J Bacteriol, 2001 Jul, 183(14), 4269 - 77 trans-3-Chloroacrylic acid dehalogenase from Pseudomonas pavonaceae 170 shares structural and mechanistic similarities with 4-oxalocrotonate tautomerase; Poelarends GJ et al.; The genes (caaD1 and caaD2) encoding the trans-3-chloroacrylic acid dehalogenase (CaaD) of the 1,3-dichloropropene-utilizing bacterium Pseudomonas pavonaceae 170 were cloned and heterologously expressed in Escherichia coli and Pseudomonas sp . strain GJ1 . CaaD is a protein of 50 kDa that is composed of alpha-subunits of 75 amino acid residues and beta-subunits of 70 residues . It catalyzes the hydrolytic cleavage of the beta-vinylic carbon-chlorine bond in trans-3-chloroacrylic acid with a turnover number of 6.4 s(-1) . On the basis of sequence similarity, oligomeric structure, and subunit size, CaaD appears to be related to 4-oxalocrotonate tautomerase (4-OT) . This tautomerase consists of six identical subunits of 62 amino acid residues and catalyzes the isomerization of 2-oxo-4-hexene-1,6-dioate, via hydroxymuconate, to yield 2-oxo-3-hexene-1,6-dioate . In view of the oligomeric architecture of 4-OT, a trimer of homodimers, CaaD is postulated to be a hexameric protein that functions as a trimer of alpha beta-dimers . The sequence conservation between CaaD and 4-OT and site-directed mutagenesis experiments suggested that Pro-1 of the beta-subunit and Arg-11 of the alpha-subunit are active-site residues in CaaD . Pro-1 could act as the proton acceptor/donor, and Arg-11 is probably involved in carboxylate binding . Based on these findings, a novel dehalogenation mechanism is proposed for the CaaD-catalyzed reaction which does not involve the formation of a covalent enzyme-substrate intermediate. Crit Rev Oncol Hematol, 2001 Jul-Aug, 39(1-2), 87 - 98 Molecular targeting of malignant gliomas with novel multiply-mutated interleukin 13-based cytotoxins; Nash KT et al.; A vast majority of high-grade gliomas over-express a receptor for interleukin 13 (IL13) . This glioma-associated receptor for IL13 is interleukin 4 (IL4)-independent . This is in contrast to the physiological and IL4-shared receptor for the IL13, IL13/4 receptor, which is found on many normal organs . IL13-based Pseudomonas exotoxin (PE)-containing cytotoxic fusion proteins have been shown to be very potent anti-glioma agents . However, native IL13-based cytotoxins interact with both forms of the IL13 receptor . Therefore, mutations in IL13 were made in order to diminish/eliminate IL13's interaction with the shared IL13/4 receptor of normal tissue . These mutations encompassed amino acids located on alpha-helix A and C of IL13 . We have engineered double or triple mutants of IL13 linked to various forms of PE . We found that these mutations could be successfully incorporated into IL13 without the loss of the protein's ability to selectively deliver the toxin to glioma cells while reducing their toxicity. Microbios, 2001, 105(412), 133 - 40 Effect of culture medium pH on bacterial gellan production; West TP et al.; The effect of the initial pH of the culture medium used in the production of the exopolysaccharide gellan by the bacterium Pseudomonas species ATCC 31461, when glucose or corn syrup served as the carbon source, was investigated . With glucose as the carbon source, exopolysaccharide formation was highest after 72 h of growth when the initial pH of the culture medium was 6.8 to 7.4 . Polysaccharide production by the bacterial cells grown on corn syrup for 72 h was maximal when the initial pH of the medium was 7.0 or 7.2 . Cell weights of the strain after 72 h tended to be higher for the glucose-grown cells than for the corn syrup-grown cells. Biochemistry, 2001 Jun 26, 40(25), 7404 - 9 Influence of the aglycone region of the substrate binding cleft of Pseudomonas xylanase 10A on catalysis; Armand S et al.; Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substrate binding cleft comprising three glycone (-1 to -3) and four aglycone (+1 to +4) subsites and, typical of retaining glycoside hydrolases, exhibits transglycosylation activity at elevated substrate concentrations . In a previous study {Charnock, S . J., et al . (1997) J . Biol . Chem . 272, 2942-2951}, it was demonstrated that the -2 subsite mutations E43A and N44A caused a 100-fold reduction in activity against xylooligosaccharides, but did not influence xylanase activity . This led to the proposal that the low activity of these mutants against xylooligosaccharides was due to nonproductive complex formation between these small substrates and the extended aglycone region of the active site . To test this hypothesis, key residues at the +2 (Asn182), +3 (Tyr255), and +4 (Tyr220) subsites were substituted for alanine, and the activity of the mutants against polysaccharides and oligosaccharides was evaluated . All the aglycone mutants exhibited greatly reduced or no transglycosylating activity, and the triple mutants, E43A/Y220A/Y255A and E43A/N182A/Y255A, had activity against xylotriose similar to that of E43A . The aglycone mutations caused an increase in both k(cat) and K(m) against xylan, with N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and 15-fold higher k(cat) values, respectively, than wild-type Pc Xyn10A . These data indicate that Glu43 plays a role in binding xylooligosaccharides, but not xylan, suggesting that the mechanisms by which Pc Xyn10A binds polysaccharides and oligosaccharides are distinct . The increased k(cat) of the mutants against xylan indicates that the aglycone region of wild-type Pc Xyn10A restricts the rate of catalysis by limiting diffusion of the cleaved substrate, generated at the completion of the k(2) step, out of the active site. Curr Microbiol, 2001 Sep, 43(3), 176 - 81 Adaptive and cross-protective responses of Pseudomonas sp . DJ-12 to several aromatics and other stress shocks; Park SH et al.; Pseudomonas sp . DJ-12 cells were subjected to mild treatments of stress such as exposure to biphenyl, 4-chlorobiphenyl (4CB), 4-hydroxybenzoate (4HBA), ethanol, and heat, and then were examined for production of stress-shock proteins and morphological changes . The adapted cells were then subjected to lethal stress conditions such as 200 mm 4CB, 100 mm biphenyl, 10 mm 4HBA, 20% ethanol, and 46 degrees C to examine crossly protective responses to the stresses . Several stress-shock proteins including DnaK and GroEL were newly synthesized in the adapted cells . Some of them were commonly produced by those stresses separately treated . The cells treated with these aromatic hydrocarbons showed destructive openings on the cell envelopes . On the other hand, those cells treated with ethanol or heat displayed irregular rod shapes with wrinkled surfaces . The adapted cells to each stress under sublethal conditions exhibited increased resistance to the same stress of lethal conditions . The cells adapted with 5 mm 4HBA showed greater protection for survival than those adapted by other stresses . In addition, those adapted cells showed increased resistance to other stresses as a cross-protection phenomenon . The cells adapted to 42 degrees C exhibited markedly increased resistance to the lethal stresses of 46 degrees C as well as to 20% ethanol. J, Exp . Mar . Biol . Ecol. . 2001 Jul 1, 261(2), 225 - 235 Isolation and identification of an ice-nucleating bacterium from the gills of the intertidal bivalve mollusc Geukensia demissa; Loomis SH et al.; In the fall, freeze tolerant intertidal invertebrates usually produce ice-nucleating proteins that are secreted into the hemolymph . These proteins help protect against freeze damage by insuring that ice formation is limited to extracellular spaces . Geukensia demissa, a freeze tolerant, salt marsh bivalve mollusc was examined for the presence of ice nucleating proteins . The ice-nucleating temperature (INT) of the hemolymph was not significantly different from artificial seawater of the same salinity indicating the lack of an ice nucleating protein in the hemolymph . The palial fluid did have an elevated INT, indicating the presence of an ice nucleator . The INT of the palial fluid was significantly reduced by boiling and filtration through a 0.45-&mgr;m filter . High INT was also observed in the seawater associated with the bivalves, and was demonstrated in water samples collected from salt marshes but not sand and pebble beaches . Moreover, the INT of water samples collected from a salt marsh decreased in the summer . All of these data suggest that the ice-nucleating agents in the hemolymph and the seawater are ice-nucleating bacteria . One species of ice-nucleating bacteria, Pseudomonas fulva was isolated from the gills of Geukensia . These bacteria could perform the same function as hemolymph ice-nucleating proteins by limiting ice formation to extracellular compartments. Antonie Van Leeuwenhoek, 2001 Jan, 79(1), 61 - 71 Identification and expression of the Pseudomonas syringae pv . aptata hrpZ(Psa) gene which encodes an harpin elicitor; Musa AR et al.; A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv . syringae hrpZ gene was identified in Pseudomonas syringae pv . aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants . and in E . coli DH10B (pCCP1069) containing the P . syringae pv . aptata hrp gene cluster . PCR with oligonucleotides, based on the hrpZ(Pss) gene and used as primers with the total genomic DNA of P . syringae pv . aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions . The amplicon was cloned into the pGEM-T plasmid vector, amplified in E . coli DH5alpha and sequenced . The sequence showed 95%, 83% and 61% identity with those of hrpZ(Pss), hrpZ(Psg and hrpZ(Pst) genes encoding the harpins of the P . syringae pv . syringae, glycinea and tomato, respectively . The amplicon was cloned into the pMAL expression system . The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography . On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P . syringae pv . aptata harpin. Toxicon, 2001 Sep, 39(9), 1283 - 90 Divergent Pseudomonas exotoxin A sensitivity in normal and transformed liver cells is correlated with low-density lipoprotein receptor-related protein expression; Laithwaite JE et al.; Pseudomonas exotoxin A (PEA) is an extracellular virulence factor produced by the opportunistic human pathogen Pseudomonas aerguinosa . PEA intoxification begins when PEA binds to the low-density lipoprotein receptor-related protein (LRP) . The liver is the primary target of systemic PEA, due largely to the high levels of functional LRP expressed by liver cells . Using a 3H-leucine incorporation assay to measure inhibition of protein synthesis we have demonstrated that normal (BNL CL.2) and transformed (BNL 1ME A7R.1) liver cells exhibit divergent PEA sensitivity; with BNL 1ME A7R.1 cells demonstrating greater PEA sensitivity than their non-transformed counterparts . The receptor-associated protein, a LRP antagonist, decreased PEA toxicity in BNL 1ME A7R.1 cells, confirming the importance of the LRP in PEA intoxification in this cell type . Increased PEA sensitivity in BNL 1ME A7R.1 cells was associated with increased functional cell surface LRP expression, as measured by alpha2-macroglobulin binding and internalization studies, and increased LRP mRNA levels, as determined by Northern blot analysis . Interestingly, BNL CL.2 cells were more sensitive than BNL 1ME A7R.1 cells to conjugate and mutant PEA toxins that do not utilize the LRP for cellular entry . These data demonstrate that increased LRP expression is an important mechanism by which PEA sensitivity is increased in BNL 1ME A7R.1 transformed liver cells. J Biol Chem, 2001 Aug 17, 276(33), 31186 - 92 Epub 2001 May 29. Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysis of residues involved in substrate binding; Hogg D et al.; The crystal structure of Pseudomonas cellulosa mannanase 26A has been solved by multiple isomorphous replacement and refined at 1.85 A resolution to an R-factor of 0.182 (R-free = 0.211) . The enzyme comprises (beta/alpha)(8)-barrel architecture with two catalytic glutamates at the ends of beta-strands 4 and 7 in precisely the same location as the corresponding glutamates in other 4/7-superfamily glycoside hydrolase enzymes (clan GH-A glycoside hydrolases) . The family 26 glycoside hydrolases are therefore members of clan GH-A . Functional analyses of mannanase 26A, informed by the crystal structure of the enzyme, provided important insights into the role of residues close to the catalytic glutamates . These data showed that Trp-360 played a critical role in binding substrate at the -1 subsite, whereas Tyr-285 was important to the function of the nucleophile catalyst . His-211 in mannanase 26A does not have the same function as the equivalent asparagine in the other GH-A enzymes . The data also suggest that Trp-217 and Trp-162 are important for the activity of mannanase 26A against mannooligosaccharides but are less important for activity against polysaccharides. Kidney Int, 2001 Jun, 59(6), 2309 - 15 Clinical course of peritonitis due to Pseudomonas species complicating peritoneal dialysis: a review of 104 cases; Szeto CC et al.; BACKGROUND: Peritonitis due to Pseudomonas species is a serious complication in continuous ambulatory peritoneal dialysis (CAPD) patients . The clinical course of peritonitis due to Pseudomonas complicating CAPD remains unclear . METHODS: All of the Pseudomonas species episodes of peritonitis in our dialysis unit were studied from 1995 to 1999 . During this period, there were 859 episodes of peritonitis recorded, 113 of which were caused by the Pseudomonas species . Nine episodes were excluded because they were mixed growth . The remaining 104 episodes in 68 patients were reviewed . RESULTS: The underlying renal diagnosis and prevalence of comorbid conditions of the 68 patients were similar to those found in our entire dialysis population . There was a history of antibiotic therapy within 30 days of the onset of peritonitis due to the Pseudomonas species in 69 episodes (66.3%) . In 47 episodes (45.2%) there was a concomitant exit site infection . The overall primary response rate was 60.6% and the complete cure rate was 22.1% . The presence of exit site infection was associated with a lower primary response rate (22 in 47 vs . 41 in 57 episodes, P < 0.01) and a lower complete cure rate (5 in 47 vs . 18 in 57 episodes, P < 0.02) . The episodes that had received recent antibiotic therapy had a significantly lower complete cure rate than the de novo cases (8 in 69 vs . 15 in 35 episodes, P < 0.001) . Episodes receiving third-generation cephalosporin as part of the initial antibiotic regimen had a significantly higher primary response rate than the ones that initially received aminoglycoside (54 in 81 episodes vs . 8 in 22 episodes, P < 0.05), but their complete cure rates were similar . Twenty-four cases failed to respond to antibiotics and the Tenckhoff catheter was removed . The chance of returning to CAPD was higher when the Tenckhoff catheter was removed on day 10 than on day 15 (9 in 14 cases vs . 5 in 10 cases), although the result was not statistically significant . The Tenckhoff catheter was removed and replaced at another site simultaneously in another 14 cases after the effluent cleared up . None of these patients had a relapse of peritonitis within three months . CONCLUSIONS: Recent antibiotic therapy is the major risk factor for peritonitis due to the Pseudomonas species . Exit site infection and recent antibiotic therapy are associated with poor therapeutic response to antibiotics . When the therapeutic response is suboptimal, early Tenckhoff catheter removal may help preserve the peritoneum for further peritoneal dialysis . Elective Tenckhoff catheter exchange after clearing up the peritoneal dialysis effluent may also reduce the likelihood of relapse . It is desirable to use third-generation cephalosporin in the initial antibiotic regimen for peritonitis treatment in localities with a high incidence of peritonitis due to the Pseudomonas species. Water Sci Technol, 2001, 43(2), 261 - 9 Kinetics of azoreductase and assessment of toxicity of metabolic products from azo dyes by Pseudomonas luteola; Hu TL; This is a continuous study on a decolorization strain, Pseudomonas luteola, which involves treating seven azo dyes with different structures . This study focuses mainly on determining both the mechanism of decolorization by P . luteola and the activity of azoreductase from P . luteola as well as identifying and assessing the toxicity of metabolic products of azo dyes . The growth of P . luteola reached the stationary phase after shaking incubation for 24 hours . Then, while being kept static, the color of seven tested azo dyes (100 mg/l) could be removed . The proportion of color removal was between 59-99%, which figure is related to the structure of the dye . Monoazo dyes (RP2B, V2RP and Red 22) showed the fastest rate of decolorization, i.e . from 0.23-0.44 mg dye-mg cell-1 hr-1 . P . luteola could remove the color of V2RP and a leather dye at a concentration of 200 mg/l, and as to the rest of the azo dyes, it could remove at a concentration of up to 100 mg/l . Decolorization of RP2B and Red 22 required activation energy of 7.00 J/mol and 6.63 J/mole, respectively, indicating that it was easier for azoreductase to decolorize structurally simple dyes . The kinetics of azoreductase towards seven azo dyes suggested a competitive inhibition model be applied . Microtox was used to analyze the toxicity of the metabolic products of azo dyes . EC50 showed differences in toxicity before and after the azo dyes had been metabolized . Analysis revealed significant differences between the results obtained by EC50 with Blue 15 and those obtained with the leather dye, indicating that the toxicities of the metabolic products were increased . The differences obtained by EC50 with Red 22, RP2P and V2RP were small, and Black 22 showed no such difference . Sulfanic acid and orthanilic acid may be the intermediate products of Violet 9 and RP2B, respectively . However, according to FT-IR analysis, aromatic amines were present in the metabolic product. J Bacteriol, 2001 Jun, 183(12), 3752 - 60 Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp . strain BH72; Egener T et al.; The endophytic diazotroph Azoarcus sp . strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there . In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced . The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R . capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located . Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a sigma(54)-type promoter . Shorter transcripts sequentially missing genes of the 3' part of the full-length mRNA were more abundantly detected . Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase . An in-frame deletion of fdxN resulted in reduced growth rates (59% +/- 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type . Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans . Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level . Surprisingly, the fdxN deletion also had an effect on the rapid "switch-off" of nitrogenase activity in response to ammonium . Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene . In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process. J Bacteriol, 2001 Jun, 183(12), 3663 - 79 Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp . strain CA10; Nojiri H et al.; The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp . strain CA10 were determined . Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate . The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE . This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus . In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found . Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation . Therefore, these ORFs were designated antABC . Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes . IS5car2 and IS5car3 constituted the putative composite transposon containing antABC . One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9 . In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed . In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed . In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1. Biophys J, 2001 Jun, 80(6), 2928 - 34 Solution conformation of the Met 61 to His 61 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551; Miller GT et al.; The gene encoding for bacterial cytochrome c-551 from Pseudomonas stutzeri substrain ZoBell has been mutated to convert the invariant sixth ligand methionine residue into histidine, creating the site-specific mutant M61H . Proton NMR resonance assignments were made for all main-chain and most-side chain protons in the diamagnetic, reduced form at pH 9.2 and 333 K by two-dimensional NMR techniques . Distance constraints (1074) were determined from nuclear Overhauser enhancements and main-chain torsion-angle constraints (72) from scalar coupling estimates . Solution conformations for the protein were computed by the simulated annealing approach . For 28 computed structures, the root mean squared displacement from the average structure excluding the terminal residues 1, 2, 81, and 82 was 0.52 A (sigma = 0.096) for backbone atoms and 0.90 A (sigma = 0.122) for all heavy atoms . The global folding of the mutant protein is the same as for wild type . The biggest changes are localized in a peptide span over residues 60-65 . The most striking behavior of the mutant protein is that at room temperature and neutral pH it exists in a state similar to the molten globular state that has been described for several proteins under mild denaturing conditions, but the mutant converts to a more ordered state at high pH and temperature. Z Naturforsch {C}, 2001 Mar-Apr, 56(3-4), 308 - 10 Ferribactins as the biosynthetic precursors of the Pseudomonas siderophores pyoverdins; Hohlneicher U et al.; By a feeding experiment with a 15N-labelled precursor it is shown that ferribactins are transformed into pyoverdins and thus are their biogenetic precursors. Biometals, 2001 Mar, 14(1), 81 - 4 The structure of a pyoverdine produced by a Pseudomonas tolaasii-like isolate; Fernandez DU et al.; Cultures of Agaricus bisporus, the most extensively cultivated mushroom, can be infected severely by Pseudomonas tolaasii . This pathogen is characterized by the so-called white line reaction, a precipitate formed on agar plates between its colonies and those of P . reactans, both belonging to the collective species P . fluorescens . A recent study has shown that a group of P . tolaasii isolates can be subdivided into two groups or 'siderovars', based on the pyoverdines they produce (Munsch et al . 