonas putida have been determined . Our results confirm that the P . putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately from overlapping genes . The P . putida ATCase does not possess dissociable regulatory and catalytic functions but instead apparently contains the regulatory nucleotide binding site within a unique N-terminal extension of the pyrB-encoded subunit . The first gene, pyrB, is 1,005 bp long and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the enzyme . The second gene is 1,275 bp long and encodes a 424-residue polypeptide which bears significant homology to dihydroorotase (DHOase) from other organisms . Despite the homology of the overlapping gene to known DHOases, this 44.2-kDa polypeptide is not considered to be the functional product of the pyrC gene in P . putida, as DHOase activity is distinct from the ATCase complex . Moreover, the 44.2-kDa polypeptide lacks specific histidyl residues thought to be critical for DHOase enzymatic function . The pyrC-like gene (henceforth designated pyrC') does not complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs . The proposed function for the vestigial DHOase is to maintain ATCase activity by conserving the dodecameric assembly of the native enzyme . This unique assembly of six active pyrB polypeptides coupled with six inactive pyrC' polypeptides has not been seen previously for ATCase but is reminiscent of the fused trifunctional CAD enzyme of eukaryotes. Arch Microbiol, 1995 Apr, 163(4), 282 - 5 Cytological aspects of resistance to potassium tellurite conferred on Pseudomonas cells by plasmids; Suzina NE et al.; The ultrastructure of strains Pseudomonas putida BS228 and Pseudomonas aeruginosa ML4262 harboring plasmids pBS10, pBS31, and pBS221, which determine resistance to potassium tellurite, was studied . Bacteria were grown in media containing increasing concentrations of potassium tellurite . Crystalline structures containing tellurium appeared in their periplasmic space . The dynamics of crystal growth was studied . Crystals were released into the medium by pinching off of the outer membrane vesicles containing growing crystals . A possible mechanism of this process was described; cytobiochemical peculiarities were discussed. Eur J Biochem, 1995 Apr 1, 229(1), 284 - 90 Purification and characterization of an ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, from Pseudomonas putida 77; Ogawa J et al.; N-Methylhydantoin amidohydrolase, an ATP-dependent amidohydrolase involved in microbial degradation of creatinine, was purified 70-fold to homogeneity, with a 62% overall recovery, and was crystallized from Pseudomonas putida 77 . The enzyme has a relative molecular mass of 300,000 . It is a tetramer of two identical small subunits (M(r) 70,000) and two identical large subunits (M(r) 80,000) . The enzyme requires ATP for the amidohydrolysis of N-methylhydantoin and vice versa . Mg2+, Mn2+ or Co2+, and K+, NH4+, Rb+ or Cs+, were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively . The Km and Vmax values for N-methylhydantoin were 32 microM and 9.0 mumol.min-1.mg protein-1 . The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, DL-5-methylhydantoin, glutarimide and succimide in addition to N-methylhydantoin . 2-Pyrrolidone, 2-oxazolidone, delta-valerolactam, 2,4-thiazolidinedione, 2-imidazolidone, D-5-oxoproline methyl ester, DL-5-oxoproline methyl ester, and naturally occurring pyrimidine compounds, i.e . dihydrouracil, dihydrothymine, uracil, and thymine, effectively stimulated ATP hydrolysis by the enzyme without undergoing detectable self-hydrolysis. Eur J Biochem, 1995 Apr 1, 229(1), 113 - 8 Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida; Cerdan P et al.; The substrate specificities of two catechol 2,3-dioxygenases, one encoded by xylE on the TOL plasmid pWW0 and the other encoded by nahH on the NAH7 plasmid, were investigated . The XylE catechol 2,3-dioxygenase catalyzes the ring-cleavage of catechol, 3-methylcatechol and 4-methylcatechol . The NahH catechol 2,3-dioxygenase was partially deficient in oxidizing 3-methylcatechol due to defects in two catalytic properties . First, NahH has a lower kcat value for 3-methylcatechol compared to XylE, and secondly, NahH is more susceptible than XylE to suicide inhibition by 3-methylcatechol . To identify the amino acid residues of XylE and NahH responsible for the differences in the efficacy of the 3-methylcatechol oxidation, kcat and kinact (the rate constant for suicide inhibition) for 3-methylcatechol were determined for several NahH-XylE hybrid proteins, each of which consisted of the NahH sequence in the N-terminal region and the XylE sequence in the C-terminal region . It is shown that a single amino acid substitution