Pseudomonas

Appl Microbiol Biotechnol, 2003 Apr, 61(2), 140 - 9 Epub 2002 Oct 29.
Molecular cloning, sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, Pseudomonas sp PE2, and its domain structure; Kitamura E et al.; The pchA gene encoding chitinase A (PchA) from a Pythium porphyrae cell-wall-degrading marine bacterium, Pseudomonas sp . PE2, was cloned and characterized . The deduced PchA was a modular enzyme composed of an N-terminal signal peptide, a glycoside hydrolase family 18 catalytic domain that was responsible for the chitinase activity, the chitin-binding domains (ChBDs), and the carbohydrate-binding modules (CBM) . The amino acid sequence of ChBD(PchA) was highly conserved in the CBM family 12 that also accommodates ChBDs without an AKWWTQG motif, a domain commonly found in bacterial chitinase and Streptomyces griseus protease C . Interestingly, CBM(PchA) showed significant sequence homology to the C-terminal region of endoglucanase B from Cellvibrio mixtus, which is a member of CBM family 6 . This is the first report of a chitinase possessing a domain with high similarity to CBM family 6 . Deletion analysis indicated clearly that ChBD(PchA) might play an important role in the binding of native chitin and chitosan, but not processed chitin . CBM(PchA) also appeared to play such a role in the binding of xylan and Avicel . These results suggest that the C-terminal region of PchA might be a key component in the binding of chitin in the cell walls of P . porphyrae or other structural components of marine organisms.

Plant Physiol, 2003 Mar, 131(3), 1239 - 49
Pto mutants differentially activate Prf-dependent, avrPto-independent resistance and gene-for-gene resistance; Xiao F et al.; Pto confers disease resistance to Pseudomonas syringae pv tomato carrying the cognate avrPto gene . Overexpression of Pto under the cauliflower mosaic virus 35S promoter activates spontaneous lesions and confers disease resistance in tomato (Lycopersicon esculentum) plants in the absence of avrPto . Here, we show that these AvrPto-independent defenses require a functional Prf gene . Several Pto-interacting (Pti) proteins are thought to play a role in Pto-mediated defense pathways . To test if interactions with Pti proteins are required for the AvrPto-independent defense responses by Pto overexpression, we isolated several Pto mutants that were unable to interact with one or more Pti proteins, but retained normal interaction with AvrPto . Overexpression of two mutants, Pto(G50S) and Pto(R150S), failed to activate AvrPto-independent defense responses or confer enhanced resistance to the virulent P . s . pv tomato . When introduced into plants carrying 35S::Pto, 35S::Pto(G50S) dominantly suppressed the AvrPto-independent resistance caused by former transgene . 35S::Pto(G50S) also blocked the induction of a number of defense genes by the wild-type 35S::Pto . However, 35S::Pto(G50S) and 35S::Pto(R150S) plants were completely resistant to P . s . pv tomato (avrPto), indicating a normal gene-for-gene resistance . Furthermore, 35S::Pto(G50S) plants exhibited normal induction of defense genes in recognition of avrPto . Thus, the AvrPto-independent defense activation and gene-for-gene resistance mediated by Pto are functionally separable.

J Biol Chem, 2003 May 16, 278(20), 18606 - 16 Epub 2003 Mar 07.
Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA; Diaz-Lopez T et al.; RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10 . RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication . We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators . To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif . We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect . Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH . We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers . These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.

Biotechnol Appl Biochem, 2003 Apr, 37(Pt 2), 187 - 94
Overexpression of the pectin lyase gene of Pseudomonas marginalis in Escherichia coli and purification of the active enzyme; Papi R et al.; A pectin lyase gene (pnl) of Pseudomonas marginalis was cloned and overexpressed in Escherichia coli BL21(DE3) . The pnl gene was amplified by PCR, inserted into pET29c with a six-His tag and the overproduced active enzyme was purified almost to homogeneity using a Ni(2+)-nitrilotriacetate-agarose column . The purified pectin lyase (PNL; EC 4.2.2.10, family 1) is inhibited by NAD+ (at concentrations above 0.25 mM), NADH or dithiothreitol . Evidence for the existence of a heat-labile protein inhibitor of PNL is also reported . The DNA-binding ability of PNL was demonstrated by DNA-retardation experiments . The partially purified enzyme was incubated with plasmid DNA and the complex was shifted to a higher molecular mass . Analysis of the electroeluted proteins from the protein-DNA complex revealed that one of the electroeluted protein bands was PNL . Antibodies against the overexpressed PNL were also prepared and partially purified.

J Pharmacol Toxicol Methods, 2002 May-Jun, 47(3), 169 - 75
An ELISA method for detection of human antibodies to an immunotoxin; Benbrook DM; INTRODUCTION: The use of biological molecules, such as immunotoxins, as pharmaceuticals is limited by the presence and development of human antibodies to these agents . This immune response can cause significant inflammatory-related toxicities and can interfere with the efficacy of the biological agent . Therefore, a clinically applicable method to detect these human antibodies is needed for screening patients prior to enrollment and for monitoring patients during treatment . The SS1(dsFv)-PE38 immunotoxin currently in clinical trials is a hybrid molecule targeted against mesothelin-expressing cancer cells via the Fv portion of a murine antibody linked to the Pseudomonas exotoxin (PE), which can inhibit protein synthesis leading to cell death . The objective of this study was to determine if an enzyme-linked immunosorbent assay (ELISA)-based method could be used to detect human anti-SS1(dsFv)-PE38 antibodies in patient serum . METHODS: Human antibodies to the immunotoxin in serially diluted serum specimens were captured on immunotoxin-coated ELISA plates, and detected using a secondary goat antihuman antibody linked to biotin in combination with horseradish peroxidase linked to avidin D (HRP-Avidin) . The color was developed with tetramethyl benzidine (TMB) . Curves of optical density (OD(630)) versus dilution for 44 serum specimens were compared with positive and negative control serum specimens to classify the serum as positive or negative for anti-immunotoxin antibodies . RESULTS: Ten out of the 40 patients screened were positive for anti-immunotoxin antibodies . Repeated testing of seven samples produced the same results in two independent experiments . The first two patients treated with the immunotoxin developed anti-immunotoxin antibodies during treatment . The results were in perfect concordance with a tissue culture-based neutralization assay performed by an independent laboratory . DISCUSSION: An ELISA-based strategy using an immunotoxin to capture human anti-immunotoxin antibodies provides a consistently accurate technology for screening and monitoring patient serum specimens in clinical trials.

J Mol Biol, 2003 Mar 21, 327(2), 445 - 52
Design of a modular immunotoxin connected by polyionic adapter peptides; Kleinschmidt M et al.; Immunotoxins are genetically engineered fusion proteins of an antibody Fv fragment and a toxin from bacteria or plants, which function as anti-cancer therapeutics . Here, we describe a new generation of immunotoxins in which both proteins do not form a single fusion protein but are coupled specifically via cysteine-containing polyionic fusion peptides . The engineered Pseudomonas exotoxin PE38 was N-terminally fused to the peptide E(8)C . In combination with the disulfide-stabilized Fv fragment of the tumor-specific antibody B3, which was extended by the peptide R(8)CP, the fusion peptides ensured a specific and covalent coupling of the Fv fragment and the toxin . The resulting immunotoxin was as active and as specific as an immunotoxin consisting of a fusion protein of the same antibody fragment connected to the toxin.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3519 - 24 Epub 2003 Mar 07.
Interplay of the Arabidopsis nonhost resistance gene NHO1 with bacterial virulence; Kang L et al.; It is poorly understood why a particular plant species is resistant to the vast majority of potential pathogens that infect other plant species, a phenomenon referred to as "nonhost" resistance . Here, we show that Arabidopsis NHO1, encoding a glycerol kinase, is required for resistance to and induced by Pseudomonas syringae isolates from bean and tobacco . NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance . Strikingly, P . s . pv . tomato DC3000, an isolate fully virulent on Arabidopsis, actively suppressed the NHO1 expression . This suppression is abolished in coi1 plants, indicating that DC3000 required an intact jasmonic acid signaling pathway in the plant to suppress NHO1 expression . Constitutive overexpression of NHO1 led to enhanced resistance to this otherwise virulent bacterium . The presence of avrB in DC3000, which activates a cultivar-specific "gene-for-gene" resistance in Arabidopsis, restored the induction of NHO1 expression . Thus, NHO1 is deployed for both general and specific resistance in Arabidopsis and targeted by the bacterium for parasitism.

Biomacromolecules, 2003 Mar-Apr, 4(2), 424 - 8
First-order kinetics analysis of monomer composition dependent polyhydroxyalkanoic acid degradation in Pseudomonas spp; Choi MH et al.; The intracellular degradation of polyhydroxyalkanoic acid (PHA) in pseudomonads was investigated by first-order kinetics analysis using the initial rate method . One type of PHA was accumulated in five Pseudomonas spp., P . oleovorans, P . aeruginosa, P . fluorescens, P . citronellolis, and P . putida, by growing them on octanoic acid . The monomer compositions of the five PHA were not significantly different from one another: 85-90 mol % 3-hydroxyoctanoic acid (3HO), 7-12 mol % 3-hydorxycaproic acid (3HC), and 3-6 mol % 3-hydroxydecanoic acid (3HD) . The first-order degradation rate constants (k(1)) for the octanoate-derived PHA (designated P(3HO)) in the five species were in a similar range between 0.060 and 0.088 h(-1) . This may indicate the similar specificities of the five intracellular depolymerases . In addition, the similar k(1) among the different species may correlate with the high degree of amino acid sequence identities (over 85%) among the intracellular PHA depolymerase phaZ genes . Six other chemically different types of PHA were accumulated in P . putida from n-nonanoic acid, n-decanoic acid, 5-phenyvaleric acid, or 11-phenoxyundecanoic acid as a single or a mixed carbon source . The calculated k(1) values were characteristic to each PHA, reflecting their chemical structures . In comparison with P(3HO), an increase in the levels of the two minor monomers 3HC and 3HD as in P(21 mol % 3HC-co-56 mol % 3HO-co-23 mol % 3HD) significantly slowed the rate of intracellular degradation . From the comparison of k(1) values, it is suggested that the P . putida intracellular depolymerase is most active against P(3HO).

Biomacromolecules, 2003 Mar-Apr, 4(2), 204 - 10
Solid-phase handling of hydrophobins: immobilized hydrophobins as a new tool to study lipases; Palomo JM et al.; Hydrophobins are fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach . This attribute can be used to introduce hydrophobic foci on the surface of hydrophilic supports where hydrophobins are attached by covalent binding . In this paper, we report the binding of Pleurotus ostreatus hydrophobins to a hydrophilic matrix (agarose) to construct a support for noncovalent immobilization and activation of lipases from Candida antarctica, Humicola lanuginosa, and Pseudomonas flourescens . Lipase immobilization on agarose-bound hydrophobins proceeded at very low ionic strength and resulted in increased lipase activity and stability . The enzyme could be desorbed from the support using moderate concentrations of Triton X-100, and its enantioselectivity was similar to that of lipases interfacially immobilized on conventional hydrophobic supports . These results suggest that lipase adsorption on hydrophobins follows an "interfacial activation" mechanism; immobilization on hydrophobins offers new possibilities for lipase study and modulation and reveals a new application for fungal hydrophobins.