2000) . One group of strains is characterized by the pyoverdine described by Demange et al . (1990) . A representative of the second group (strain Ps3a) was found to produce the same pyoverdine as a strain which had been classified before as P . aureofaciens . However, based mainly on 16S rRNA gene sequence comparisons and REP-PCR generated fingerprints, the two strains are not identical . They are also distinguishable from the P . tolaasii type strain. Plant J, 2001 Apr, 26(1), 101 - 12 The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily; Swiderski MR et al.; Specific recognition of Pseudomonas syringae strains that express the avirulence gene avrPphB requires two genes in Arabidopsis, RPS5 and PBS1 . Previous work has shown that RPS5 encodes a member of the nucleotide binding site-leucine rich repeat class of plant disease resistance genes . Here we report that PBS1 encodes a putative serine-threonine kinase . Southern blot analysis revealed that the pbs1-1 allele contained a deletion of the 3' end of the PBS1 open reading frame . DNA sequence analysis of the pbs1-2 allele showed it to be a missense mutation that caused a glycine to arginine substitution in the activation segment of PBS1, a region known to regulate substrate binding and catalytic activity in many protein kinases . The identity of PBS1 was confirmed using both transient transformation and stable transformation of mutant pbs1 plants . Comparison of the predicted PBS1 amino acid sequence with other plant protein kinases revealed that PBS1 belongs to a distinct subfamily of protein kinases that contains no other members of known function . The Pto kinase of tomato, which is required for specific resistance to P . syringae strains expressing avrPto, did not fall in the same subfamily as PBS1 and is only 42% identical in the kinase domain . These data suggest that PBS1 and Pto may fulfil different functions in the recognition of pathogen avirulence proteins . We discuss several possible models for the roles of PBS1 and RPS5 in AvrPphB recognition. FEMS Microbiol Lett, 2001 May 15, 199(1), 119 - 23 Response of electrically stimulated cells of Pseudomonas oleovorans strain ATCC 29347 suspended in silicone oil; Anglade J et al.; A high intensity direct current was applied for more than 10 min onto a bacterial suspension of Pseudomonas oleovorans ATCC 29347 suspended in silicone oil . The application of a gradually increased electric field from 0 to 2500 V x cm(-1) resulted in a decrease of the optical density of the bacterial suspension and the occurrence of a peak current of several hundred microA for living cells instead of a linear increase (few microA) for killed or lyophilised cells . This procedure is not only a rapid way of investigating the living state of cell cultures but also an efficient experimental tool to study the cellular effects of a controlled electrical stress. Bioorg Med Chem, 2001 Apr, 9(4), 847 - 52 Homology model of the closed, functionally active, form of the amino terminal domain of mGlur1; Costantino G et al.; The amino terminal domain (ATD) of metabotropic glutamate receptors (mGluRs) contains the neurotransmitter binding site and is related in sequence to leucine/isoleucine/valine binding proteins (LIVBP) . It has been proposed that the ATD of mGluRs shares with periplasmic binding proteins a common mechanism of ligand binding and processing which involves the equilibrium between closed and open forms . The availability of the X-ray structure of LIVBP in its open, unliganded form, has allowed the construction of homology models of the ATD of mGluR1 which have been instrumental in clarifying the mode of binding of agonists and antagonists . We propose in this paper the use of the X-ray structure of AmiC . the controller of transcription antitermination in the amidiase operon of Pseudomonas aerugimosa as suitable template for the construction of the closed form of the ATD of mGluR1 . The resulting model of the closed form of the ATD of mGluR1 indicates that several interdomain hydrogen bonds and salt bridges may be formed upon domain contraction and that the ligand directly participates to this interdomain network. J Biol Chem, 2001 Jul 6, 276(27), 25114 - 20 Epub 2001 May 14. Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor alpha 2 chain; Kawakami K et al.; Interleukin (IL)-13 receptor alpha2 (IL-13Ralpha2) chain is an essential binding component for IL-13-mediated ligand binding . Recently, we have demonstrated that this receptor chain also plays an important role in the internalization of IL-13 . To study the mechanism of IL-13 internalization, we generated mutated IL-13Ralpha2 chains that targeted trileucine residues (Leu(335), Leu(336), and Leu(337)) in the transmembrane domain and a tyrosine motif (Tyr(343)) in the intracellular domain and transfected these cDNAs in COS-7 cells . Cells that expressed a C-terminally truncated IL-13Ralpha2 chain (Delta335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding . Truncation of IL-13Ralpha2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Ralpha2 chain transfected cells . COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Ralpha2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Ralpha2 transfectants . These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR) to cells that expressed IL-13Ralpha2 chain mutants . We further demonstrate that the IL-13Ralpha2 chain is not ubiquitinated and that internalization of IL-13Ralpha2 did not depend on ubiquitination . Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Ralpha2 internalization in distinct manners. Environ Sci Technol, 2001 Feb 1, 35(3), 552 - 9 Carbon tetrachloride dechlorination by the bacterial transition metal chelator pyridine-2,6-bis(thiocarboxylic acid); Lewis TA et al.; A reaction pathway is proposed to explain the formation of end products during defined chemical reactions between carbon tetrachloride (CCl4) and either metal complexes of pyridine-2,6-bis(thiocarboxylic acid) (PDTC) or pure cultures of Pseudomonas stutzeri KC . The pathway includes one-electron reduction of CCl4 by the Cu(II):PDTC complex, condensation of trichloromethyl and thiyl radicals, and hydrolysis of a labile thioester intermediate . Products detected were carbon dioxide, chloride, carbonyl sulfide, carbon disulfide, and dipicolinic acid . Spin-trapping and electrospray MS/MS experiments gave evidence of trichloromethyl and thiyl radicals generated by reaction of CCl4 with PDTC and copper . Experiments testing the effects of transition metals showed that dechlorination by PDTC requires copper and is inhibited by cobalt but not by iron or nickel . PDTC was shown to react stoichiometrically rather than catalytically without added reducing equivalents . With added reductants, an increased turnover was seen along with increased chloroform production. Int J Cancer, 2001 Jun 15, 92(6), 861 - 70 Isolation of new anti-CD30 scFvs from DNA-immunized mice by phage display and biologic activity of recombinant immunotoxins produced by fusion with truncated pseudomonas exotoxin; Rozemuller H et al.; To target CD30 on Hodgkin's disease and anaplastic large-cell lymphoma, anti-CD30 single-chain antibodies were obtained by DNA immunization of mice with the complete human CD30 cDNA . Spleens were isolated from mice with high anti-CD30 titer, and the RNA was used for the production of an scFv-displaying phage library . Specific phages were enriched by 3 rounds of panning on soluble CD30 or CD30+ K562 cells . Recombinant immunotoxins (rITs) were made from 3 ELISA-positive scFv phages by fusion to a 38 kDa truncated mutant of Pseudomonas exotoxin (PE38) with or without a KDEL mutant sequence at the C terminus . In vitro cytotoxicity of purified anti-CD30 rITs was measured on CD30-transfected A431 cells . IC50 values ranged from 3 to 7 ng/ml (50-110 pM) for PE38 rITs and 0.1 ng/ml (2 pM) for the PE38-KDEL IT on A431-CD30 cells . The parental A431 cells were resistant, indicating that the cytotoxicity was specific and CD30-mediated . rITs were tested for anti-tumor activity in a nude mouse model . A431-CD30 cells were injected s.c . on day 0; then, mice bearing measurable tumors were treated beginning on day 4 with 3 alternate daily doses i.v . Anti-tumor activity was dose-dependent and not found when irrelevant ITs were administered or when CD30- tumors were treated . Our data show that DNA immunization and antibody phage display may be useful in producing new rITs against hematologic malignancies . Published 2001 Wiley-Liss, Inc. Vet Microbiol, 2001 Jun 22, 80(4), 347 - 57 A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs; Chen JR et al.; Using the binding and translocation domain of Pseudomonas exotoxin A {domain III deleted PE termed PE(DeltaIII)} as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen . PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M . hyopneumoniae 97kDa adhesion were successfully fused to the downstream of PE(DeltaIII) to create a subunit vaccine, i.e . PE(DeltaIII)-RR1 . This chimeric protein, over-expressed in inclusion bodies of E . coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97kDa adhesin and was readily purified . The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(DeltaIII)-RR1 . Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(DeltaIII)-RR1 was found more marked than that in mice immunized with the M . hyopneumoniae whole cells . Similarly, PE(DeltaIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M . hyopneumoniae vaccine . The PE(DeltaIII)-RR1 would be potentially useful for the future development of a M . hyopneumoniae adhesin vaccine. Folia Microbiol (Praha), 2000, 45(4), 321 - 4 Development of immunomagnetic separation technique for isolation of Pseudomonas syringae pv . phaseolicola; Guven K et al.; Immunomagnetic separation technique was developed for specific detection and selective isolation of Pseudomonas syringae pv . phaseolicola, the agent of halo-blight disease of beans . Whole-cell and exopolysaccharide fraction of the bacterium was used for polyclonal antibody production in rabbits . High specificity of the antisera was determined in agglutination reactions . The optimum immunocapture time for both antisera was determined as 1 h by using 1/nL CFU (i.e . 10(6) CFU per mL) . No significant difference was observed in the binding capacity of cells to immunomagnetic particles with different antisera. Planta, 2001 Apr, 212(5-6), 792 - 8 Suppression of the ribosomal L2 gene reveals a novel mechanism for stress adaptation in soybean; Ludwig A et al.; Pseudomonas syringae pv . glycinea bacteria or zoospores of the fungus Phytophthora sojae were used to trigger a hypersensitive reaction (HR) in cell cultures of soybean (Glycine max {L.} Merr . cv . Williams 82) . During a screen for genes that show an altered expression as a response to dying neighbour cells we have identified a gene fragment that is specifically but transiently down-regulated in an HR . The corresponding cDNA codes for the ribosomal protein L2 (rpL2) of 80S ribosomes, which is essential for the peptidyl-transferase activity . Two gene copies of rpL2 exist in soybean and both genes are transcribed . The temporary down-regulation of the rpL2 genes is followed by a transient block in the synthesis of new proteins as visualised by pulse-labelling experiments using 35S-amino acids . The same basic phenomenon was also found after treatment of soybean cells with other stress-causing compounds such as elicitors or heavy metals . It is suggested that the transient block in protein synthesis allows a more rapid depletion of, for example, signal molecules with a short half-life time and thus leads to a faster adaptation of the cellular protein inventory to the new environmental conditions. J Bacteriol, 2001 Jun, 183(11), 3282 - 92 Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae; Li H et al.; In the plant pathogen Pseudomonas syringae pv . glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase . The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC . In this study, lscB and lscC were cloned from a genomic library of strain PG4180 . Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene . lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180 . Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis . Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC . Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180 . The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC . Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase . Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction . LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter . PCR screening in various P . syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P . syringae pathovars. Appl Microbiol Biotechnol, 2001 Apr, 55(3), 311 - 6 Enhanced degradation of naphthalene by immobilization of Pseudomonas sp . strain NGK1 in polyurethane foam; Manohar S et al.; A Pseudomonas sp . strain NGKI (NCIM 5120) capable of degrading naphthalene was immobilized in polyurethane foam . The naphthalene-degrading activity of the freely suspended cells was compared with that of immobilized cells in batches in shaken culture and in a continuous culture system in a packed-bed reactor . Increasing concentrations of naphthalene were better tolerated and more quickly degraded by immobilized cell cultures than by free cells . An initial naphthalene concentration of 25 mM was completely degraded by freely suspended cells (4 x 10(10) cfu ml(-1)) and polyurethane-foam-immobilized cells (0.8-1 x 10(12) cfu g(-1) foam cubes) after 4 days and 2 days of incubation, respectively . Free cells degraded a maximum of 30 mM naphthalene after 4 days of incubation with 50 mM naphthalene, and no further degradation was observed even after 15 days of incubation, whereas foam-immobilized cells brought about the complete degradation of 50 mM initial naphthalene after 6 days of incubation . Furthermore, with 25 mM naphthalene, the polyurethane-foam-immobilized cells were re-used 45 times over a period of 90 days without losing naphthalene-degrading activity . By contrast, with the same amount of naphthalene, alginate-, agar-, and polyacrylamide-entrapped cells could be reused for 18, 12, and 23 times over a period of 44, 28, and 50 days, respectively . During continuous degradation in a packed-bed reactor, foam-immobilized cells degraded 80 mM naphthalene at a rate of 150 ml(-1) h(-1) . With the same flow rate and 40 mM naphthalene, this system operated efficiently and continuously for about 120 days, whereas the packed-bed reactor with alginate-, agar-, and polyacrylamide-entrapped cells could be operated only for 45, 40, and 60 days respectively . Thus, more efficient degradation of naphthalene could be achieved by immobilizing cells of Pseudomonas sp . strain NGK1 in polyurethane foam, rather than in the other matrices tested. Plant Cell, 2001 May, 13(5), 1079 - 93 A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity; Lee J et al.; Harpin from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola (harpin(Psph)) elicits the hypersensitive response and the accumulation of pathogenesis-related gene transcripts in the nonhost plant tobacco . Here, we report the characterization of a nonproteinaceous binding site for harpin(Psph) in tobacco plasma membranes, which is assumed to mediate the activation of plant defense responses in a receptor-like manner . Binding of 125I-harpin(Psph) to tobacco microsomal membranes (dissociation constant = 425 nM) and protoplasts (dissociation constant = 380 nM) was specific, reversible, and saturable . A close correlation was found between the abilities of harpin(Psph) fragments to elicit the transcript accumulation of the pathogenesis-related tobacco gene HIN1 and to compete for binding of 125I-harpin(Psph) to its binding site . Another elicitor of the hypersensitive response and HIN1 induction in tobacco, the Phytophthora megasperma-derived beta-elicitin beta-megaspermin, failed to bind to the putative harpin(Psph) receptor . In contrast to activation by beta-megaspermin, harpin(Psph)-induced activation of the 48-kD salicylic acid-responsive mitogen-activated protein kinase (MAPK) and HIN1 transcript accumulation were independent of extracellular calcium . Moreover, use of the MAPK kinase inhibitor U0126 revealed that MAPK activity was essential for pathogenesis-related gene expression in harpin(Psph)-treated tobacco cells . Thus, a receptor-mediated MAPK-dependent signaling pathway may mediate the activation of plant defense responses induced by harpin(Psph). Enzyme Microb Technol, 2001 May 7, 28(7-8), 653 - 660 The enantioselective hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester in supercritical carbon dioxide using lipases; Hartmann T et al.; A new experimental high-pressure-unit was constructed for the enantioselective enzymatic hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester (a precursor for biological interesting substances) in a biphasic buffer/SCCO(2)-system . One objective is to take advantage of the solubility differences of the substrate and the produced acid . Thus the different solubilities of the substrates and the products in the different phases were studied regarding to an overall process integration . One ester enantiomer is preferably hydrolyzed, the other remains in the supercritical phase . And the produced acid enantiomer is concentrated in the buffer phase . The decrease in pressure is followed by an extraction process of the remaining substrate-enantiomer, in consequence it will be possible to combine an enzymatic reaction with a separation step . The catalysis was optimized in regard to enantioselectivity, enantiomeric excess, conversion and reaction time . A high enantioselectivity is achieved for the aromatic substrate using the lipase of Pseudomonas cepacia . The results show that this unconventional reaction system offers tremendous advantages for enzyme process development. Enzyme Microb Technol, 2001 May 7, 28(7-8), 617 - 624 Purification and characterization of a novel bromoperoxidase-catalase isolated from bacteria found in recycled pulp white water; Kuusk H et al.; A bacterial strain, Pseudomonad EF group 70B, containing a high catalase-like activity was found in process water (white water) from pulp using recycled fibers . The enzyme was purified and characterized, and found to be a hydroperoxidase . The active enzyme has an apparent molecular mass of about 153 kDa with two identical subunits and a pI value of 4.7 . It has a rather sharp pH optimum for catalase activity at 6.0 but exhibits catalase, peroxidase and brominating activities over a broad pH range from 4 to 8 . It was not inhibited by 3-amino-1,2,4-triazole . Peroxidase-like activity was found when adding o-dianisidine, pyrogallol, guaiacol and 4-aminoantipyrine . Brominating activity was noticed using monochlorodimedone as a substrate . The absorption spectrum exhibited a Soret band at 404 nm . Upon reduction with dithionite the Soret peak decreased and shifted to 436 nm . Pyridine hemochrome spectra indicated the presence of a protophorfyrin IX heme group and the enzyme was inhibited by the known heme ligands cyanide and azide . N-terminal amino acid analysis gave the sequence STEVKLPYAVAGGGTTILDAFPGE, which showed no homology with those of known catalases or peroxidases . It is concluded that the enzyme is a novel type of catalase-peroxidase or, more specifically, a bromoperoxidase-catalase, and that future developments of inhibitors of hydrogen peroxide-degrading activities in white water may be based on this enzyme and other catalase-peroxidases. Genetics, 2001 May, 158(1), 439 - 50 The leucine-rich repeat domain can determine effective interaction between RPS2 and other host factors in arabidopsis RPS2-mediated disease resistance; Banerjee D et al.; Like many other plant disease resistance genes, Arabidopsis thaliana RPS2 encodes a product with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains . This study explored the hypothesized interaction of RPS2 with other host factors that may be required for perception of Pseudomonas syringae pathogens that express avrRpt2 and/or for the subsequent induction of plant defense responses . Crosses between Arabidopsis ecotypes Col-0 (resistant) and Po-1 (susceptible) revealed segregation of more than one gene that controls resistance to P . syringae that express avrRpt2 . Many F(2) and F(3) progeny exhibited intermediate resistance phenotypes . In addition to RPS2, at least one additional genetic interval associated with this defense response was identified and mapped using quantitative genetic methods . Further genetic and molecular genetic complementation experiments with cloned RPS2 alleles revealed that the Po-1 allele of RPS2 can function in a Col-0 genetic background, but not in a Po-1 background . The other resistance-determining genes of Po-1 can function, however, as they successfully conferred resistance in combination with the Col-0 allele of RPS2 . Domain-swap experiments revealed that in RPS2, a polymorphism at six amino acids in the LRR region is responsible for this allele-specific ability to function with other host factors. Lett Appl Microbiol, 2001 May, 32(5), 346 - 8 The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea; Walker R et al.; AIMS: Antifungal metabolites of Pseudomonas antimicrobica have previously been shown to inhibit conidial germination of the grey mould pathogen Botrytis cinerea . In this study, metabolites of the bacterium have been tested at different stages of Botrytis germination to determine their effects on germ tube production and extension . METHODS AND RESULTS: Metabolites were added to conidia that had been pre-incubated for either 120 or 255 min . Pseudomonas antimicrobica inhibited B . cinerea conidial germination and caused a significant reduction in germ tube extension, irrespective of the stage of germination . Abnormal germination and a reduction in the frequency of lateral branching of the germ tubes in the presence of the metabolites were also reported, suggesting interference with normal hyphal development . CONCLUSION: The bacterium can inhibit germination of conidia and extension of germ tubes at different stages of Botrytis development . SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonistic activity of the bacterium has promising implications for its use as a biocontrol agent. Nat Struct Biol, 2001 May, 8(5), 442 - 6 Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes; Wlodawer A et al.; The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp . 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions . Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0-1.4 A resolution . The structure of PSCP comprises a single compact domain with a diameter of approximately 55 A, consisting of a seven-stranded parallel beta-sheet flanked on both sides by a number of helices . The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue . Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287. Environ Microbiol, 2001 Mar, 3(3), 176 - 86 The structure of a local population of phytopathogenic Pseudomonas brassicacearum from agricultural soil indicates development under purifying selection pressure; Sikorski J et al.; Among the isolates of a bacterial community from a soil sample taken from an agricultural plot in northern Germany, a population consisting of 119 strains was obtained that was identified by 16S rDNA sequencing and genomic fingerprinting as belonging to the recently described species Pseudomonas brassicacearum . Analysis of the population structure by allozyme electrophoresis (11 loci) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR; four primers) showed higher resolution with the latter method . Both methods indicated the presence of three lineages, one of which dominated strongly . Stochastic tests derived from the neutral theory of evolution (including Slatkin's exact test, Watterson's homozygosity test and the Tajima test) indicated that the population had developed under strong purifying selection pressure . The presence of strains clearly divergent from the majority of the population can be explained by in situ evolution or by influx of strains as a result of migration or both . Phytopathogenicity of a P . brassicacearum strain determined with tomato plants reached the level obtained with the type strain of the known pathogen Pseudomonas corrugata . The results show that a selective sweep was identified in a local population . Previously, a local selective sweep had not been seen in several populations of different bacterial species from a variety of environmental habitats. Microbiology, 2001 May, 147(Pt 5), 1171 - 82 Highly conserved sequences flank avirulence genes: isolation of novel avirulence genes from Pseudomonas syringae pv . pisi; Arnold DL et al.; DNA sequences flanking two avr genes (avrPpiA1 and avrPpiB1) from Pseudomonas syringae pv . pisi show a high degree of similarity . Specific primers designed from the conserved regions were used in PCR amplifications with all P . syringae pv . pisi races . As well as amplifying the expected avrPpiA- and avrPpiB-containing fragments, two additional fragments were amplified: one contained a single open reading frame (ORF1) and was found in races of genomic group II (2, 3A, 4A and 6); the second fragment contained two open reading frames (ORF2 and ORF3), separated by 658 nt, and was detected in all races . All three ORFs had G+C ratios (46.9-48 mol%) that were significantly less than that for P . syringae