present in the NahH sequence, His250-->Gln, was responsible for the reduced kcat and increased kinact values for 3-methylcatechol . In addition to the substitution at residue 250, some substitution(s) at residues 77-102 were responsible for the twofold difference in the kinact values for NahH and XylE with 3-methylcatechol . We also show that the binding site of 3-methylcatechol for suicide inhibition is different from the catalytic site. J Bacteriol, 1995 Apr, 177(8), 2230 - 5 A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C; Coque JJ et al.; Two genes, cmcI and cmcJ, corresponding to open reading frames 7 and 8 (ORF7 and ORF8) of the cephamycin C cluster of Nocardia lactamdurans encode enzymes that convert cephalosporin C to 7-methoxycephalosporin C . Proteins P7 and P8 (the products of ORF7 and ORF8 expressed in Streptomyces lividans) introduce the methoxyl group at C-7 of the cephem nucleus . Efficient hydroxylation at C-7 and transfer of the methyl group from S-adenosylmethionine require both proteins P7 and P8, although P7 alone shows weak C-7 hydroxylase activity and strong cephalosporin-dependent NADH oxidase activity . Both P7 and P8 appear to be synthesized in a coordinated form by translational coupling of cmcI and cmcJ . Protein P7 contains domains that correspond to conserved sequences in cholesterol 7 alpha-monooxygenases and to the active center of O-methyltransferases by comparison with the crystal structure of catechol-O-methyltransferase . Protein P8 may act as a coupling protein for efficient hydroxylation at C-7 in a form similar to that of the two-component system of Pseudomonas putida p-hydroxyphenylacetate-3-hydroxylase. J Biochem (Tokyo), 1995 Apr, 117(4), 830 - 5 Production and application of monoclonal antibodies specific to pyrroloquinoline quinone; Narita H et al.; We produced five monoclonal antibodies (mAbs 1, 2, 6, 7, and 9) that are specific to pyrroloquinoline quinone (PQQ) . PQQ-conjugated hemocyanin was used for the immunization of mice and the hybridomas were selected using PQQ-conjugated BSA in an enzyme-linked immunosorbent assay . MAbs 2 and 9 were of the IgG1 isotype . Both could recognize free PQQ, the former probably at the o-quinone and the latter at the opposite side of the molecule . They did not bind with trihydroxyphenylalanine, dihydroxyphenylalanine, 1,2,4-trihydroxybenzene, ascorbic acid, riboflavin, or menadione . In contrast to the IgGs, mAbs 1, 6, and 7 (IgMs) did not bind with free PQQ . Using mAb 2, a competitive enzyme-linked immunosorbent assay was developed, which enabled us to determine 50 nM-1 microM free PQQ . Furthermore, we analyzed the covalently bound prosthetic groups of two quinoproteins (amine oxidase from Aspergillus niger and amine dehydrogenase from Pseudomonas putida) by Western analysis using these mAbs . However, the results was negative, indicating that the prosthetic groups are not PQQ. Mol Microbiol, 1995 Apr, 16(2), 205 - 13 Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains; Fernandez S et al.; In the presence of toluene, xylenes and other structural analogues, the regulatory protein XylR, of the family of transcriptional regulators which act in concert with the sigma 54 factor, activate the promoter Pu of the TOL (toluene degradation) plasmid pWWO of Pseudomonas putida . Amino acid changes Val-219-Asp and Ala-220-Pro, introducing a proline kink at the hinge region between the N-terminal A domain and the central portion of XylR, resulted in a semi-constitutive phenotype which mimicked the activating effect of aromatic inducers . This phenotype was further exacerbated by inserting extra amino acid residues within the same inter-domain region . A truncated XylR protein devoid of the signal-receiving, amino-terminal portion of the protein stimulated the cognate promoter Pu at high levels independently of inducer addition, both in Escherichia coli and in Pseudomonas putida . Replacement of the amino-terminal domain by a heterologous peptide derived from the MS2 virus polymerase resulted in a hybrid protein still able to bind DNA to the same extent in vivo as XylR, but unable to stimulate transcription . These data indicate that a key event in the activation of XylR by toluene/xylenes is the release of the repression caused by the A domain of the protein on surfaces located at the central domain of the regulator. Gene, 1995 Mar 21, 155(1), 83 - 8 Alginate regulatory and biosynthetic gene homologs in Pseudomonas putida WCS358: correlation with the siderophore regulatory gene pfrA; Venturi V et al.; A previous study {Venturi et al., Mol . Microbiol . 