Mol Microbiol, 2003 Mar, 47(6), 1545 - 62
Nucleotide sequence, functional characterization and evolution of pFKN, a virulence plasmid in Pseudomonas syringae pathovar maculicola; Rohmer L et al.; Pseudomonas syringae pv . maculicola strain M6 (Psm M6) carries the avrRpm1 gene, encoding a type III effector, on a 40 kb plasmid, pFKN . We hypothesized that this plasmid might carry additional genes required for pathogenesis on plants . We report the sequence and features of pFKN . In addition to avrRpm1, pFKN carries an allele of another type III effector, termed avrPphE, and a gene of unknown function (ORF8), expression of which is induced in planta, suggesting a role in the plant-pathogen interaction . The region of pFKN carrying avrRpm1, avrPphE and ORF8 exhibits several features of pathogenicity islands (PAIs) . Curing of pFKN (creating Psm M6C) caused a significant reduction in virulence on Arabidopsis leaves . However, complementation studies using Psm M6C demonstrated an obvious virulence function only for avrRpm1 . pFKN can integrate and excise from the chromosome of Psm M6 at low frequency via homologous recombination between identical sequence segments located on the chromosome and on pFKN . These segments are part of two nearly identical transposons carrying avrPphE . The avrPphE transposon was also detected in other strains of P . s . pv . maculicola and in P . s . tomato strain DC3000 . The avrPphE transposon was found inserted at different loci in different strains . The analysis of sequences surrounding the avrPphE transposon insertion site in the chromosome of Psm M6 indicates that pFKN integrates into a PAI that encodes type III effectors . The integration of pFKN into this chromosomal region may therefore be seen as an evolutionary process determining the formation of a new PAI in the chromosome of Psm M6.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 167 - 72
Genetic diversity of phlD gene from 2,4-diacetylphloroglucinol-producing Pseudomonas spp . strains from the maize rhizosphere; Picard C et al.; In biocontrol Pseudomonads, phlD is an essential gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) . HaeIII restriction of amplified phlD gene, previously proposed as the most discriminant analysis, showed no polymorphism among 144 Pseudomonas strains isolated from maize roots . However, these strains fell into three statistically significant DAPG production level groups . phlD sequences of 13 strains belonging to the three DAPG groups revealed a KspI restriction site only in good DAPG-producing strains . This result was confirmed on the 144 strains, 82 of which were identified as good-DAPG producers by both biochemical and amplified phlD KspI restriction analysis . They are candidates as potential biocontrol agents.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 207 - 10
Differential scanning calorimetry of the effects of Ca2+ on the thermal unfolding of Pseudomonas cepacia lipase; Tanaka A et al.; Thermal unfolding of P . cepacia lipase was observed by adiabatic differential scanning microcalorimetry in the absence and presence of calcium ions at pH 8, and thermodynamic parameters of unfolding were evaluated to analyze the unfolding mechanism of the enzyme . The temperature of unfolding was higher at higher concentrations of Ca2+ . From the Ca2+ concentration-dependence of the unfolding temperature, the number of calcium ions that dissociated from the enzyme molecule upon unfolding was estimated to be one . These results confirmed the validity of the unfolding mechanism proposed previously: NCa2+ < = => D + Ca2+, where N and D represent the native and denatured states, respectively, of the enzyme.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 36 - 45
Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans strain CA10; Nojiri H et al.; 2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid {6-(2'-aminophenyl)-HODA} hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain . The enzyme was dimeric, and its optimum pH was 7.0-7.5 . Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde . The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C) . The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg) . The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.

Genetics, 2003 Feb, 163(2), 735 - 46
Natural selection for polymorphism in the disease resistance gene Rps2 of Arabidopsis thaliana; Mauricio R et al.; Pathogen resistance is an ecologically important phenotype increasingly well understood at the molecular genetic level . In this article, we examine levels of avrRpt2-dependent resistance and Rps2 locus DNA sequence variability in a worldwide sample of 27 accessions of Arabidopsis thaliana . The rooted parsimony tree of Rps2 sequences drawn from a diverse set of ecotypes includes a deep bifurcation separating major resistance and susceptibility clades of alleles . We find evidence for selection maintaining these alleles and identify the N-terminal part of the leucine-rich repeat region as a probable target of selection . Additional protein variants are found within the two major clades and correlate well with measurable differences among ecotypes in resistance to the avirulence gene avrRpt2 of the pathogen Pseudomonas syringae . Long-lived polymorphisms have been observed for other resistance genes of A . thaliana; the Rps2 data suggest that the long-term maintenance of phenotypic variation in resistance genes may be a general phenomenon and are consistent with diversifying selection acting in concert with selection to maintain variation.

Teratog Carcinog Mutagen, 2003, Suppl 1, 283 - 94
Customized cDNA microarray for expression profiling of environmentally important genes of Pseudomonas stutzeri strain KC; Musarrat J et al.; DNA microarray is a powerful tool for parallel detection of multiple target genes in biological systems . In this study, a low-density DNA microarray has been custom designed by using Pseudomonas stutzeri strain KC ORFs that are implicated in carbon tetrachloride degradation . PCR amplified strain KC probes of varying lengths were obtained using ORF-specific primers . Purified short probes (80-120 bp) and full-length amplicons were directly immobilized on gamma-aminosilane coated and superaldehyde trade mark glass substrates without any chemical modification . The full-length amplicons exhibited a much higher signal compared to the shorter probes upon hybridization with the Cy5/Cy3-labeled unfragmented cDNA targets . The meager signal with the shorter probes limits the advantage of using the multiple probes of the same genes for enhancing the specificity of hybridization with environmental samples . Nevertheless, expression analysis of strain KC genome, under controlled laboratory conditions, revealed the constitutive expression of at least 11 putative ORFs of the pdt operon . Comparatively weaker hybridization signals with the cDNA from mutant cells suggested a low abundance of mRNA transcripts in the KC 1896 mutant . Similar expression levels of the pdt ORFs I, J, K, M, N, O, P, and fur gene both under iron-limiting conditions and in presence of iron (20 micro M Fe(3+)) suggested metal ion-independent regulation of the pdt operon . The tailor-made array with strain KC gene-specific probes served as a model for demonstrating the utility of cDNA microarray technology in monitoring the expression of environmentally important genes in bacteria .

Plant Cell, 2003 Mar, 15(3), 760 - 70
NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol; Spoel SH et al.; Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles . In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling . Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling . Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1 . Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.

Curr Opin Microbiol, 2003 Feb, 6(1), 20 - 8
Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells; Greenberg JT et al.; Many bacterial pathogens cause disease by injecting virulence proteins (effectors) into host cells via the specialized type III secretion system . Recently, exceptional progress in identifying effectors was made in the phytopathogen Pseudomonas syringae using a novel genetic screen and bioinformatic approach . These studies, along with localization experiments, suggest that most P . syringae effectors function by targeting the plasma membrane, chloroplasts or mitochondria of host cells . The type III secretome of P . syringae is highly variable and dynamic, a lesson gleaned from a comparative genomic analysis . Variation in the effector repertoire is likely to facilitate the adaptation of P . syringae to different hosts.

Arch Microbiol, 2003 Mar, 179(3), 151 - 9 Epub 2003 Feb 08.
Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase; Sonawane A et al.; The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen . All key enzymes of glutamate metabolism were detected in P . fluorescence, with glutaminase and asparaginase showing the highest specific activities . A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains . The enzyme was purified and shown to be identical with the ansB gene product described previously . In addition to PGA, P . fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn . PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu) . In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites . A mutant of P . putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.

Plant J, 2003 Feb, 33(4), 733 - 42
Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv . phaseolicola is due to degradation products of salicylic acid; van Wees SC et al.; In plants carrying the NahG transgene, salicylate hydroxylase converts salicylic acid (SA) to catechol . Arabidopsis NahG plants are defective in non-host resistance to Pseudomonas syringae pv . phaseolicola strain 3121 (Psp), suggesting that resistance requires SA signaling . However, several mutants with defects in SA signaling, including eds1, pad4, eds5, sid2, and npr1, remain resistant to Psp, demonstrating that susceptibility of NahG plants is not due to absence of SA . SA synthesis is blocked in sid2NahG double mutants, but resistance to Psp is retained . Therefore, it must be the degradative action of NAHG on SA that causes the loss of resistance of NahG to Psp . Treatment of plants with catechol compromised Psp resistance suggesting that the effect of NahG on resistance results from catechol production . Application of catalase to NahG or catechol-treated wild-type plants partially restored resistance to Psp, suggesting that the deleterious effect of catechol results from inappropriate production of hydrogen peroxide . These results indicate that conclusions about SA requirements based solely on phenotypes of NahG plants should be re-evaluated.

Plant J, 2003 Feb, 33(4), 665 - 76
Expression profiling of the host response to bacterial infection: the transition from basal to induced defence responses in RPM1-mediated resistance; de Torres M et al.; Changes in transcription in leaves of Arabidopsis thaliana were characterised following challenge with strains of Pseudomonas syringae pv . tomato DC3000 allowing differentiation of basal resistance (hrpA mutants), gene-specific resistance (RPM1-specified interactions) and susceptibility (wild-type pathogen) . In planta avirulence gene induction, changes in host {Ca2+}cyt and leaf collapse were used to delineate the transition from infection to induced resistance . The plant responds rapidly, dynamically and discriminately to infection by phytopathogenic bacteria . Within the first 2 h host transcriptional changes are common to all challenges indicating that Type III effector function does not contribute to early events in host transcriptome re-programming . The timing of induction for specific transcripts was reproducible, hierarchical and modulated at least in part through EDS1 function . R gene-specific transcripts were not observed until 3 h after inoculation . Intriguingly, the R gene-specific response proteins are expected to localise to diverse cellular addresses indicative of a global impact on cellular homeostasis . The altered transcriptional response rapidly manifests into initial symptoms of leaf collapse within 2 h, although establishment of the full macroscopic HR occurs significantly later.

Microbiol Res, 2003, 158(1), 47 - 54
Molecular diversity in the bacterial community and the fluorescent pseudomonads group in natural and chlorobenzoate-stressed peat-forest soil; Ramirez-Saad HC et al.; Bacterial community shifts in a soil microcosm spiked with 3-chlorobenzoate or 2,5-dichlorobenzoate were monitored . The V6-V8 variable regions of soil bacterial 16S rRNA and rDNA were amplified and separated by temperature gradient gel electrophoresis (TGGE) profiling . Culturing in the presence of 2.5 mM chlorinated benzoates suppressed 10 to 100 fold the total aerobic bacterial community but had no effect on the diversity within the group of fluorescent pseudomonads . In contrast, the uncultured bacterial community showed a decrease in the number of bands in the TGGE profiles of the chlorobenzoate-spiked treatments . Accordingly, the Shannon's diversity and equitability indices of these treatments reflected a decreasing trend in time . The approach allowed a direct assessment of community shifts upon contamination of soil.

Dis Aquat Organ, 2003 Jan 22, 53(1), 33 - 9
Bacteriophage control of Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis; Park SC et al.; Two previously isolated phages were used to examine the therapeutic effects against Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis . Phage PPp-W4 (Podoviridae) inhibited the in vitro growth of P . plecoglossicida more effectively than Phage PPpW-3 (Myoviridae), and a mixture (PPpW-3/W-4) of the 2 phages exhibited the highest inhibitory activity . In phage therapy experiments, ayu were fed P . plecoglossicida-impregnated feed (10(7) CFU fish(-1)) and then fed phage-impregnated feed (10(7) PFU fish(-1)) . Mortalities of fish receiving PPpW-3, PPpW-4, PPpW-3/W-4, and a control fish receiving no phages were 53.3, 40.0, 20.0 and 93.3%, respectively . Phage (PPpW-3/W-4)-receiving fish also showed high protection against water-borne infection with P . plecoglossicida . In a field trial, when phage (PPpW-3/W-4)-impregnated feed was administered to ayu in a pond where the disease occurred naturally, daily mortality of fish decreased at a constant level (5% d(-1)) to one-third after a 2 wk period . The causal relationship of phages in this phenomenon was verified by the long-lasting appearance of administered phages in the kidneys of the fish, and a disappearance of P . plecoglossicida from apparently healthy fish . Neither phage-resistant organisms nor phage-neutralizing antibodies were detected in diseased fish or apparently healthy fish, respectively . These results indicate the potential for phage control of the disease.