and each was preceded by a potential hrp box promoter . In P . syringae pv . phaseolicola, ORF1 and ORF2 each elicited a strong non-host hypersensitive reaction on bean leaves; ORF1 was designated avrPpiG, the product of which had strong similarity to AvrRxv, AvrBsT and YopP . ORF2 was identical to a gene, designated avrPpiC, previously isolated from P . syringae pv . pisi race 5 . ORF3 was always found in association with avrPpiC and both were detected in a wide range of P . syringae pathovars . In contrast, avrPpiG was only detected in strains of P . syringae pv . pisi genomic group II and P . syringae pv . coronafaciens (ICMP 3113) . In P . syringae pv . pisi, avrPpiG was plasmid-borne and avrPpiC and ORF3 were chromosomal . This conservation of flanking sequences has implications for the horizontal transfer of avirulence and virulence genes, suggesting that specific regions of the bacterial genome act as sites for their integration/excision. Blood, 2001 May 1, 97(9), 2673 - 9 The interleukin-13 receptor alpha2 chain: an essential component for binding and internalization but not for interleukin-13-induced signal transduction through the STAT6 pathway; Kawakami K et al.; The interleukin-13 receptor (IL-13R) complex is composed of 2 different chains, IL-13Ralpha1 (also known as IL-13Ralpha') and IL-13Ralpha2 (also known as IL-13Ralpha) . For a functional IL-13 receptor, the IL-13Ralpha1 chain forms a productive complex with the primary IL-4 binding protein (IL-4Ralpha also known as IL-4Rbeta) . However, the function of the IL-13Ralpha2 chain is not clear even though this chain binds IL-13 with high affinity . This study demonstrates that IL-13Ralpha2 can undergo internalization after binding to ligand without causing activation of its signaling pathways . These conclusions were drawn on the basis of (1) internalization of (125)I-IL-13 in Chinese hamster ovarian (CHO-K1) and T98G glioblastoma cells transiently transfected with the IL-13Ralpha2 chain; (2) a recombinant chimeric fusion protein comprising IL-13 and a mutated form of Pseudomonas exotoxin (termed IL13-PE38QQR or IL-13 toxin) is specifically cytotoxic to IL-13Ralpha2-transfected CHO-K1 cells in a gene dose-dependent manner, whereas cells transfected with vector alone were not sensitive; and (3) IL-13 did not cause activation of signal transduction and activation of transcription 6 (STAT6) in IL-13Ralpha2-transfected cells . IL-13 efficiently caused activation of STAT6 protein in cells transfected with the IL-13Ralpha1 and IL-4Ralpha chains, and IL-13Ralpha2 inhibited this activation . Taken together, these observations indicate that internalization of IL-13Ralpha2 is signal independent and that this property of IL-13Ralpha2 can be exploited for receptor-directed cancer therapy. J Biol Chem, 2001 Jun 22, 276(25), 22500 - 6 Epub 2001 Apr 18. Emergence of multifunctional oxygenase activities by random priming recombination; Suenaga H et al.; Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl . The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs) . Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo . Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox . The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene . These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners . Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation. Mol Plant Microbe Interact, 2001 Apr, 14(4), 545 - 54 Isolation and characterization of the gene coding for the amidinotransferase involved in the biosynthesis of phaseolotoxin in Pseudomonas syringae pv . phaseolicola; Hernandez-Guzman G et al.; Pseudomonas syringae pv . phaseolicola is the causal agent of the "halo blight" disease of beans . A key component in the development of the disease is a nonhost-specific toxin, Ndelta-(N'-sulphodiaminophosphinyl)-ornithyl-alanyl-homoarginine, known as phaseolotoxin . The homoarginine residue in this molecule has been suggested to be the product of L-arginine:lysine amidinotransferase activity, previously detected in extracts of P . syringae pv . phaseolicola grown under conditions of phaseolotoxin production . We report the isolation and characterization of an amidinotransferase gene (amtA) from P . syringae pv . phaseolicola coding for a polypeptide of 362 residues (41.36 kDa) and showing approximately 40% sequence similarity to L-arginine:inosamine-phosphate amidinotransferase from three species of Streptomyces spp . and 50.4% with an L-arginine:glycine amidinotransferase from human mitochondria . The cysteine, histidine, and aspartic acid residues involved in substrate binding are conserved . Furthermore, expression of the amtA and argK genes and phaseolotoxin production occurs at 18 degrees C but not at 28 degrees C . An amidinotransferase insertion mutant was obtained that lost the capacity to synthesize homoarginine and phaseolotoxin . These results show that the amtA gene isolated is responsible for the amidinotransferase activity detected previously and that phaseolotoxin production depends upon the activity of this gene. Mol Plant Microbe Interact, 2001 Apr, 14(4), 451 - 9 Functional studies of the bacterial avirulence protein AvrPto by mutational analysis; Chang JH et al.; Pseudomonas syringae pathovars expressing avrPto are avirulent on plants expressing the resistance gene Pto . Over 85 mutants of avrPto were generated with multiple strategies, and several assays were used to characterize AvrPto function . Only a core of 95 amino acids of the 164 residues was sufficient for binding Pto in the yeast two-hybrid system . Only nine of 65 mutant proteins of AvrPto with amino acid substitutions, created in planta and in vitro, did not interact with Pto in the Gal4 yeast two-hybrid system, suggesting that AvrPto can tolerate many nonconservative substitutions and still interact with Pto . These nine and 12 additional substitution mutants of AvrPto were characterized further . The ability to elicit a hypersensitive response and the effect on pathogenesis in planta for these 21 mutants of AvrPto were strongly correlated with recognition by Pto in the yeast two-hybrid system . Analyses of two proteins with substitutions H54P or D52G/L65P indicated that these residues may be required for delivery into the host cell and protein stability in the bacterial cytoplasm, respectively . The mutants that no longer interacted with Pto and had modified activities in planta were predicted to have changes in their secondary structure. Plant J, 2001 Mar, 25(5), 563 - 74 A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens; Shah J et al.; The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance . However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance . Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated . The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s) . In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes {PR-1, BGL2 (PR-2) and PR-5}; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica . The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv . tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway . Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes . However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance . Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway. J Biol Chem, 2001 Jun 22, 276(25), 22507 - 13 Epub 2001 Apr 16. Translational repression and specific RNA binding by the coat protein of the Pseudomonas phage PP7; Lim F et al.; PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta . Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase . Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages . Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established . Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm . Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified. Prostate, 2001 Apr, 47(1), 21 - 8 Antitumor effect of an HER2-specific antibody-toxin fusion protein on human prostate cancer cells; Wang L et al.; BACKGROUND: HER2/neu has been implicated in the oncogenesis of human prostate cancer . Clinical studies have suggested that overexpression of HER2 may be one of the indicators of poor prognosis in prostate cancer patients . METHODS: We used Western blot analysis to examine the expression of HER2 in a panel of established human prostate cancer cell lines and used an MTT assay to evaluate the cytotoxicity on these cells of a recombinant fusion protein consisting of an HER2-specific single-chain antibody and the Pseudomonas exotoxin A, scFv(FRP5)-ETA . RESULTS: LNCaP cells express high levels of HER2 protein . Exposure of LNCaP cells to scFv(FRP5)-ETA caused remarkable cell death . In contrast, PC3M cells, which express an undetectable level of HER2 protein, were resistant to scFv(FRP5)-ETA-induced cytotoxicity . MDA PCa 2a, MDA PCa 2b, and DU145 cells express low-to-medium levels of HER2 protein and showed an HER2 level-dependent response to scFv(FRP5)-ETA-induced cytotoxicity . The scFv(FRP5)-ETA-induced cytotoxicity of LNCaP cells could be inhibited by an anti-HER2 monoclonal antibody (mAb), which downregulated the levels of HER2 protein, indicating the specificity of scFv(FRP5)-ETA in inducing cytotoxicity in LNCaP cells . Using an apoptosis ELISA, we demonstrated that scFv(FRP5)-ETA induced apoptosis in LNCaP cells . The apoptosis was inhibited by the presence of dihydrotestosterone (DHT) in culture medium . Exposure of LNCaP cells to scFv(FRP5)-ETA caused reduction in the level of the prostate-specific antigen (PSA) . CONCLUSIONS: Our data suggest that scFv(FRP5)-ETA might be a useful agent for the treatment of human prostate cancer cells with high levels of HER2 expression. Tree Physiol, 2000 Jul, 20(13), 915 - 20 A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings; Laukkanen H et al.; We isolated a rapidly growing, pigment-producing mycobacterium from senescent tissue cultures derived from mature Scots pine (Pinus sylvestris L.) . The bacterium was found in three senescent suspension cultures and in a senescent protoplast culture . Growth of Scots pine cells had ceased in all of these cultures . Exogenous contamination was eliminated by rigorous surface sterilization of the buds with hypochlorite before aseptic removal of the bud scales . Based on biochemical and physiological properties and DNA sequence comparisons, the isolated mycobacterium did not belong to any known species . Its sequence most closely resembled those of Mycobacterium obuense (97%) and M . aichiense (96%) . Tissue browning was frequently observed in callus or suspension culture of Scots pine . Because the effect of the mycobacterium on growth of undifferentiated tissues that were browning was difficult to evaluate, we applied the bacterium to Scots pine seeds in aseptic conditions . Seedlings grown in the presence of the mycobacterium had shorter hypocotyls than control seedlings and seedlings cocultivated with a Pseudomonas strain known to be harmless to plants . However, hypocotyl growth of seedlings cocultivated with another mycobacterium, M . chlorophenolicum, was similar to that observed in the presence of the isolated mycobacterium . Phenylalanine ammonia-lyase activity of seedlings cocultivated with the mycobacterium isolate was significantly higher than that of control seedlings or seedlings cocultivated with M . chlorophenolicum or Pseudomotnas fluorescens . We believe that this is the first report of the isolation of mycobacteria from tissue cultures of a tree . Our finding that the mycobacterium may interfere with the growth of Scots pine in vitro warrants further study. J Pharmacol Exp Ther, 2001 May, 297(2), 479 - 88 Cytochrome P450 metabolites of arachidonic acid but not cyclooxygenase-2 metabolites contribute to the pulmonary vascular hyporeactivity in rats with acute Pseudomonas pneumonia; Yaghi A et al.; We have previously demonstrated depressed vascular contractility in intralobar pulmonary artery (PA) rings isolated from rats with acute Pseudomonas pneumonia . Here we describe the role of arachidonic acid (AA) metabolites in the regulation of pulmonary vascular tone