10 (1993) 63-73} demonstrated that the siderophore regulatory gene pfrA of Pseudomonas putida (Pp) WCS358 is highly similar and interchangeable with the alginate regulatory gene algQ (algR2) of P . aeruginosa (Pa) . The algQ gene is physically linked to two other alginate regulators in the Pa chromosome, namely algR (algR1), a response regulator, and algP (algR3), a histone-like gene . In this study, we have identified the same genes and a similar genetic organization in the Pp chromosome . The two genes linked to pfrA, designated pprA and pprB, are similar to algR and algP, respectively . Chromosomal mutants of pprA and pprB were constructed showing that unlike pfrA, the two newly identified regulators are not involved in siderophore regulation . The pprA gene complemented a Pa algR mutant phenotype, suggesting that it could be involved in alginate gene regulation . The WCS358 strain is not producing alginate, but we demonstrated by Southern analysis that it also possesses, in addition to pprA and pprB, algD and algU (algT) gene homologs, two genes essential for alginate biosynthesis . Using an algD-xylE transcriptional fusion, we observed that the algD promoter is active in strain WCS358 and absolutely requires pfrA . The possibility that all five genes of Pp WCS358 are involved in alginate biosynthesis is discussed. J Biol Chem, 1995 Mar 17, 270(11), 6403 - 411 Identification of functional residues in a 2-hydroxymuconic semialdehyde hydrolase . A new member of the alpha/beta hydrolase-fold family of enzymes which cleaves carbon-carbon bonds; Diaz E et al.; The 2-hydroxymuconic semialdehyde hydrolase, XylF, of the Pseudomonas putida TOL plasmid-encoded pathway for the catabolism of toluene and xylenes, catalyzes one of the rarest types of enzyme reaction (EC 3.7.1.9), the hydrolysis of a carbon-carbon bond in its substrate, the ring-fission product of 3-alkyl-substituted catechols . In this study, amino acid sequence comparisons between XylF and other hydrolases, and analysis of the similarity between the predicted secondary structure of XylF and the known secondary structure of the haloalkane dehalogenase from Xanthobacter autotrophicus strain GJ10, led us to identify several conserved residues likely to have a functional role in the catalytic center of XylF . Three amino acids, Ser107, Asp228, and His256, were found to be arranged in a sequential order similar to that in alpha/beta hydrolase-fold enzymes . Investigations of the potential functional role of these and other residues through amino acid modification and in vitro site-directed mutagenesis experiments provided evidence in support of the hypothesis that XylF is a serine hydrolase of the alpha/beta hydrolase-fold family of enzymes, and pointed to the residues identified above as the catalytic triad of XylF . These studies also provided information on other conserved residues in XylF-related enzymes . Interestingly, the substitution of Phe by Met in position 108 of XylF created an enzyme with increased thermostability and altered substrate specificity. J Biol Chem, 1995 Mar 10, 270(10), 5144 - 50 Single amino acids changes in the signal receptor domain of XylR resulted in mutants that stimulate transcription in the absence of effectors; Delgado A et al.; The XylR protein positively controls expression from the Pseudomonas putida TOL plasmid sigma 54-dependent "upper" pathway operon promoter (Pu) and the xylS gene promoter (Ps), in response to the presence of aromatic effectors . Two mutant XylR regulators able to stimulate transcription from Pu and Ps in the absence of effectors were isolated . These mutants exhibited single point mutations, namely Asp135-->Asn and Pro85-->Ser . Both mutations are located in the amino termini domain of XylR, which is thought to be responsible for interactions with effectors . The effector profile of XylRP85S was similar to that of wild-type XylR protein; however, XylRD135N exhibited an altered pattern of effector recognition: with m-nitrotoluene it stimulated transcription from the Pu promoter above the high basal level, whereas this nitroarene inhibited the wild-type regulator . Previous work (Delgado, A., and Ramos, J.L . (1994) J . Biol . Chem . 269, 8059-8062) showed that residue 172 was involved in effector interactions, as mutant XylRE172K also recognized m-nitrotoluene . However, double mutant XylR135N/E172K did not stimulate transcription in the absence of effector, but retained the ability to stimulate transcription with m-nitrotoluene . Transcription mediated by XylRD135N and XylRP85S from Pu::lacZ was analyzed in detail . Like the wild-type regulator, XylRD135N and XylRP85S required sigma 54 for full transcription activation, but in contrast with the wild-type regulator, XylRD135N, but not XylRP85S, stimulated transcription from Pu in the absence of the integration host factor protein . XylRD135N, also in