Plant Mol Biol, 2003 Jan, 51(1), 21 - 37
Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response; Dong J et al.; WRKY proteins are a recently identified class of DNA-binding proteins that recognize the TTGAC(C/T) W-box elements found in the promoters of a large number of plant defense-related genes . With oligo molecules containing the W-box sequences as probes, we detected a number of WRKY DNA-binding activities in Arabidopsis that were induced by salicylic acid (SA) . Search of the Arabidopsis genome identifies 72 genes encoding proteins characteristic of WRKY DNA-binding transcription factors that can be divided into three groups based on the number and structures of their WRKY zinc-finger motifs . Northern blotting analysis revealed that 49 of the 72 AtWRKY genes were differentially regulated in the plants infected by an avirulent strain of the bacterial pathogen Pseudomonas syringae or treated by SA . These pathogen- and/or SA-regulated WRKY genes can be further categorized into groups based on their expression patterns in both wild-type plants and mutants defective in defense signaling pathways . Inspection of the 5' sequences upstream of the predicated translation start sites revealed a substantial enrichment of W boxes in the promoters of pathogen- and/or SA-regulated Arabidopsis WRKY genes . These results suggest that defense-regulated expression of WRKY genes involves extensive transcriptional activation and repression by its own members of the transcription factor superfamily.

J Basic Microbiol, 2003, 43(1), 56 - 61
Induction of phenol utilization in Pseudomonas CF600 grown under varying nitrogen levels; Moharikar A et al.; This study demonstrates the effect of various nitrogen levels in the medium along with different carbon sources in the media on the utilization of phenol by Pseudomonas CF600 . Experiments were carried out using cultures derived from minimal media containing the carbon sources phenol and citrate, followed by varying nitrogen levels in the medium . Respirometric analysis under different conditions was carried out with cells using phenol and catechol as substrates . When nitrogen was limiting in the medium, phenol induced higher oxygen uptake rates, whereas catechol was independent of the nitrogen levels . These observations were also supported by the residual phenol levels in the medium with different levels of nitrogen as NH(4) ion . Results show that nitrogen-limiting conditions favor the phenol utilization by Pseudomonas CF600.

J Biol Inorg Chem, 2003 Feb, 8(3), 318 - 26 Epub 2002 Nov 07.
N-isotope effects on the Raman spectra of Fe(2)S(2) ferredoxin and Rieske ferredoxin: evidence for structural rigidity of metal sites; Rotsaert FJ et al.; The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe(2)S(2) ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins . Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe(2)S(2) ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides . We now find that uniform (15)N-labeling of Anabaena Fe(2)S(2) ferredoxin results in shifts of -1 cm(-1) in the Fe-S(t) stretching modes at 282, 340, and 357 cm(-1) . The (15)N dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen . No (15)N dependence occurs for the nu(Fe-S(b)) modes at 395 and 426 cm(-1) . Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-monooxygenase system of Pseudomonas mendocina . Upon selective (15)N-labeling of the alpha-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm(-1) all undergo shifts of -1 to -2 cm(-1), thereby identifying them as combinations of nu(Fe-S(t)) and delta(Cys) . These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with (15)N-histidine ((15)N in either the alpha1,delta1 or delta1,epsilon2 positions) . Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety . The extensive kinematic coupling of iron ligand vibrations observed in both the Fe(2)S(2) and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins . In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer.

Acta Microbiol Pol, 2002, 51(3), 247 - 54
Ability of Pseudomonas sp . to synthesize aminopeptidases in the presence of various carbon and nitrogen sources; Jankiewicz U et al.; A soil strain of Pseudomonas sp . is able to synthesize at least two aminopeptidases exhibiting high activity in the presence of Phe-beta-NA and Ala-beta-NA as substrates . Irrespective of the used substrate, total activity of studied enzymes was strongly related to concentrations of organic components (peptone, glutamic acid, glucose) in mineral media and was the higher, the higher the concentration . Tendency of changes in total activity was similar for alanyl- and phenylalanylaminopeptidase though their response to different concentrations of organic components was different . Specific activity measured in the presence of Phe-beta-NA and Ala-beta-NA as the substrates was not strictly dependent on increasing concentrations of organic components in the media . The highest specific activity of aminopeptidase was obtained in the presence of Phe-beta-NA as a substrate on the fifth day of culture in medium containing 1% glucose . The obtained results seem to indicate the inductive character of the studied aminopeptidases . On the other hand, however, they do not exclude other regulatory mechanisms of their synthesis, including catabolic repression.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 271 - 6
Growth promotion of the edible fungus Pleurotus ostreatus by fluorescent pseudomonads; Cho YS et al.; Bacteria were isolated from the mycelial surface of Pleurotus ostreatus and their role in fruiting body induction (fructification) of the edible mushroom P . ostreatus was investigated . Analysis of the bacterial community that colonized the mycelium showed that the species composition and numbers of culturable bacteria differed according to the developmental stage of P . ostreatus . In particular, the population size of fluorescent pseudomonads increased during fruiting body induction . An experiment showed that inoculation of pure cultures of the mycelium with strains of fluorescent Pseudomonas spp . isolated from the mycelial plane of commercially produced mushrooms promoted the formation of primordia and enhanced the development of the basidiome of P . ostreatus . Results of this research strongly suggest that inoculation of the mycelium with specific bacteria may have beneficial applications for mushroom production.

Syst Appl Microbiol, 2002 Dec, 25(4), 513 - 9
Isolation and characterization of a new type of aerobic, oxalic acid utilizing bacteria, and proposal of Oxalicibacterium flavum gen . nov., sp . nov; Tamer AU et al.; A mesophilic, aerobic oxalic acid utilizing yellow-pigmented bacterium has been isolated from litter of oxalate producing plants in the region of Izmir (Turkey) . It is motile by means of 1-3 polar flagella . Optimal growth occurred between 25-30 degrees C at pH 6.9 . The G+C content of DNA is 62-64 mol % (Tm) . Based on its morphological and biochemical features the organism belongs to the genus Pseudomonas, but differs from all the previously described species . The taxonomic relationships among strains described as or previously tentatively assigned to the genus Pseudomonas were investigated using numerical classification, DNA base composition and DNA-DNA hybridization . 16S rDNA sequences were determined for the strain TA17 . On the basis of 16S rDNA sequence comparisons, physiological and biochemical characteristics, it is proposed to classify TA17T in a new genus and species for which the name Oxalicibacterium flavum gen . nov., sp . nov . is proposed . The type strain is TA17T (= NEU98T, = LMG 21571T).

Cell, 2003 Feb 7, 112(3), 379 - 89
Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance; Mackey D et al.; Type III pili deliver effector proteins (virulence factors) from bacterial pathogens to host cells . Plants express disease resistance (R) proteins that respond specifically to a particular type III effector by activating immune responses . We demonstrated previously that two unrelated type III effectors from Pseudomonas syringae target and modify the Arabidopsis RIN4 protein . Here, we show that AvrRpt2, a third, unrelated type III effector, also targets RIN4 and induces its posttranscriptional disappearance . This effect is independent of the presence of RPS2, the Arabidopsis R protein that senses AvrRpt2 . RIN4 overexpression inhibits multiple phenotypes associated with AvrRpt2 function . Conversely, disruption of RIN4 results in RPS2-dependent lethality . RPS2 and RIN4 physically associate in the plant . We suggest that RIN4 is the target of the AvrRpt2 virulence function, and that perturbation of RIN4 activates RPS2 . Thus, RIN4 is a point of convergence for the activity of at least three unrelated P . syringae type III effectors.

Cell, 2003 Feb 7, 112(3), 369 - 77
Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4; Axtell MJ et al.; Plants have evolved a sophisticated innate immune system to recognize invading pathogens and to induce a set of host defense mechanisms resulting in disease resistance . Pathogen recognition is often mediated by plant disease resistance (R) proteins that respond specifically to one or a few pathogen-derived molecules . This specificity has led to suggestions of a receptor-ligand mode of R protein function . Delivery of the bacterial effector protein AvrRpt2 by Pseudomonas syringae specifically induces disease resistance in Arabidopsis plants expressing the RPS2 R protein . We demonstrate that RPS2 physically interacts with Arabidopsis RIN4 and that AvrRpt2 causes the elimination of RIN4 during activation of the RPS2 pathway . AvrRpt2-mediated RIN4 elimination also occurs in the rps2, ndr1, and Atrar1 mutant backgrounds, demonstrating that this activity can be achieved independent of an RPS2-mediated signaling pathway . Therefore, we suggest that RPS2 initiates signaling based upon perception of RIN4 disappearance rather than direct recognition of AvrRpt2.

Mol Plant Microbe Interact, 2003 Jan, 16(1), 43 - 52
A pseudomonas syringae pv . tomato DC3000 Hrp (Type III secretion) deletion mutant expressing the Hrp system of bean pathogen P . syringae pv . syringae 61 retains normal host specificity for tomato; Fouts DE et al.; The plant pathogenic species Pseudomonas syringae is divided into numerous pathovars based on host specificity . For example, P . syringae pv . tomato DC3000 is pathogenic on tomato and Arabidopsis, whereas P . syringae pv . syringae 61 is pathogenic on bean . The ability of P . syringae strains to elicit the hypersensitive response (HR) in non-hosts or be pathogenic (or parasitic) in hosts is dependent on the Hrp (type III secretion) system and effector proteins this system is thought to inject into plant cells . To test the role of the Hrp system in determining host range, the hrp/hrc gene cluster (hrpK through hrpR) was deleted from DC3000 and complemented in trans with the orthologous cluster from strain 61 . Mutant CUCPB5114 expressing the bean pathogen Hrp system on plasmid pCPP2071 retained the ability of wild-type DC3000 to elicit the HR in bean, to grow and cause bacterial speck in tomato, and to elicit a cultivar-specific (gene-for-gene) HR in tomato plants carrying the Pto resistance gene . However, the symptoms produced in compatible tomato plants involved markedly reduced chlorosis, and CUCPB5114(pCPP2071) did not grow or produce symptoms in Arabidopsis Col-0 although it was weakly virulent in NahG Arabidopsis . A hypersensitive-like collapse was produced by CUCPB5114(pCPP2071) in Arabidopsis Col-0 at 1 x 10(7) CFU/ml, but only if the bacteria also expressed AvrB, which is recognized by the RPM1 resistance gene in Col-0 and confers incompatibility . These observations support the concept that the P . syringae effector proteins, rather than secretion system components, are the primary determinants of host range at both the species and cultivar levels of host specificity.

Proteins, 2003 Mar 1, 50(4), 636 - 47
Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases; Aghajari N et al.; Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures . It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs . Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomonas species (PAP), solved to 2.1- and 1.96-A resolution, respectively . Comparative studies of PAP structures with mesophilic counterparts show that the overall structures are similar but that the conformation of the substrate-free active site in PAP resembles that of the substrate-bound region of the mesophilic homolog, with both an active-site tyrosine and a substrate-binding loop displaying a conformation as in the substrate-bound form of the mesophilic proteases . Further, a region in the catalytic domain of PAP undergoes a conformational change with a loop movement as large as 13 A, induced by the binding of an extra calcium ion . Finally, the active site is more accessible due to deletions occurring in surrounding loop regions .