in inflammation . Pneumonia was induced by intratracheal injection of P . aeruginosa organisms . Rats were sacrificed 44 h later . EETs and 20-HETE were formed at significantly lower rates in pneumonia compared with control lung microsomes . Vasoactive effects of CYP metabolites (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and 20-HETE) on small PA rings from control or pneumonia rats were assessed in vitro . All four EETs and 20-HETE were more potent PA vasoconstrictors than KCl or phenylephrine (PE) . However, this potency was attenuated in PA rings from pneumonia lungs compared with control . In contrast, pneumonia had no effect on COX activity {total pulmonary prostaglandin (PG), PGE(2), and 6-keto-PGF(1 alpha)} . In vitro vascular contractility to KCl, PE, or PGF(2 alpha) was assessed in small PA rings from control and pneumonia rats in the presence and absence of the COX-2 inhibitor NS-398 (10 microM) . NS-398 did not reverse the attenuated contractile responses to KCl, PE, or PGF(2 alpha) in pneumonia rats . Nitrite/nitrate levels, inducible nitric-oxide synthase and heme oxygenase activities were all significantly elevated in pneumonia lungs . In conclusion, vasodilator PGs produced by COX-2 do not contribute to the depressed PA contractility in this model of pneumonia . Depressed pulmonary production and vasoconstrictor effects of CYP metabolites of AA (possibly due to increased NO and/or carbon monoxide) indicate a potential role for these vasoactive metabolites in this model of acute pneumonia. Biochemistry, 2001 Apr 3, 40(13), 3967 - 73 15N NMR relaxation studies of backbone dynamics in free and steroid-bound Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni; Yun S et al.; The backbone dynamics of Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N relaxation measurements . The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S(2), tau(e), and R(ex)) and the overall rotational correlation time (tau(m)) . The rotational correlation times were 19.23 +/- 0.08 and 17.08 +/- 0.07 ns with the diffusion anisotropies (D( parallel)/D( perpendicular)) of 1.26 +/- 0.03 and 1.25 +/- 0.03 for the free and steroid-bound KSI, respectively . The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in helix A1 and strand B4 . However, the majority of the residues exhibit reduced order parameters upon ligand binding . In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme . Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding. Biochemistry, 2001 Mar 27, 40(12), 3512 - 24 Solution structure of the toluene 4-monooxygenase effector protein (T4moD); Hemmi H et al.; Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH- and O(2)-dependent hydroxylation of toluene to form p-cresol . The complex consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a diiron hydroxylase {T4moH, with (alphabetagamma)(2) quaternary structure}, and a catalytic effector protein (T4moD) . The solution structure of the 102-amino acid T4moD effector protein has been determined from 2D and 3D (1)H, (13)C, and (15)N NMR spectroscopic data . The structural model was refined through simulated annealing by molecular dynamics in torsion angle space (DYANA software) with input from 1467 experimental constraints, comprising 1259 distance constraints obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80 hydrogen bond constraints . Of 60 conformers that met the acceptance criteria, the 20 that best satisfied the input constraints were selected to represent the solution structure . With exclusion of the ill-defined N- and C-terminal segments (Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20 conformers with respect to the mean coordinates was 0.71 A for the backbone and 1.24 A for all non-hydrogen atoms . The secondary structure of T4moD consists of three alpha-helices and seven beta-strands arranged in an N-terminal betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology . Although the published NMR structures of the methane monooxygenase effector proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) have a similar secondary structure topology, their three-dimensional structures differ from that of T4moD . The major differences in the structures of the three effector proteins are in the relative orientations of the two beta-sheets and the interactions between the alpha-helices in the two domains . The structure of T4moD is closer to that of the methane monooxygenase effector protein from M . capsulatus (Bath) than that from M . trichosporium OB3b . The specificity of T4moD as an effector protein was investigated by replacing it in reconstituted T4MO complexes with effector proteins from monooxygenases from other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene 3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase) . The results showed that the closely related TbuV effector protein (55% sequence identity) provided partial activation of the complex, whereas the more distantly related TbmC (34% sequence identity) and S1 (29% sequence identity) did not . The (1)H NMR chemical shifts of the side-chain amide protons of Asn34, a conserved, structurally relevant amino acid, were found to be similar in spectra of effector proteins T4moD and TbuV but not in the spectrum of TbmC . This suggests that the region around Asn34 may be involved in structural aspects contributing to functional specificity. Microbiol Res, 2001 Mar, 155(4), 309 - 14 Antifungal activity of chitinases produced by some fluorescent pseudomonads against Colletotrichum falcatum Went causing red rot disease in sugarcane; Viswanathan R et al.; Chitinase production and growth of certain fluorescent pseudomonads isolated from sugarcane rhizosphere on different subtrates were studied . When chitin was substituted for glycerol in King's B medium, 3 of the 4 strains showed enhanced bacterial multiplication . Bacterial cells grown on chitin-containing medium showed enhanced antifungal activity against Colletotrichum falcatum Went causing red rot disease in sugarcane . Chitinase production was significantly higher when chitin was amended to King's B medium . Higher chitinase production was also recorded when fluorescent pseudomonad strains were grown in the medium containing crab-shell chitin . Cell-free bacterial culture filtrate from chitin-containing medium significantly inhibited mycelial growth of the pathogen . These cell-free conditioned media contained 3 to 7 polypeptides . Western blot analysis revealed five isoforms of chitinase with molecular masses of 47, 36, 32, 20 and 18.5 kDa . A possible role of chitinases in red rot disease management is discussed. J Bacteriol, 2001 May, 183(9), 2842 - 51 In vivo and in vitro effects of integration host factor at the DmpR-regulated sigma(54)-dependent Po promoter; Sze CC et al.; Transcription from the Pseudomonas CF600-derived sigma(54)-dependent promoter Po is controlled by the aromatic-responsive activator DmpR . Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro . In vivo, the Po promoter exhibits characteristics that typify many sigma(54)-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output . IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites . In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription . This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at sigma(54) promoters such as aiding binding of the regulator or recruitment of sigma(54)-RNA polymerase via UP element-like DNA . The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA . Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter. Int J Cancer, 2001 Apr 15, 92(2), 168 - 75 Interleukin-13 receptor as a unique target for anti-glioblastoma therapy; Husain SR et al.; Surgery, radiotherapy and chemotherapy have minimally altered survival of glioblastoma patients . We explored a specific approach for glioblastoma therapy in which cellular interleukin-13 (IL-13) receptors were targeted by an IL-13 cytotoxin . A wide array of human glioblastoma cell lines expressing the receptor for IL-13 were effectively killed by an IL-13 cytotoxin, a chimeric protein composed of human IL-13 and a mutated form of Pseudomonas exotoxin (termed IL13-PE38QQR or IL-13 toxin) . Daily (qd) intratumoral injections of IL-13 toxin (50 and 100 microg/kg/day) for 5 consecutive days into subcutaneous human U251 glioblastoma tumors (approx . 30 mm(2)) in nude mice resulted in complete regression of tumors in 4/5 and 5/5 mice, respectively . Tumor regression persisted for at least 221 days postimplantation . Three alternate day injections (qod) of IL-13 toxin (250 microg/kg/day) into other subcutaneous U87 glioblastoma tumors also produced durable complete responses (CR) in all 5 mice . Twice daily (bid) intraperitoneal injections of IL-13 toxin at 25 or 50 microg/kg/dose for 5 days (total doses = 10) regressed U251 tumors by 45% and 58% with 1/5 and 2/5 CRs, respectively, on day 54 . Intraperitoneal administration of IL-13 toxin with an identical schedule at a dose of 50 microg/kg injected into mice bearing U87 xenografts reduced tumor burden by one-half on day 36 . Similar doses (25 or 50 microg/kg) with a daily schedule (qd x 5) by the intravenous route also suppressed growth of U251 subcutaneous tumors by 75% and 81% with 1/6 CR in either group by day 34 . All mice tolerated therapy well without any visible signs of toxicity . On the basis of these studies, we have initiated a Phase I clinical trial using IL13-PE38QQR in patients with recurrent glioblastoma . Published 2001 Wiley-Liss, Inc. Arch Microbiol, 2001 Feb, 175(2), 79 - 85 The clc element of Pseudomonas sp . strain B13 and other mobile degradative elements employing phage-like integrases; van der Meer JR et al.; Genes for metabolic pathways in bacteria that degrade aromatic or aliphatic pollutants have mostly been confined to either plasmid DNAs or to the chromosome . For a few pathways, including classical pathways for chlorocatechol and biphenyl degradation, recent evidence has been obtained for location of the pathway genes on mobile DNA elements which employ phage-like integrases . This enables the DNA elements to integrate into specific sites on the chromosome and yet to excise and transfer to other host bacteria . This mini-review gives an overview of those elements and their relationship to an increasing number of phage-like elements associated with bacterial virulence. Appl Environ Microbiol, 2001 Apr, 67(4), 1945 - 8 Growth interactions during bacterial colonization of seedling rootlets; De Bellis P et al.; Rootlet elongation and bacterial growth on rootlets were determined after inoculation of cucumber and spinach seedlings with Pseudomonas strains differing in production of siderophores and HCN . Siderophore producers grew more profusely than nonproducers on both species and promoted rootlet elongation on cucumber . Coinoculation of siderophore producers and nonproducers resulted in restricted growth of the latter . The total populations of nonproducers of HCN in the presence of HCN producers were not decreased, but the tenacity of their association with the rootlet surface was altered. Appl Environ Microbiol, 2001 Apr, 67(4), 1718 - 27 Characterization of fluorescent and nonfluorescent peptide siderophores produced by Pseudomonas syringae strains and their potential use in strain identification; Bultreys A et al.; Nonfluorescent highly virulent strains of Pseudomonas syringae pv . aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains . The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P . syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units . The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively . The nonfluorescent siderophore is used by fluorescent and nonfluorescent P . syringae strains . These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue . When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in beta-hydroxyaspartic acid residues of the peptide chains . These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically . These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P . syringae and P . viridiflava strains and to distinguish between the two siderovars in P . syringae pv . aptata. J Biol Chem, 2001 May 18, 276(20), 17429 - 36 Epub 2001 Feb 22. Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88; Costas AM et al.; The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and overproduced in Escherichia coli . The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses . Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized . The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite . The reverse reaction was not observed . Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer . PtxD has a high affinity for its substrates with Km values of 53.1 +/- 6.7 microm and 54.6 +/- 6.7 microm, for phosphite and NAD, respectively . Vmax and kcat were determined to be 12.2 +/- 0.3 micromol x min(-1) x mg(-1) and 440 min(-1) . NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite . Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last . Amino acid sequence comparisons place PtxD as a new member of the d-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorganic substrate . To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxidation of a reduced phosphorus compound. Biochim Biophys Acta, 2001 Mar 30, 1531(1-2), 1 - 3 The use of Pseudomonas acyl-CoA synthetase to form acyl-CoAs from dicarboxylic fatty acids; Milne KG et al.; Pseudomonas acyl-CoA synthetase is shown to act on saturated dicarboxylic acids with a chain length of C10 or greater to produce conjugates containing a single CoA unit . The synthetase can, therefore, be used to generate novel acyl-CoA analogues for studies on proteins that utilise, bind to, or are modulated by acyl-CoAs. Mol Plant Microbe Interact, 2001 Mar, 14(3), 394 - 404 Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv . tomato across the host plant cell wall; Brown IR et al.; The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae . We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P . syringae pv . tomato strain DC3000 grown under hrp-inducing conditions on EM grids . Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media . Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella . The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro . Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana . The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ . Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ . A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall. Mol Plant Microbe Interact, 2001 Mar, 14(3), 336 - 48 The contribution of syringopeptin and syringomycin to virulence of Pseudomonas syringae pv . syringae strain B301D on the basis of sypA and syrB1 biosynthesis mutant analysis; Scholz-Schroeder BK et al.; Sequencing of an approximately 3.9-kb fragment downstream of the syrD gene of Pseudomonas syringae pv . syringae strain B301D revealed that this region, designated sypA, codes for a peptide synthetase, a multifunctional enzyme involved in the thiotemplate mechanism of peptide biosynthesis . The translated protein sequence encompasses a complete amino acid activation module containing the conserved domains characteristic of peptide synthetases . Analysis of the substrate specificity region of this module indicates that it incorporates 2,3-dehydroaminobutyric acid into the syringopeptin peptide structure . Bioassay and high performance liquid chromatography data confirmed that disruption of the sypA gene in strain B301D resulted in the loss of syringopeptin production . The contribution of syringopeptin and syringomycin to the virulence of P . syringae pv . syringae strain B301D was examined in immature sweet cherry with sypA and syrB1 synthetase mutants defective in the production of the two toxins, respectively . Syringopeptin (sypA) and syringomycin (syrB1) mutants were reduced in virulence 59 and 26%, respectively, compared with the parental strain in cherry, whereas the syringopeptin-syringomycin double mutant was reduced 76% in virulence . These data demonstrate that syringopeptin and syringomycin are major virulence determinants of P . syringae pv . syringae. J Bacteriol, 2001 Apr, 183(8), 2405 - 10 Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different; Seffernick JL et al.; The gene encoding melamine deaminase (TriA) from Pseudomonas sp . strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity . Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products . The first deamination reaction occurred more than 10 times faster than the second . Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition . Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from Pseudomonas sp . strain ADP . Each enzyme consists of 475 amino acids and differs by only 9 amino acids . AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline . Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates . Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom . Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively . These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively. Biosci Biotechnol Biochem, 2001 Jan, 65(1), 222 - 5 Crystallization and structural analysis of intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri; Mezaki Y et al.; The intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri (G4-1), which has a raw starch binding domain, has been crystallized . The structure was identified (PDB entry 1GCY) by the molecular replacement method using the structure of its catalytic domain (G4-2) . The result showed that the raw starch binding domain is in a disordered state, the corresponding electron densities being almost invisible . Superposition of these two enzyme forms showed evidence for the possible location of the raw starch binding domain (SBD) . This crystal is a novel case, in that it forms a regular lattice incorporating flexibly bound SBD in the channel of crystal packing of the catalytic domains. Curr Microbiol, 2001 Mar, 42(3), 160 - 7 Effect of genomic location on horizontal transfer of a recombinant gene cassette between Pseudomonas strains in the rhizosphere and spermosphere of barley seedlings; Sengelov G et al.; The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria . However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids . To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated . Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 x 10(-3) transconjugants/(donors x recipients)1/2 in the rhizosphere and 4.57 x 10(-2) transconjugants/(donors x recipients)1/2 in the spermosphere . Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 x 10(-3) transconjugants/(donors x recipients)1/2 . Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation . However, transformation did occur by using the same bacterial strains under laboratory conditions . The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Carbohydr Res, 2001 Feb 28, 330(4), 505 - 10 Location of the O-methyl groups in the O polysaccharide of Pseudomonas syringae pv . phaseolicola; Zdorovenko EL et al.; The O-methylation pattern of the O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv . phaseolicola GSPB 1552 was revealed by methylation (CD3I) analysis, Smith degradation, and NMR spectroscopy . Together with the major O repeats consisting of D-rhamnopyranose (D-Rhap) and D-fucofuranose (D-Fucf), there are minor repeats (approximately 30%) containing 3-O-methyl-D-rhamnose (D-acofriose), which is 2-substituted in the interior repeats and occupies the terminal non-reducing end of the OPS . It was suggested that the methylated O repeats are linked to each other nearby the non-reducing end of the OPS and that the 'biological' O repeat of the OPS has the following structure: {molecular structure: see text}. J Immunother, 2001 Mar-Apr, 24(2), 144 - 50 Cytotoxicity of antiosteosarcoma recombinant immunotoxins composed of TP-3 Fv fragments and a truncated Pseudomonas exotoxin A; Onda M et al.; Regrowth of drug-resistant tumor cells is responsible for approximately half of an unselected osteosarcoma population still dying of the disease despite aggressive combination therapy . Two monoclonal antibodies, TP-1 (immunoglobulin 2a) and TP-3 (immunoglobulin 2b) are available, which specifically recognize an antigen on osteosarcoma cells . In this work, we have fused the variable (V) genes of TP-3 to a truncated fragment of Pseudomonas exotoxin A, referred to as PE38 . Two immunotoxins were made that differed in the Fv portion: TP-3(scFv)-PE38, which contains a peptide linker, and TP-3(dsFv)-PE38, which contains a disulfide bond for stabilization of the association between the V domains . Recombinant TP-3 immunotoxins were expressed in Escherichia coli and purified from inclusion bodies . We describe the design and expression of these immunotoxins, and their properties with regard to antigen binding, stability, and cytotoxicity . Toxicity studies were done in mice . We found that the immunotoxins exhibited very similar in vitro properties, whereas in vivo TP-3(dsFv)-PE38 was much better tolerated than TP-3(scFv)-PE38. Lett Appl Microbiol, 2001 Mar, 32(3), 166 - 70 Detection of tabtoxin-producing strains of Pseudomonas syringae by PCR; Lydon J et al.; AIMS: The present study describes a system based on PCR to distinguish tabtoxin-producing strains of Pseudomonas syringae from other Ps . syringae plant pathogens that produce chlorosis-inducing phytotoxins . METHODS AND RESULTS: Thirty-two strains of Ps . syringae and related species were examined . Two sets of PCR primers were developed to amplify genes (tblA and tabA) required for tabtoxin production . Only a PCR product of 829 bp or 1020 bp was produced in PCR reactions with the tblA or tabA primer sets, respectively, and cells from tabtoxin-producing pathovars of Pseudomonas syringae . All known non-tabtoxin producing bacterial species failed to produce an amplification product with either primer set . CONCLUSIONS: PCR of genes required for tabtoxin production is a simple, rapid and reliable method for identifying tabtoxin-producing strains of Ps . syringae . SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol can effectively distinguish tabtoxin-producing strains of Ps . syringae from other Ps . syringae pathovars and Ps . syringae pv . tabaci strains from other tabtoxin-producing Ps . syringae pathovars. Acta Crystallogr D Biol Crystallogr, 2001 Apr, 57(Pt 4), 582 - 5 Crystallization and preliminary X-ray analysis of dmpFG-encoded 4-hydroxy-2-ketovalerate aldolase--aldehyde dehydrogenase (acylating) from Pseudomonas sp . strain CF600; Manjasetty BA et al.; The final two steps of the meta-cleavage pathway for catechol degradation in Pseudomonas sp . strain CF600 involve the conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A by the enzymes 4-hydroxy-2-ketovalerate aldolase and NAD(+)-dependent acylating aldehyde dehydrogenase . Biochemical studies indicate that these two enzymes comprise a bifunctional heterodimer (DmpFG, molecular mass 71 kDa) and suggest that the product of the aldolase reaction is transferred to the dehydrogenase active site via a channeling mechanism . Crystals of the DmpFG complex grow in multiple fan-like clusters of thin plates by the hanging-drop method and are improved by streak-seeding . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 102.0, b = 140.7, c = 191.3 A, and diffract to 2.1 A resolution . The asymmetric unit contains four DmpFG heterodimers . Heavy-atom derivative screening identified three isomorphous derivatives. Ann Oncol, 2001 Jan, 12(1), 115 - 8 Paraneoplastic pemphigus as the initial presentation of chronic lymphocytic leukemia; van Mook WNK et al.; The case history of a 61-year-old male patient is described, who presented with severe stomatitis, conjunctivitis and leukocytosis . The diagnosis chronic lymphocytic leukemia (CLL) stage A (0) was made, for which no treatment was necessary . Progression of stomatitis and conjunctivitis and erythosquamous skin lesions with bullae and vesiculae formation developed . Under the diagnosis of bullous pemphigoid the patient was treated with corticosteroids . The histologic and immunofluorescence examination of a skin biopsy was compatible with this diagnosis, and antibodies to skin could not be detected in a first serum sample . Pseudomonas was cultured from all lesions, the corticosteroids were stopped and antibiotic treatment was started, without clear effect . Because of progression of skin lesions and debilitation, the patient finally declined all treatment and died five weeks after admission . Post-mortem examination showed enlarged lymphnodes in the cervical, aortal en iliacal areas, with histology confirming the diagnosis of CLL . Indirect immunofluorescence with the second serum sample showed auto-antibodies in high titer directed against the intercellular epithelial substance . Immunoblot studies showed binding with the classic target antigens in paraneoplastic pemphigus . Re-examination of the histologic skin specimen and the result of direct immunofluorescence were in retrospect compatible with the diagnosis of paraneoplastic pemphigus. Mikrobiol Z, 2000 Sep-Oct, 62(5), 18 - 22 {Properties of bacteria of pathovars of Pseudomonas syringae affecting cereals}; Pasichnyk LA; Antigenic properties of Pseudomonas syringae pv . coronafaciens isolated from the oats have been studied for the first time, and serologic grouping of the strains of P . syringae pv . coronafaciens and P . syringae pv . atrofaciens has been carried out . It has been determined that the strains of P . syringae pv . coronafaciens, as to availability of specific antigenic complexes belong to two serological groups I and V, and thus they are differed from the strains of the pathovar atrofaciens which includes four serological groups of the strain isolated from wheat (II, IV, V, VI) and five serological groups of the strains, isolated from rye (I, II, IV, V, VI) . It has been shown that the overwhelming majority of strains of P . syringae pv . coronafaciens isolated from the affected plants of the oats belong to serogroup V, while P . syringae pv . atrofaciens from the rye--to serogroup I . The strains of these bacteria, isolated as epiphytes, dominate in the other serological groups. Biometals, 2000 Dec, 13(4), 301 - 9 The structure of a pyoverdine from Pseudomonas sp . CFML 96.188 and its relation to other pyoverdines with a cyclic C-terminus; Weber M et al.; From Pseudomonas sp . CFML 96.188 a pyoverdine was isolated and its primary structure was elucidated by spectroscopic methods and degradation reactions . This strain is of interest as it accepts the structurally different pyoverdines from several other Pseudomonas strains . They all have in common as a specific structural feature a C-terminal cyclic substructure, the importance of which for the recognition of a pyoverdine at the cell surface of a given strain will be discussed. Zhongguo Yao Li Xue Bao, 1999 Sep, 20(9), 795 - 9 Inhibitory effect of recombinant TGF alpha-PE40 on vascular smooth muscle cell proliferation; Wang B et al.; AIM: To study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the cultured vascular smooth muscle cells (SMC) . METHODS: Expression of epidermal growth factor receptor (EGFR) mRNA and EGFR in cultured proliferating and quiescent SMC was analyzed with Northern blot and immunohistochemistry . Inhibitory effects of TP40 on SMC proliferation and protein synthesis were analyzed with crystal violet staining and {3H}leucine incorporation . Competition assays were performed by the addition of 100-fold excess of EGF . RESULTS: Expression of EGFR mRNA and EGFR in rapidly proliferating SMC increased than that in quiescent SMC . When the concentration of TP40 was 10 or 100 micrograms.L-1, inhibitory effects of TP40 on rapidly proliferating SMC proliferation and protein synthesis were much higher than that on quiescent SMC (P < 0.01), and the IC50 of {3H}leucine incorporation against rapidly proliferating and quiescent SMC were 8.01 (5.05-12.69) and 121.95 (90.98-163.47) micrograms.L-1 . Excess EGF completely blocked inhibitory effects of TP40 . CONCLUSION: The rapidly proliferating SMC express EGFR at a high level . TP40 selectively inhibited the proliferation of rapidly proliferating SMC . The cytotoxic effects of TP40 were specifically mediated by EGFR. Braz J Biol, 2000 Nov, 60(4), 637 - 44 Population dynamics of Pseudomonas sp . along a spatial gradient of phosphate: an experimental approach for spatial ecology; Codeco CT et al.; Many theoretical models have been proposed to study the effect of space on population dynamics and interactions, but most of them are difficult to translate into experimental setups due to their abstract nature . Here we defend the gradostat as a valuable experimental tool for testing such theories . The gradostat is a culture system with bi-directional flow that forms nutrient gradients at steady state . In this study, we use a 3-vessel gradostat with a phosphate gradient to study the effect of spatial heterogeneity on the spatial distribution of Pseudomonas sp., an heterotrophic aquatic bacterium . The observed distributions partially agree with theoretical predictions, obtained from a mathematical model. Enzyme Microb Technol, 2001 Mar 8, 28(4-5), 333 - 338 Novel route for the resolution of both enantiomers of dropropizine by using oxime esters and supported lipases of Pseudomonas cepacia; Salunkhe MM et al.; Resolution of (R)- and (S)-dropropizine which is an antitussive and central sedative therapeutic agent in high optical and chemical yields was achieved by lipases of Pseudomonas cepacia supported on ceramic particles (lipase PS-C) and on diatomite (lipase PS-D) with oxime esters in organic solvents . The influence of several factors (lipase source, structural variations in oxime esters, the amount of lipase and its recyclability) on the enantioselectivity have been investigated . Different properties were used to describe the solvents, namely the hydrophobicity (quantified by log P) and the dielectic constant (epsilon) . This enzymatic acylation using oxime esters was significant as only (S)-dropropizine and (R)-dropropizine monoacetate was obtained . (R)-Dropropizine monoacetate was chemically hydrolyzed to obtain (R)-dropropizine . The highest enantioselectivity was observed when O-acetyl benzophenone oxime was used . This enzymatic resolution provides a versatile method for getting the pure enantiomers of dropropizine by effectively optimizing the various reaction parameters. Microbiology, 2001 Mar, 147(Pt 3), 745 - 56 An inducible 1-butanol dehydrogenase, a quinohaemoprotein, is involved in the oxidation of butane by "Pseudomonas butanovora"; Vangnai AS et al.; Butane-grown "Pseudomonas butanovora" expressed two soluble alcohol dehydrogenases (ADHs), an NAD(+)-dependent secondary ADH and an NAD(+)-independent primary ADH . Two additional NAD(+)-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate . The inducible NAD(+)-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway . BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1.0 mol pyrroloquinoline quinone (PQQ) and 0.25 mol haem c as prosthetic groups . BDH was synthesized as a monomer of approximately 66 kDa . It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C(4) diols and aromatic alcohols . It exhibited the lowest K:(m) (7+/-1 microM) and highest k(cat)/K:(m) (72x10(4) M(-1) s(-1)) value towards 1-butanol . BDH exhibited ferricyanide-dependent ADH activity . Calcium ions (up to 10 mM) increased BDH activity substantially . Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni . The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown "P . butanovora". South Med J, 2001 Feb, 94(2), 244 - 6 Aerosolized amikacin in the treatment of Pseudomonas pneumonia in the nursing home setting; Standridge JB et al.; Studies have shown that aerosolized aminoglycosides represent a safe and effective means of treating pneumonia due to Pseudomonas sp . Aerosolized aminoglycosides have been shown to improve clinical outcome, with less risk of nephrotoxicity relative to parenteral aminoglycosides . Apparently, less drug resistance is associated with the use of aerosolized aminoglycosides . Cost factors favor aerosolized delivery methods . The full recovery experienced by the patient in this case study suggests that aerosolized amikacin may be a safe, efficacious, and cost-effective means of treating pseudomonal pneumonia in geriatric patients . Controlled clinical trials should be conducted to further investigate this treatment regimen. Appl Microbiol Biotechnol, 2001 Jan, 55(1), 112 - 6 Development and utilisation of a medium to isolate phenanthrene-degrading Pseudomonas spp; Andersen SM et al.; In this study, we isolated phenanthrene degraders belonging to Pseudomonas spp . by combining the selective force of two previously described media . The two compounds, sodium lauryl sarcosine and trimethoprim, from the Gould S1 medium, were added to minimal agar plates sprayed with phenanthrene . Pseudomonas spp . that could produce clearing zones were isolated in one step from the rhizosphere without first selecting for Pseudomonas spp . and subsequently screening for degraders or vice versa . Enumeration and isolation of Pseudomonas spp . attached to the rhizosphere showed clear differences between two types of soil . Rhizosphere-attached phenanthrene degraders (from Pseudomonas spp.) were isolated from a former coal gasification site, but were absent in an agricultural soil subjected to organic farming . We isolated 23 phenanthrene degraders producing clearing zones from the rhizosphere of barley roots . All of these 23 isolates (of which 16 were fluorescent in UV light) proved to be members of the Pseudomonas RNA homology group I, on the basis of results of the analytical profile index (API) test system and classic taxonomic tests. Environ Microbiol, 2000 Aug, 2(4), 407 - 16 A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid); Lewis TA et al.; A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl4) transformation ability of wild-type KC . Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA . CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl4 dechlorination in cultures of strain KC . Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spel fragment absent in strain CTN1 . Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt -) phenotype . One cosmid (pT31) complemented the Pdt- phenotype of CTN1 and conferred CCl4 transformation activity and PDTC production upon other pseudomonads . Southern analysis showed that none of three other P . stutzeri strains representing three genomovars contained DNA that would hybridize with the 25,746 bp insert of pT31 . Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background . These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion . The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P . stutzeri strains. J Mol Evol, 2001 Feb, 52(2), 117 - 28 Phylogeny, genome evolution, and host specificity of single-stranded RNA bacteriophage (family Leviviridae); Bollback JP et al.; Bacteriophage of the family Leviviridae have played an important role in molecular biology where representative species, such as Q beta and MS2, have been studied as model systems for replication, translation, and the role of secondary structure in gene regulation . Using nucleotide sequences from the coat and replicase genes we present the first statistical estimate of phylogeny for the family Leviviridae using maximum-likelihood and Bayesian estimation . Our analyses reveal that the coliphage species are a monophyletic group consisting of two clades representing the genera Levivirus and Allolevivirus . The Pseudomonas species PP7 diverged from its common ancestor with the coliphage prior to the ancient split between these genera and their subsequent diversification . Differences in genome size, gene composition, and gene expression are shown with a high probability to have changed along the lineage leading to the Allolevivirus through gene expansion . The change in genome size of the Allolevivirus ancestor may have catalyzed subsequent changes that led to their current genome organization and gen