Microbiology, 2003 Jan, 149(Pt 1), 37 - 46
Surface motility in Pseudomonas sp . DSS73 is required for efficient biological containment of the root-pathogenic microfungi Rhizoctonia solani and Pythium ultimum; Andersen JB et al.; Pseudomonas sp . DSS73 was isolated from the rhizoplane of sugar beet seedlings . This strain exhibits antagonism towards the root-pathogenic microfungi Pythium ultimum and Rhizoctonia solani . Production of the cyclic lipopeptide amphisin in combination with expression of flagella enables the growing bacterial culture to move readily over the surface of laboratory media . Amphisin is a new member of a group of dual-functioning compounds such as tensin, viscosin and viscosinamid that display both biosurfactant and antifungal properties . The ability of DSS73 to efficiently contain root-pathogenic microfungi is shown to arise from amphisin-dependent surface translocation and growth by which the bacterium can lay siege to the fungi . The synergistic effects of surface motility and synthesis of a battery of antifungal compounds efficiently contain and terminate growth of the microfungi.

J Immunol, 2003 Feb 15, 170(4), 2236 - 41
In vivo efficacy of anti-glycoprotein 41, but not anti-glycoprotein 120, immunotoxins in a mouse model of HIV infection; Pincus SH et al.; Immunotoxins (ITs) targeting the HIV envelope protein are among the most efficacious antiviral therapies when tested in vitro . Yet a first-generation IT targeted to gp120, CD4-PE40 (chimeric immunotoxin using CD4 and the translocation and enzymatic domains of Pseudomonas exotoxin A), showed limited promise in initial clinical testing, highlighting the need for improved ITs . We have used a new mouse model of HIV infection to test the comparative efficacy of anti-HIV ITs targeted to gp120 or to gp41 . Irradiated SCID/nonobese diabetic mice are injected with a tumor of human CD4(+) cells susceptible to infection and at a separate site persistently HIV-infected cells . The spread of infection from infected to susceptible tumor is monitored by plasma p24 and the presence of HIV-infected cells in the spleen . Anti-gp41 ITs in combination with tetrameric CD4-human Ig fusion protein have pronounced anti-HIV effects . Little if any anti-HIV efficacy was found with either CD4-PE40 or an Ab-targeted anti-gp120 IT . These data support continued exploration of the utility of ITs for HIV infection, particularly the use of anti-gp41 ITs in combination with soluble CD4 derivatives.

J Immunol, 2003 Feb 15, 170(4), 2129 - 37
Parenchymal, but not leukocyte, TNF receptor 2 mediates T cell-dependent hepatitis in mice; Schumann J et al.; TNF-alpha is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA) . In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs . Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear . TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines . In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model . Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process . Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type --> tnfr2 degrees and the reciprocal mouse bone marrow chimeras . Furthermore, PEA induced NF-kappaB activation and cytokine production in the livers of both wild-type and tnfr2 degrees mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2 degrees, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs . Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo . The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.

Bioresour Technol, 2003 May, 88(1), 41 - 6
Determination of organochlorine pesticides in agricultural soil with special reference to gamma-HCH degradation by Pseudomonas strains; Nawab A et al.; Soil samples were taken from different agricultural fields and analyzed for organochlorine pesticide residues by gas chromatography . The analysis indicated that the soil samples contained some common organochlorine pesticides DDT, DDD, DDE, HCH and Aldrin . gamma-HCH was detected as 47.35 ppb whereas the concentrations of alpha-HCH, beta-HCH, p('),p(')-DDE, o('),p(')-DDT were 38.81, 1.79, 7.10 and 13.30 ppb, respectively, in the same soil . Two Pseudomonas strains isolated from agricultural soil were found to possess gamma-hexachlorocyclohexane degrading ability when the isolates were grown in a mineral salt medium containing gamma-HCH as the sole source of carbon and a number of metabolites were produced and detected by the gas chromatography . These bacterial isolates were further tested for carbohydrate and amino acid utilization as well as for their susceptibility against 10 commonly used antibiotics namely amoxycillin, chloramphenicol, cloxacillin, doxycycline, methicillin, nalidixic acid, neomycin, nitrofurantoin, streptomycin and tetracycline . Both the isolates were also screened for plasmid DNA and found to harbour a single plasmid.

Appl Environ Microbiol, 2003 Feb, 69(2), 1290 - 4
Enhancement of population size of a biological control agent and efficacy in control of bacterial speck of tomato through salicylate and ammonium sulfate amendments; Ji P et al.; Sodium salicylate and ammonium sulfate were applied to leaf surfaces along with suspensions of the biological control agents Pseudomonas syringae Cit7(pNAH7), which catabolizes salicylate, and Cit7, which does not catabolize salicylate, to determine whether enhanced biological control of bacterial speck of tomato could be achieved . Foliar amendment with salicylate alone significantly enhanced the population size and the efficacy of Cit7(pNAH7), but not of Cit7, on tomato leaves . Application of ammonium sulfate alone did not result in enhanced population size or biological control efficacy of either Cit7(pNAH7) or Cit7; however, when foliar amendments with both sodium salicylate and ammonium sulfate were applied, a trend toward further increases in population size and biological control efficacy of Cit7(pNAH7) was observed . This study demonstrates the potential of using a selective carbon source to improve the efficacy of a bacterial biological control agent in the control of a bacterial plant disease and supports previous conclusions that the growth of P . syringae in the phyllosphere is primarily carbon limited and secondarily nitrogen limited.

Appl Environ Microbiol, 2003 Feb, 69(2), 1220 - 8
Differences between Pseudomonas syringae pv . syringae B728a and Pantoea agglomerans BRT98 in epiphytic and endophytic colonization of leaves; Sabaratnam S et al.; The leaf colonization strategies of two bacterial strains were investigated . The foliar pathogen Pseudomonas syringae pv . syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed . The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves . The P . agglomerans strain exclusively colonized epiphytic sites on the two plant species . Under favorable conditions, the P . agglomerans strain formed aggregates that often extended over multiple epidermal cells . The P . syringae pv . syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P . syringae pv . syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period . The epiphytic P . syringae pv . syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites . The endophytic P . syringae pv . syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize . A rainstorm involving a high raindrop momentum was associated with rapid growth of the P . agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P . syringae pv . syringae strain on bean but not with growth of the P . syringae pv . syringae strain on maize . These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P . syringae pv . syringae strain were dependent on the plant species, whereas those of the nonpathogenic P . agglomerans strain were not.

Appl Environ Microbiol, 2003 Feb, 69(2), 1143 - 53
High-performance liquid chromatography analyses of pyoverdin siderophores differentiate among phytopathogenic fluorescent Pseudomonas Species; Bultreys A et al.; The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated . A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins . Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P . syringae . Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins . Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species . Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P . syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine . The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin . The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two beta-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based . The peptide chain influenced the chelation of iron more in atypical pyoverdins . Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.

Appl Environ Microbiol, 2003 Feb, 69(2), 1004 - 12
Comparative genetic diversity of the narG, nosZ, and 16S rRNA genes in fluorescent pseudomonads; Delorme S et al.; The diversity of the membrane-bound nitrate reductase (narG) and nitrous oxide reductase (nosZ) genes in fluorescent pseudomonads isolated from soil and rhizosphere environments was characterized together with that of the 16S rRNA gene by a PCR-restriction fragment length polymorphism assay . Fragments of 1,008 bp and 1,433 bp were amplified via PCR with primers specific for the narG and nosZ genes, respectively . The presence of the narG and nosZ genes in the bacterial strains was confirmed by hybridization of the genomic DNA and the PCR products with the corresponding probes . The ability of the strains to either reduce nitrate or totally dissimilate nitrogen was assessed . Overall, there was a good correspondence between the reductase activities and the presence of the corresponding genes . Distribution in the different ribotypes of strains harboring both the narG and nosZ genes and of strains missing both genes suggests that these two groups of strains had different evolutionary histories . Both dissimilatory genes showed high polymorphism, with similarity indexes (Jaccard) of between 0.04 and 0.8, whereas those of the 16S rRNA gene only varied from 0.77 to 0.99 . No correlation between the similarity indexes of 16S rRNA and dissimilatory genes was seen, suggesting that the evolution rates of ribosomal and functional genes differ . Pairwise comparison of similarity indexes of the narG and nosZ genes led to the delineation of two types of strains . Within the first type, the similarity indexes of both genes varied in the same range, suggesting that these two genes have followed a similar evolution . Within the second type of strain, the range of variations was higher for the nosZ than for the narG gene, suggesting that these genes have had a different evolutionary rate.

Appl Environ Microbiol, 2003 Feb, 69(2), 878 - 83
Tin-carbon cleavage of organotin compounds by pyoverdine from Pseudomonas chlororaphis; Inoue H et al.; The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT . Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized . The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P . chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads . Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I . Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants . F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively . The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production . The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine . On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu(2+) and Sn(4+), respectively . These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.

Plant Cell, 2003 Feb, 15(2), 365 - 79
HLM1, an essential signaling component in the hypersensitive response, is a member of the cyclic nucleotide-gated channel ion channel family; Balague C et al.; The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance . A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele . Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato . In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen . The HLM1 gene encodes a cyclic nucleotide-gated channel, CNGC4 . Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K(+) and Na(+) and is activated by both cGMP and cAMP . HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals . Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.

Plant Cell, 2003 Feb, 15(2), 317 - 30
Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae; Tao Y et al.; We performed large-scale mRNA expression profiling using an Affymetrix GeneChip to study Arabidopsis responses to the bacterial pathogen Pseudomonas syringae . The interactions were compatible (virulent bacteria) or incompatible (avirulent bacteria), including a nonhost interaction and interactions mediated by two different avirulence gene-resistance (R) gene combinations . Approximately 2000 of the approximately 8000 genes monitored showed reproducible significant expression level changes in at least one of the interactions . Analysis of biological variation suggested that the system behavior of the plant response in an incompatible interaction was robust but that of a compatible interaction was not . A large part of the difference between incompatible and compatible interactions can be explained quantitatively . Despite high similarity between responses mediated by the R genes RPS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1-mediated responses were not . This finding is consistent with the resistance phenotypes of these plants . We propose a simple quantitative model with a saturating response curve that approximates the overall behavior of this plant-pathogen system.

Toxicon, 2003 Mar 1, 41(3), 339 - 47
GTX(4) imposters: characterization of fluorescent compounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1, symbionts of saxitoxin-producing Alexandrium spp; Baker TR et al.; Saxitoxins, the etiological agent of paralytic shellfish poisoning, are synthesized by dinoflagellates and cyanobacteria . Several reports indicate that bacteria are capable of saxitoxin synthesis . Two bacterial strains were isolated from saxitoxin-producing dinoflagellates, Alexandrium tamarense and A . lusitanicum (=Alexandrium minutum), and grown under a variety of culture conditions including those previously reported to induce saxitoxin synthesis in bacteria . Five fluorescent compounds were accumulated by the bacteria that had HPLC-FLD retention times similar to a reference standard of GTX(4), one of the saxitoxin congeners . However, we were unable to detect GTX(1), the epimeric partner of GTX(4), in the bacterial samples . The GTX(4) standard was hydrolyzed by NaOH/heat treatment but four of the bacterial compounds were stable . Unlike GTX(4), none of the five bacterial compounds were detectable by HPLC-FLD following electrochemical oxidation . The fluorescence emission spectrum of each of the five bacterial compounds was unique and readily discernable from the spectrum of GTX(4) . None of the samples containing the putative GTX(4) toxin yielded positive results when analyzed by a 3H-saxitoxin receptor-binding assay for saxitoxin-like activity . We cannot rule out the possibility that these bacteria produce saxitoxins, however, our data clearly demonstrate that they accumulate at least five different fluorescent compounds that could be easily mistaken for GTX(4) . We conclude that these five fluorescent compounds are GTX(4) imposters and that fluorescence scanning and chemical/heat stability should, at a minimum, be incorporated into HPLC-FLD protocols for identification of saxitoxins.

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 490 - 7
{Study on Pseudomonas sp . WBC-3 capable of complete degradation of methylparathion}; Chen Y et al.; A bacterial strain was isolated from the polluted soil around Shashi Pesticides Factory, which was capable of complete degradation of methylparathion . Through chemotaxonomic characterizations and phylogenetic inference based on 16S rDNA sequence analysis, the strain was identified as a member of the genus and was named as Pseudomonas sp . WBC-3 . It can tolerate high-concentration methylparathion up to 800 mg/L in basic medium and up to 2000 mg/L in 0.1% glucose medium . Using 300 mg/L methylparathion as its sole carbon and nitrogen sources, the strain was able to degrade 15 mg of parathion per liter per hour and reached its stationary phase in about 22 hours . The strain possessed broad-spectrum degradative capability to kinds of organophosphorus pesticides and aromatic compounds . Its organophosphate hydrolase was purified from the periplasm of WBC-3 to homogeneity, through a whose process consisting of ammonium sulfate precipitation, Sephadex G-75 gel filtration, Q Sepharose FF ionexchange column chromatography . The purified enzyme showed a single band on SDS-PAGE gel with an approximate molecular weight of 33.5 kD.

Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 19 - 26
{Isolation and characterization of a p-nitrophenol degradation Pseudomonas sp . strain P3 and construction of a genetically engineered bacterium}; Cui Z et al.; A Pseudomonas strain P3 was isolated in this work . P3 can grow with p-nitrophenol as the sole carbon and nitrogen source . Growth in media with nitrogen, P3 can degrade p-nitrophenol to accumulate nitrite in the culture media . P3 can utilize a series of aromatic compounds as sole carbon sources . Different heavy metal ions have different effects on the degradation of p-Nitrophenol by P3 . Glucose had no effect on the degradation of p-nitrophenol, while trace yeast extract greatly increased the degradation rate . Methyl parathion hydrolase gene mpd was clone into P3 by conjugation and genetically engineered bacterium PM was obtained . Methyl parathion hydrolase was expressed by PM . PM could grew on methyl parathion as sole carbon source . PM could degrade methyl parathion with relatively high activity and stability.

Proteins, 2003 Feb 15, 50(3), 423 - 36
Structure of the Clade 1 catalase, CatF of Pseudomonas syringae, at 1.8 A resolution; Carpena X et al.; Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined . The structure of CatF has been refined at 1.8 A resolution by using X-ray synchrotron data collected from a crystal flash-cooled with liquid nitrogen . The crystallographic agreement factors R and R(free) are, respectively, 18.3% and 24.0% . The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry . The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence . Mass spectrometry analysis confirmed the absence of 26 N-terminal residues, possibly removed by a periplasmic transport system . The core structure of the CatF subunit was closely related to seven other catalases with root-mean-square deviations (RMSDs) of 368 core Calpha atoms of 0.99-1.30 A . The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180 degrees with respect the orientation of the heme in bovine liver catalase . NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model . Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150 . A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases .

Wei Sheng Wu Xue Bao, 1999 Oct, 39(5), 475 - 7
{Purification and properties of glutamate dehydrogenase from Pseudomonas pseudoalcaligenes}; Ding S et al.; Glutamate dehydrogenase was purified from the crude extract of Pseudomonas pseudoal-caligenes . The enzyme had a molecular weight of 290,000 and was composed of six subunits with identical molecular weight of 47,000 . The enzyme was highly specific for NADP(H) and the substrates . The biochemical properties such as kinetic parameters and heat stability were also examined . The purified GDH showed considerable loss of activity upon freezing.

Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 132 - 6
{Purification and characterization of alkaline xylanases from Pseudomonas G6-2}; Liu R et al.; Pseudomonas G6-2 produced two extracellular xylanases, named XynA and XynB . The enzymes were purified by ammonium sulfate fractionation, Sephadex G-100, DEAE-Sephadex, CM-Sephadex and Bio-gel P-10 chromatographies . Both enzymes were indicated to be endoxylanases, which produced oligomers of xylose from xylan and did not hydrolyze it to xylose . They had same temperature optimum(50 degrees C) and different pH optimum(pH 7.0-9.8 for XynA and pH 7.0-8.0 for XynB) . At pH 7.6 and 65 degrees C, XynA and XynB possessed the half life of 6 min and 140 min, respectively . Their activities were strongly inhibited by Cu2+, Fe3+, Pb2+, Zn2+ and Hg2+ . The results of chemical modification indicated that tryptophan and carboxy group were related to active center.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 345 - 7 Epub 2003 Jan 23.
Crystallization and preliminary X-ray diffraction analysis of the di-haem cytochrome c peroxidase from Pseudomonas stutzeri; Bonifacio C et al.; Crystals of cytochrome c peroxidase from Pseudomonas stutzeri were obtained using sodium citrate and PEG 8000 as precipitants . A complete data set was collected to a resolution of 1.6 A under cryogenic conditions using synchrotron radiation at the ESRF . The crystals belong to space group P2(1), with unit-cell parameters a = 69.29, b = 143.31, c = 76.83 A, beta = 100.78 degrees . Four CCP molecules were found in the asymmetric unit, corresponding to a pair of dimers related by local dyads . The crystal packing in the structure shows that the functional dimers can dimerize, as suggested by previous biochemical studies.

Wei Sheng Wu Xue Bao, 2001 Dec, 41(6), 699 - 703
{Melanin properties studies of the recombinant of Pseudomonas pseudoalcaligenes containing tyrosinase gene}; Cai X; Pseudomonas pseudoalcaligenes is a notably killing maggots bacterium, which was isolated from natural dead maggots . Tyrosinase gene of P . maltophilia AT18 has been introduced into P . pseudoalcaligenes, and enabled it the ability of producing melanin steadily . Antiradiation effect of the melanin is quite strong . The melanin of the recombinant is nonfixiform material . It's solubility is very little in many sorts of organic or inorganic solvent . It's solubility is big in alkaline solution, but little in neutral or weak acid solution . It is deposited when pH < 4 . It is oxidized by H2O2 or NaC10 . It is also reduced by Ag+ or H2S . It has free radical and it can absorbs free radical generated by ultra-violent ray . It can absorbs ray of all sorts of wave length . The absorption rate of ultra-violet ray is the biggest in all sorts of wave length . It can effectively protects protein, DNA and other biomacromolecule matter against the damages by ultra-violet ray.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 642 - 5
{N-terminal analysis and antibody preparation of insecticidal protein form Pseudomonas pseudoalcaligenes}; Ding S et al.; The insecticidal protein from Pseudomonas pseudoaligenes was and exotoxin which had toxicity on locusts . In order to elucidate its molecular properties an amino acid sequence, the insecticidal protein was purified from the culture supernatant by ultrafiltration, ion-exchange chromatography and gel filtration, and showed a single band on SDS-PAGE . Analysis of the purified insecticidal protein dentified N-terminal sequence of ten amino acid residues . Its polyclonal antibody was also obtained by immunizing rabbit with the insecticidal protein recovered form SDS-PAGE gel . The antibody titer determined by ELISA method was 1:12,800, indicating that it has high reactivity . Western blot analysis revealed that the antibody was spectific to 26 kD insecticidal protein, and did not cross-react with other proteins produced by the bacterium, suggesting that a specific antibody with high titer was obtained and could be used for further investigations of the gene cloning and expression of insecticidal protein.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 605 - 10
{Purification and some properties of D-hydantoinase produced by Pseudomonas 2262}; Shi Y et al.; A D-hydantoinase produced by Pseudomonas 2262 was purified to electrophoretic homogeneity by the steps of thermal treatment, (NH4)2SO4 fractionation and column chromatography with Q-Sepharose fast flow, phenyl-Sepharose fast flow and Superose 12 . Purification of about 60 fold was achieved with an overall yield of 16% . The relative molecular mass of the native enzyme is 109 kD and that of subunit is 53.7 kD by the analysis of Native and SDS-PAGE as well as gel filtration respectively . Some properties of the enzyme such as the sensitivity to thiol reagent and the effects of metal ions, for instance inhibited by Zn2+ and activited by Mn2+, Mg2+ are identical to dihydropyrimidinase . The optimum temperature and pH for enzymatic catalysis are 70 degrees C and 8.0 respectively . The enzyme activity is stable under 60 degrees C and in the pH range of 6-10 . The N-terminal sequence for 10 amino acid residues is MDKLIKNGTI.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 553 - 8
{Transfer and expression of tyrosinase gene of Pseudomonas maltophilia in Pseudomonas pseudoalcaligenes}; Cai X et al.; Pseudomonas pseudoalcaligenes is a notably killing maggots bacterium, which was isolated from natural dead maggots in the manure pits in the countryside of Yancheng . It easily die from effect of ultra-violet ray when it is exposed in the sun . Antiradiation effect of melanin is quite strong . Mel gene of P . maltophilia AT18 has been introduced into P . pseudoalcaligenes, and enabled it the ability of producing melanin steadily . Southern hybridization studies confirmed that the small fragment cloned in the P . pseudoalcaligenes comes from P . maltophilia DNA . SDS-PAGE analysis also revealed that an additional protein of 18 kD, which was equal to the size of the putative tyrosinase according to mel fragment, was exressed in the P . pseudoalcaligenes recombinant carrying the mel gene, The results of assay show that antiradiation effect of recombinant is quites strong, the effect time of killing maggats is longer than the recipient, the recombinant can't infect animals and fowls.

Biochemistry, 2003 Feb 4, 42(4), 940 - 50
Protein and DNA residue orientations in the filamentous virus Pf1 determined by polarized Raman and polarized FTIR spectroscopy; Tsuboi M et al.; The Pseudomonas bacteriophage Pf1 is a long ( approximately 2000 nm) and thin ( approximately 6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue alpha-helical subunit . The coat subunits are arranged as a superhelix of C(1)()S(5.4)() symmetry (class II) . Polarized Raman and polarized FTIR spectroscopy of oriented Pf1 fibers show that the packaged single-stranded DNA genome is ordered specifically with respect to the capsid superhelix . Bases are nonrandomly arranged along the capsid interior, deoxynucleosides are uniformly in the C2'-endo/anti conformation, and the average DNA phosphodioxy group (PO(2)(-)) is oriented so that the line connecting the oxygen atoms (O.O) forms an angle of 71 degrees +/- 5 degrees with the virion axis . Raman and infrared amide band polarizations show that the subunit alpha-helix axis is inclined at an average angle of 16 degrees +/- 4 degrees with respect to the virion axis . The alpha-helical symmetry of the capsid subunit is remarkably rigorous, resulting in splitting of Raman-active helix vibrational modes at 351, 445 and 1026 cm(-)(1) into apparent A-type and E(2)()-type symmetry pairs . The subunit tyrosines (Tyr 25 and Tyr 40) are oriented with phenoxyl rings packed relatively close to parallel to the virion axis . The Tyr 25 and Tyr 40 orientations of Pf1 are surprisingly close to those observed for Tyr 21 and Tyr 24 of the Ff virion (C(5)()S(2)() symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed alpha-helical subunits, irrespective of capsid symmetry . The polarized Raman spectra also provide information on the orientations of subunit alanine, valine, leucine and isoleucine side chains of the Pf1 virion.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 57 - 62
{Isolation and characterization of a insecticidal protein from Pseudomonas pseudoalcaligenes}; Zhang W et al.; A insecticidal protein was purified by gel-filtration chromatography on Sephadex G-100 and anion-exchange chromatography on DEAE-Sephadex A-50 from the suspension of Pseudomonas pseudoalcaligenes culture . Certain biophysical and biochemical properties were also studied . The molecular weight of the subunit of the insecticidal protein is 25,100 and pI is 5.16 . Amino acid composition analysis showed that it is an acidic protein.

Shi Yan Sheng Wu Xue Bao, 1999 Mar, 32(1), 63 - 71
{Vascular bundle specific expression of iaaL gene affects the generation frequencies of transgenic tobacco}; Xu Y et al.; A vascular bundles specific expressing vector pBAL1 with a promoter AQ630 from rice phenylalanine ammonialyase gene and a gene encoding indoleacetic-lysine synthytase from Pseudomonas syringae subsp . savastanoi was constructed . Affirmed by Southern blotting and RTPCR analysis, the AQ630-iaaL transgenic plants show increasing shoots-regeneration frequency of young stem explants on hormone-free 1/2 MS medium and lower sensibility to IAA when roots were induced from the root explants on the media containing different concentrations of IAA compared to untransformed plants.

J Ind Microbiol Biotechnol, 2003 Jan, 30(1), 6 - 12 Epub 2003 Jan 03.
Recombinant carbazole-degrading strains for enhanced petroleum processing; Riddle RR et al.; Biotechnological upgrading of fossil fuels is of increasing interest as remaining stocks of petroleum show increasing levels of contaminants such as heavy metals, sulfur and nitrogen-containing heteroaromatic compounds . Carbazole is of particular interest as a major petroleum component known to reduce refining yields through catalyst poisoning . In this study, the biotransformation of carbazole was successfully demonstrated in a liquid two-phase system, when solubilized in either 1-methylnaphthalene or in diesel fuel . The effects of solvent toxicity were investigated by expressing the carbazole-transformation genes from MB1332, a rifampicin-resistant derivative of Pseudomonas sp . LD2, in a solvent-resistant heterologous host, P . putida Idaho {1} . This solvent-resistant strain successfully degraded carbazole solubilized in 1-methylnaphthalene and in the presence of 10 vol% xylenes similar to the non-recombinant strain Pseudomonas sp . LD2 . Identification of a suitable recombinant host, however, was essential for further investigations of partial pathway transformations . Recombinant P . putida Idaho expressing only the initial dioxygenase enzymes transformed carbazole to an intermediate well retained in the oil phase . Partial carbazole transformation converts carbazole to non-aromatic species; their effect is unknown on refinery catalyst poisoning, but would allow almost complete retention of carbon content and fuel value.

Appl Microbiol Biotechnol, 2003 Jan, 60(5), 534 - 40 Epub 2002 Dec 04.
Optimization of isonovalal production from alpha-pinene oxide using permeabilized cells of Pseudomonas rhodesiae CIP 107491; Fontanille P et al.; Optimization studies on the synthesis of isonovalal from alpha-pinene oxide by Pseudomonas rhodesiae CIP 107491 operated in a biphasic medium are presented . Three key parameters are identified . The first is the need for a permeabilization of cells by freezing them and then treating the thawed material with an organic solvent such as chloroform, toluene or diethyl ether . This operation allows both enzyme release into the aqueous phase outside the cells and an improvement in the transport properties of both substrate and product across the cell membrane, strongly increasing reaction rates . The second is that the enzyme alpha-pinene oxide lyase, which exhibits an irreversible inactivation by isonovalal (or a by-product), presents a constant turn-over, i.e., the total product synthesis is proportional to the biomass loading and is close to 108 mmol (16.4 g) isonovalal l(-1) g(-1) biomass . The third phenomenon is that the biphasic system used is not phase-transfer-limited, a feature attributed to the spontaneous formation of an oil-in-water emulsion . It is thus possible to carry out a very efficient process, allowing the recovery of 2.63 mol isonovalal l(-1) (400 g l(-1)) from 25 g biomass l(-1) in 2.5 h, corresponding to an average reaction rate as high as 0.70 mmol min(-1) g(-1) cells (160 g l(-1) h(-1)).

J Bacteriol, 2003 Feb, 185(3), 918 - 28
Recombination activity of a distinctive integron-gene cassette system associated with Pseudomonas stutzeri populations in soil; Holmes AJ et al.; Class 1 integrons have strongly influenced the evolution of multiple antibiotic resistance . Diverse integrons have recently been detected directly in a range of natural environments . In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation of Pseudomonas stutzeri strain Q . Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM) . 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar of P . stutzeri . Only strains Q and BAM were found to contain an integron and an associated gene cassette array . The intI and attI components of these strains showed 99 and 90% identity, respectively . The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes . The two integrons contained nonoverlapping sets of cassette-associated genes . In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (the Pseudomonas subfamily) . The recombination activity of P . stutzeri integron components was tested in cointegrate assays . IntIPstQ was shown to catalyze site-specific recombination between its cognate attI site and 59-be sites from antibiotic resistance gene cassettes . While IntIPstQ did not efficiently mediate recombination between members of the Pseudomonas 59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1 . We concluded that integrons present in P . stutzeri possess recombination activity and represent a hot spot for genomic diversity in this species.

FEBS Lett, 2003 Jan 16, 534(1-3), 143 - 50
Pseudomonas stutzeri soluble nitrate reductase alphabeta-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound alphabetagamma holoenzyme; Hettmann T et al.; A two-subunit (alphabeta) form of dissimilatory nitrate reductase from Pseudomonas stutzeri strain ZoBell was separated from the membrane-residing gamma-subunit by a heat solubilization step . Here we present an optimized purification protocol leading to a soluble alphabeta form with high specific activity (70 U/mg) . The soluble form has the stoichiometry alpha(1)beta(1) consisting of the 130 kDa alpha-subunit and the 58 kDa beta-subunit . We did not observe any proteolytic cleavage in the course of the heat solubilization . The enzyme is competively inhibited by azide, but not by chlorate . It exhibits a K(M) value of 3.2 mM for nitrate . We compare the enzymatic and electron paramagnetic resonance (EPR) spectroscopic properties of the alphabeta form with the alphabetagamma holoenzyme which resides in the membrane and can be prepared by detergent extraction . The nearly identical EPR spectra for the Mo(V) signal of both enzyme preparations show that the active site is unaffected by the heat step . The factors influencing the binding of the alpha- and beta-subunit to the gamma-subunit are discussed.

J Mol Biol, 2003 Jan 31, 325(5), 1019 - 30
Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation; He H et al.; Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins . Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution . The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs) . A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate . We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

Enferm Intensiva, 2002 Oct-Dec, 13(4), 155 - 63
{Development of a quality guarantee system for mechanical ventilation (register in a multi-purpose CCU)}; Penalta Sanchez RM et al.; OBJECTIVE: 1 . Obtaining information about the demographic distribution of patients undergoing long-term mechanical ventilation . 2 . Defining our reference standards for mechanical ventilation, length of UCC and complications related to mechanical ventilation (MV), comparable with the international standards . Detailed follow-up of pneumonias associated to mechanical ventilation and incidence of accidental extubation (AE) . DESIGN: Prospective, descriptive . Period between July 1998 and December 2000 . AREA OF STUDY: Multi-purpose critical care unit (12 beds) . INDIVIDUALS UNDER STUDY: Patients hospitalized in the critical care unit with any pathology in need of mechanical ventilation . RESULTS: During the period of study 1058 patients were hospitalized in the critical care unit (CCU), 287 (27%) of which needed mechanical ventilation (MV) . 29% of the patients were women . The age and APACHE II were as median (percentile 25 and 75) 68 (57-76) and 26 (20-31) respectively . The reasons that made MV necessary were: acute respiratory failure 70%, intensified acute exacerbation of chronic respiratory failure 11%, coma 18% and neuromuscular illness 1% . The density of average incidence of accidental extubation (AE) was 15.7/1000 days of MV, the AE was associated to a longer duration of MV, longer stay in CCU and in the hospital and a greater incidence of pneumonia associated to MV, but it was not associated to an increment in mortality . The density of incidence of pneumonia associated to MV was 12.6/1000 days of MV, being the germ more frequently responsible the pseudomona aeruginosa.

Curr Microbiol, 2003 Feb, 46(2), 120 - 3
Morphological changes of Pseudomonas pseudoalcaligenes in response to temperature selection; Shi B et al.; Adaptation to novel environments usually entails morphological changes . The cell morphology of six experimental populations of Pseudomonas pseudoalcaligenes and their common ancestor were examined with scanning electron microscopy (SEM) . The six experimental populations were propagated under different temperatures for 10 months: three of them cultured at constant normal temperature (35 degrees C) forming the control group, and the other three cultured at incremental higher temperatures (from 41 degrees to 47 degrees C) as the HT group . SEM showed the deformed and elongated cells in the 6-h cultures of both ancestral and control populations at 45 degrees C, indicating that 45 degrees C is stressful for the ancestral and the control populations . In contrast, the HT populations retained normal cell shape in the 6-h cultures at both 35 degrees C and 45 degrees C . The mean cell volumes of control and HT populations increased 29% and 34%, respectively, relative to the ancestor at their respective thermal regimens, suggestion that the culturing conditions might favor larger cells.

Biochem J, 2003 Apr 15, 371(Pt 2), 557 - 64
Intersubunit interaction and catalytic activity of catechol 2,3-dioxygenases; Okuta A et al.; Catechol 2,3-dioxygenases (C23Os; EC 1.3.11.2) form a large protein family that is divided into several subgroups . Amino acid sequences of C23Os belonging to subgroup I.2.A and those belonging to I.2.B are found to be approx . 50% identical . When the central parts of the C23O sequences belonging to I.2.B were fused with the N-terminal and C-terminal sequences of I.2.A C23O, the hybrid enzymes were not active . To understand why these hybrid C23Os were inactive, hybrids between XylE(P) (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylE(S) (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed . HB3-C23O consisted mostly of the XylE(S) sequence, except that its C-terminal end was derived from XylE(P) . While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylE(P) sequences than HB3-C23O, was active . This observation indicated that certain amino acid residues at the C-terminus were crucial for C23O activity in the hybrid forms of enzymes between XylE(P) and XylE(S) . According to the crystal structure of XylE(P), the C-terminal region is involved in the formation of a quaternary structure . Amino acid differences between HB3-C23O and HB4-C23O included the specific beta-strand for oligomerization . Thus the quaternary structures of active C23Os, XylE(S), XylE(P) and HB4-C23O, as well as that of inactive HB3-C23O, were examined . Active enzymes XylE(S), XylE(P) and HB4-C23O were homotetrameric, while HB3-C23O existed only as a monomer . We concluded that hybrids of subgroups I.2.A and I.2.B were often inactive because of a defect in their oligomerization.

Biochem J, 2003 Apr 15, 371(Pt 2), 541 - 8
Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH; Lee BR et al.; A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae . Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH 4.0 . The specificity of aorsin appeared to require a basic residue at the P(1) position and to prefer paired basic residues . Aorsin activated plasminogen and converted trypsinogen to trypsin . The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases . To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established . The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases . Several putative catalytic residues were mutated . The k (cat)/ K(m) values of the mutant enzymes Glu(86)-->Gln, Asp(211)-->Asn and Ser(354)-->Thr were 3-4 orders of magnitude lower and Asp(90)-->Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis . Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin.

Appl Environ Microbiol, 2003 Jan, 69(1), 130 - 8
Genetic diversity and spoilage potentials among Pseudomonas spp . isolated from fluid milk products and dairy processing plants; Dogan B et al.; Degradation of milk components through various enzymatic activities associated with the contamination of dairy products by Pseudomonas spp . can reduce the shelf life of processed milk . Reliable methods for differentiating among Pseudomonas spp . strains are necessary to identify and eliminate specific sources of bacterial contamination from dairy processing systems . To that end, we assessed the genetic diversity and dairy product spoilage potentials among a total of 338 Pseudomonas spp . isolates from raw and pasteurized milk and from environmental samples collected from four dairy processing plants . The majority of isolates were identified as P . fluorescens and P . putida by API 20 NE . A total of 42 different ribotype patterns were identified among a subset of 81 isolates . The presence of many different ribotypes within this collection indicates high genetic diversity among the isolates and suggests multiple origins of contamination within the processing plant and in dairy products . The extracellular enzyme activity patterns among Pseudomonas isolates appeared to be associated with ribotypes . Isolates with the same ribotype frequently had the same extracellular protease, lecithinase, and lipase activities . For example, isolates grouped in ribotype 55-S-6 had the highest extracellular protease activity, while those in ribotypes 50-S-8 and 72-S-3 had the highest extracellular lipase activities . We conclude that ribotyping provides a reliable method for differentiating Pseudomonas strains with dairy food spoilage potential.

Protein Expr Purif, 2003 Jan, 27(1), 85 - 9
Expression and purification of recombinant immunotoxin--a fusion protein stabilizes a single-chain Fv (scFv) in denaturing condition; Kim SH; Carcinoembryonic antigen (CEA) is expressed at greatly increased levels in nearly all human colorectal carcinomas . Anti-CEA antibodies have been proved to be useful for targeting several cancer types known to express CEA . A recombinant immunotoxin was constructed, in which the cell-binding domain of Pseudomonas exotoxin (PE) was replaced with the single-chain Fv (scFv) of anti-CEA monoclonal antibody for targeting to colorectal carcinomas . This single-chain immunotoxin was expressed in E . coli and purified under denaturing condition of 6M guanidine hydrochloride (GuHCl) . It was found that the immunotoxin maintains a binding activity in denaturing condition of 6M GuHCl and the fused PE contributes to the stability of immunotoxin in such condition . Dialysis against PBS buffer after purification under 6M GuHCl keeps the binding activity of immunotoxin.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2065 - 74
Pseudomonas salomonii sp . nov., pathogenic on garlic, and Pseudomonas palleroniana sp . nov., isolated from rice; Gardan L et al.; A total of 26 strains, including 15 strains isolated from garlic plants with the typical symptoms of 'Cafe au lait' disease and 11 strains isolated from diseased or healthy rice seeds and sheaths infested by Pseudomonas fuscovaginae, were compared with 70 type or reference strains of oxidase-positive pathogenic or non-pathogenic fluorescent pseudomonads . The strains were characterized by using a polyphasic taxonomic approach . Numerical taxonomy of phenotypic characteristics showed that the garlic and rice strains were related to each other . However, they clustered into separate phenons, distinct from those of the other strains tested, and were different in several nutritional tests . On the basis of DNA-DNA hybridization, the garlic and rice strains constituted two distinct DNA hybridization groups, indicating that they belonged to separate species . The two groups of strains were also well differentiated by siderotyping . Garlic strains were pathogenic to garlic plants and either weakly pathogenic or non-pathogenic on rice; rice strains were either weakly pathogenic or non-pathogenic on rice and non-pathogenic on garlic . A phylogenetic analysis of 16S rRNA gene sequences confirmed that the two groups of strains belonged to the y-Proteobacteria and to the genus Pseudomonas . The names Pseudomonas salomonii sp . nov . and Pseudomonas palleroniana sp . nov . are respectively proposed for the garlic strains and the rice strains . The type strains are P . salomonii CFBP 2022(T) ( = ICMP 14252(T) = NCPPB 4277(T)) and P . palleroniana CFBP 4389(T) (= ICMP 14253(T) = NCPPB 4278(T)).

EMBO J, 2003 Jan 2, 22(1), 60 - 9
Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death; Abramovitch RB et al.; The AvrPtoB type III effector protein is conserved among diverse genera of plant pathogens suggesting it plays an important role in pathogenesis . Here we report that Pseudomonas AvrPtoB acts inside the plant cell to inhibit programmed cell death (PCD) initiated by the Pto and Cf9 disease resistance proteins and, remarkably, the pro-apoptotic mouse protein Bax . AvrPtoB also suppressed PCD in yeast, demonstrating that AvrPtoB functions as a cell death inhibitor across kingdoms . Using truncated AvrPtoB proteins, we identified distinct N- and C-terminal domains of AvrPtoB that are sufficient for host recognition and PCD inhibition, respectively . We also identified a novel resistance phenotype, Rsb, that is triggered by an AvrPtoB truncation disrupted in the anti-PCD domain . A Pseudomonas syringae pv . tomato DC3000 strain with a chromosomal mutation in the AvrPtoB C-terminus elicited Rsb-mediated immunity in previously susceptible tomato plants and disease was restored when full-length AvrPtoB was expressed in trans . Thus, our results indicate that a type III effector can induce plant susceptibility to bacterial infection by inhibiting host PCD.

Carbohydr Res, 2003 Jan 2, 338(1), 109 - 12
A simple method for preparation of D-rhamnose; Ramm M et al.; A rapid procedure for the preparation of D-rhamnose from bacterial lipopolysaccharide (LPS) has been developed . It involves purification of LPS from Pseudomonas syringae pv . phaseolicola by phenol extraction and hydrophobic interaction chromatography (HIC), followed by mild hydrolysis and cleavage of the O-antigen into D-fucose and D-rhamnose . The monosaccharides were separated by column chromatography, and D-rhamnose recovered after filtration over Sephadex-LH 20.

Carbohydr Res, 2003 Jan 2, 338(1), 19 - 27
Synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-{beta-L-Xylp-(1-->4)}-alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942; Zhang J et al.; An efficient synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-{beta-L-Xylp-(1-->4)}-alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942 and its isomer beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->4)-{beta-L-Xylp-(1-->2)}-alpha-L-Rhap-(1-->3)-alpha-L-Rhap was achieved via sequential assembly of the building blocks, allyl 2,3-O-isopropylidene-alpha-L-rhamnopyranoside (2), allyl 3,4-O-isopropylidene-alpha-L-rhamnopyranoside (3), allyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate (7), and 2,3,4-tri-O-benzoyl-beta-L-xylopyranosyl trichloroacetimidate (12) . The process was carried out in a regio- and stereoselective manner using glycosyl trichloroacetimidates as donors and unprotected or partially protected rhamnopyranosides as acceptors in the presence of a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf).

J Nat Prod, 2002 Dec, 65(12), 1793 - 7
Isolation of labradorins 1 and 2 from Pseudomonas syringae pv . coronafaciens; Pettit GR et al.; Investigation of Pseudomonas syringae pv . coronafaciens cancer cell growth inhibitory constituents led to the isolation of 2-isobutyl-5-(3-indolyl)oxazole (1) and 2-n-pentyl-5-(3-indolyl)oxazole (2f), designated labradorins 1 (1) and 2 (2f), related to pimprinine (2a) . The structures were deduced by spectroscopic techniques and X-ray crystal structure determinations . Labradorin 1 (1) afforded GI(50) microg/mL values of 9.8 and 6.2 against the human cancer cell lines NCI-H 460 (lung-NSC) and BXPC-3 (pancreas-a).

Microbiol Res, 2002, 157(4), 311 - 5
Several pseudomonads, associated with the cultivated mushrooms Agaricus bisporus or Pleurotus sp., are hemolytic; Munsch P et al.; Pseudomonas tolaasii, causing brown blotch disease on cultivated mushrooms, and yielding a white line precipitate towards P . "reactans", has been shown to induce lysis of erythrocytes . Some Finnish strains isolated from diseased mushroom fruit bodies, although harboring the typical features of P . tolaasii, proved to be distinct, and have been allocated to a nov . sp . P . costantinii . We examined in these study whether all brown blotch causing agents were hemolytic . The induction of erythrocytes lysis seemed to be a rather common feature of mushroom associated-pseudomonads, especially for strains involved in the production of a white-line-in agar.

Eur J Biochem, 2003 Jan, 270(1), 20 - 7
Structure of the O polysaccharides and serological classification of Pseudomonas syringae pv . porri from genomospecies 4; Zdorovenko EL et al.; Strains of Pseudomonas syringae pv . porri are characterized by a number of pathovar-specific phenotypic and genomic characters and constitute a highly homogeneous group . Using monoclonal antibodies, they all were classified in a novel P . syringae serogroup O9 . The O polysaccharides (OPS) isolated from the lipopolysaccharides (LPS) of P . syringae pv . porri NCPPB 3365 and NCPPB 3364T possess multiple oligosaccharide O repeats, some of which are linear and composed of l-rhamnose (l-Rha), whereas the major O repeats are branched with l-rhamnose in the main chain and GlcNAc in side chains (structures 1 and 2) . Both branched O repeats, which differ in the position of substitution of one of the Rha residues and in the site of attachment of GlcNAc, were found in the two strains studied, O repeat 1 being major in strain NCPPB 3365 and 2 in strain NCPPB 3364T . {formula: see text} . The relationship between OPS chemotype and serotype on one hand and the genomic characters of P . syringae pv . porri and other pathovars delineated in genomospecies 4 on the other hand is discussed.

Biochim Biophys Acta, 2002 Dec 23, 1567(1-2), 143 - 9
Syringotoxin pore formation and inactivation in human red blood cell and model bilayer lipid membranes; Szabo Z et al.; The effect of syringotoxin (ST), a member of the cyclic lipodepsipeptides family (CLPs) produced by Pseudomonas syringae pv . syringae on the membrane permeability of human red blood cells (RBCs) and model bilayer lipid membranes (BLMs) was studied and compared to that of two recently investigated CLPs, syringomycin E (SRE) and syringopeptin 22A (SP22A) {Biochim . Biophys . Acta 1466 (2000) 79 and Bioelectrochemistry 52 (2000) 161} . The permeability-increasing effect of ST on RBCs was the least among the three CLPs . A time-dependent ST pore inactivation was observed on RBCs at 20 and 37 degrees C but not at 8 degrees C . From the kinetic model worked out parameters as permeability coefficient of RBC membrane for 86Rb(+) and pores mean lifetime were calculated . A shorter pores mean lifetime was calculated at 37 degrees C then at 20 degrees C, which gave us an explanation for the unusual slower rate of tracer efflux measured at 37 degrees C then that at 20 degrees C . The results obtained on BLM showed that the pore inactivation was due to a decrease in the number of pores but not to a change of their dwell time or conductance.

Rev Pneumol Clin, 2002 Jun, 58(3 Pt 1), 131 - 8
{Anti-Pseudomonas aerosol therapy in cystic fibrosis: improvement with tobramycin}; Foucaud P et al.; Aerosol delivery of antibiotics offers the potential to achieve high antibiotic concentrations at the site of infection while reducing the risk of systemic untoward effects because of minimal resorption into the bloodstream . We reviewed knowledge acquired in this field over the two latter decades . While the earliest data were obtained with gentamycin, the most conclusive evidence presently regards aminoglycosides and colistin . Aerosol delivery of tobramycin was recently improved with the development of a new formulation for inhalation . Coupled with an adequate nebulization system, intermittent treatment with tobramycin for inhalation has been evaluated in randomized placebo-controlled studies . These studies have demonstrated a significant improvement of respiratory function.

J Bacteriol, 2003 Jan, 185(1), 41 - 50
Characterization of the 4-carboxy-4-hydroxy-2-oxoadipate aldolase gene and operon structure of the protocatechuate 4,5-cleavage pathway genes in Sphingomonas paucimobilis SYK-6; Hara H et al.; The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by Sphingomonas paucimobilis SYK-6 . In the 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), 4-oxalomesaconate hydratase (ligJ), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligK gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase . The ligK gene was located 1,183 bp upstream of ligI and transcribed in the same direction as ligI . We also found the ligR gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of ligK . The ligK gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da . The deduced amino acid sequence of ligK showed 57 to 88% identity with those of the corresponding genes recently reported in Sphingomonas sp . strain LB126, Comamonas testosteroni BR6020, Arthrobacter keyseri 12B, and Pseudomonas ochraceae NGJ1 . The ligK gene was expressed in Escherichia coli, and the gene product (LigK) was purified to near homogeneity . Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO(2) . LigK is a hexamer, and its isoelectric point is 5.1 . The K(m) for CHA and oxaloacetate are 11.2 and 136 micro M, respectively . Inactivation of ligK in S . paucimobilis SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that ligK encodes the essential CHA aldolase for catabolism of these compounds . Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of S . paucimobilis SYK-6 consisted of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes.

J Gen Appl Microbiol, 2001 Oct, 47(5), 247 - 261
Recharacterization of Pseudomonas fulva Iizuka and Komagata 1963, and proposals of Pseudomonas parafulva sp . nov . and Pseudomonas cremoricolorata sp . nov; Uchino M et al.; Seven Pseudomonas fulva strains obtained from culture collections were taxonomically studied . The seven strains were separated into three clusters (Clusters I to III) on the basis of 16S rRNA gene sequences, and located phylogenetically in the genus Pseudomonas sensu stricto . Further, the strains were classified into 4 groups (Groups I to IV) on the basis of DNA-DNA similarity . As a result, Cluster I was split into Groups I and II . Group I included the type strain of P . fulva and two strains, and levels of DNA-DNA similarity ranged from 88 to 100% among the strains . Group II contained two strains, and the level between the two strains ranged from 91 to 100% . Group III consisted of one strain . Group IV included one strain, and this strain showed a high level of DNA-DNA similarity with the type strain of Pseudomonas straminea NRIC 0164(T) . Clusters II and III corresponded to Groups III and IV, respectively . The four groups were separated from one another and from related Pseudomonas species at the level from 3 to 45% of DNA-DNA similarity . The strains of Groups I, II, and III had ubiquinone 9 as the major quinone . According to numerical analysis by the use of 133 phenotypic characteristics, the seven P . fulva strains were split into four phenons (Phenons I to IV) . The groups by DNA-DNA similarity corresponded well with the phenons produced by numerical taxonomy, and differential characteristics were recognized . Consequently, Group I was regarded as P . fulva because the type strain (NRIC 0180(T)) of this species was included in this group . Strains in Group II were identified as a new species, Pseudomonas parafulva sp . nov., and the type strain is AJ 2129 (=IFO 16636=JCM 11244=NRIC 0501) . NRIC 0181 in Group III was identified as a new species, Pseudomonas cremoricolorata sp . nov., and the type strain is NRIC 0181 (=IFO 16634=JCM 11246) . NRIC 0182 in Group IV was identified as P . straminea on the basis of the high level of DNA-DNA similarity with the type strain of this species.

J Gen Appl Microbiol, 2001 Dec, 47(6), 279 - 305
Bacterial degradation of aromatic compounds via angular dioxygenation; Nojiri H et al.; Dioxygenation is one of the important initial reactions of the bacterial degradation of various aromatic compounds . Aromatic compounds, such as biphenyl, toluene, and naphthalene, are dioxygenated at lateral positions of the aromatic ring resulting in the formation of cis-dihydrodiol . This "normal" type of dioxygenation is termed lateral dioxygenation . On the other hand, the analysis of the bacterial degradation of fluorene (FN) analogues, such as 9-fluorenone, dibenzofuran (DF), carbazole (CAR), and dibenzothiophene (DBT)-sulfone, and DF-related diaryl ether compounds, dibenzo-p-dioxin (DD) and diphenyl ether (DE), revealed the presence of the novel mode of dioxygenation reaction for aromatic nucleus, generally termed angular dioxygenation . In this atypical dioxygenation, the carbon bonded to the carbonyl group in 9-fluorenone or to heteroatoms in the other compounds, and the adjacent carbon in the aromatic ring are both oxidized . Angular dioxygenation of DF, CAR, DBT-sulfone, DD, and DE produces the chemically unstable hemiacetal-like intermediates, which are spontaneously converted to 2,2',3-trihydroxybiphenyl, 2'-aminobiphenyl-2,3-diol, 2',3'-dihydroxybiphenyl-2-sulfinate, 2,2',3-trihydroxydiphenyl ether, and phenol and catechol, respectively . Thus, angular dioxygenation for these compounds results in the cleavage of the three-ring structure or DE structure . The angular dioxygenation product of 9-fluorenone, 1-hydro-1,1a-dihydroxy-9-fluorenone is a chemically stable cis-diol, and is enzymatically transformed to 2'-carboxy-2,3-dihydroxybiphenyl . 2'-Substituted 2,3-dihydroxybiphenyls formed by angular dioxygenation of FN analogues are degraded to monocyclic aromatic compounds by meta cleavage and hydrolysis . Thus, after the novel angular dioxygenation, subsequent degradation pathways are homologous to the corresponding part of that of biphenyl . Compared to the bacterial strains capable of catalyzing lateral dioxygenation, few bacteria having angular dioxygenase have been reported . Only a few degradation pathways, CAR-degradation pathway of Pseudomonas resinovorans strain CA10, DF/DD-degradation pathway of Sphingomonas wittichii strain RW1, DF/DD/FN-degradation pathway of Terrabacter sp . strain DBF63, and carboxylated DE-degradation pathway of P . pseudoalcaligenes strain POB310, have been investigated at the gene level . As a result of the phylogenetic analysis and the comparison of substrate specificity of angular dioxygenase, it is suggested that this atypical mode of dioxygenation is one of the oxygenation reactions originating from the relaxed substrate specificity of the Rieske nonheme iron oxygenase superfamily . Genetic characterization of the degradation pathways of these compounds suggests the possibility that the respective genetic elements constituting the entire catabolic pathway have been recruited from various other bacteria and/or other genetic loci, and that these pathways have not evolutionary matured.

Mol Plant Microbe Interact, 2002 Dec, 15(12), 1195 - 203
Pseudomonas viridiflava and P . syringae--natural pathogens of Arabidopsis thaliana; Jakob K et al.; We report the isolation and identification of two natural pathogens of Arabidopsis thaliana, Pseudomonas viridiflava and Pseudomonas syringae, in the midwestern United States . P . viridiflava was found in six of seven surveyed Arabidopsis thaliana populations . We confirmed the presence in the isolates of the critical pathogenicity genes hrpS and hrpL . The pathogenicity of these isolates was verified by estimating in planta bacterial growth rates and by testing for disease symptoms and hypersensitive responses to A . thaliana . Infection of 21 A . thaliana ecotypes with six locally collected P . viridiflava isolates and with one P . syringae isolate showed both compatible (disease) and incompatible (resistance) responses . Significant variation in response to infection was evident among Arabidopsis ecotypes, both in terms of symptom development and in planta bacterial growth . The ability to grow and cause disease symptoms on particular ecotypes also varied for some P . viridiflava isolates . We believe that these pathogens will provide a powerful system for exploring coevolution in natural plant-pathogen interactions.

Mol Cancer Ther, 2002 Oct, 1(12), 999 - 1007
Intratumor administration of interleukin 13 receptor-targeted cytotoxin induces apoptotic cell death in human malignant glioma tumor xenografts; Kawakami M et al.; Apoptosis is not only essential for homeostasis in normal cells but also in cancer cells, in which it is associated with cell death mechanisms caused by novel therapeutics . We have previously reported that interleukin-13 receptors (IL-13R) are constitutively overexpressed on a majority of human malignant glioma cell lines and primary cell cultures . In addition, we have reported that IL-13 cytotoxin, comprised of human IL-13 and a mutated form of Pseudomonas exotoxin, is highly and specifically cytotoxic to these cells and can lead to pronounced antitumor activity in malignant glioma tumors in animal models . However, the molecular mechanisms of tumor cytotoxicity induced by IL-13 cytotoxin are poorly understood . In this study, we demonstrate that glioma tumors undergo apoptotic cell death on intratumoral administration of IL-13 cytotoxin . This conclusion was made based on (a) time-dependent induction of several proapoptotic molecules, such as caspases (caspase-3, -8, and -9) in tumors; (b) cleavage of procaspase-3 and poly(ADP-ribose) polymerase (PARP); and (c) the release of cytochrome c from mitochondria to the cytosol on injection of IL-13 cytotoxin in U251 glioblastoma tumors established in immunodeficient animals . These indicators of two major pathways of apoptosis were detected in tumors even though IL-13 cytotoxin was no longer present in tumors . In addition, we found that inducible nitric oxide was expressed in tumors in a time-dependent manner with primary localization in infiltrating phagocytes after treatment with IL-13 cytotoxin . These studies demonstrate that IL-13 cytotoxin mediates apoptotic death of glioma cells, resulting in regression of established tumors . Our studies will help unravel the molecular pathways of cell death associated with tumor regression and provide additional insight and define apoptosis as possible surrogate marker of tumor response.

Mol Cancer Ther, 2002 Jun, 1(8), 595 - 600
Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin; Fan D et al.; Several tumors, including mesothelioma and ovarian cancer, can overexpress mesothelin, a glycosylphosphatidylinositol-linked differentiation glycoprotein . The membrane-bound type of mesothelin is found in the blood of cancer patients at a very low level, which makes mesothelin a good candidate for targeted therapy of certain cancers . An antimesothelin disulfide-linked Fv (SS1 Fv) was fused to a truncated mutant of Pseudomonas exotoxin A to produce the recombinant immunotoxin SS1(dsFv)-PE38, which has a high binding affinity to mesothelin (Kd = 0.7 nM) . Our studies in vitro showed that SS1(dsFv)-PE38 is significantly more cytotoxic to the high-mesothelin-producing NCI-H226 human non-small cell lung cancer cells than to human lung adenocarcinoma PC14PE6 cells, which do not express mesothelin . When administered at a nontoxic dose of 500 microg/kg on days 7, 9, and 11 to nude mice injected i.v . with the two human lung cancer cell lines, SS1(dsFv)-PE38 selectively inhibited experimental lung metastases produced by the mesothelin-producing NCI-H226 cells . Our data indicate that mesothelin-producing squamous cell carcinoma of the lung may be a good target for this immunotoxin.

J Magn Reson, 2002 Nov, 159(1), 82 - 6
Capture and manipulation of magnetically aligned Pf1 with an aqueous polymer gel; Riley SA et al.; The magnetic alignment of the Pseudomonas bacteriophage Pf1 is captured indefinitely in a gel of the aqueous triblock copolymer Pluronic F-127 . In addition to preserving high-resolution liquids NMR spectra for dissolved solutes, the gel prevents the reorientation of the phage allowing mechanical manipulation of the angle between the axis of the phage alignment and the static magnetic field . The residual 2H quadrupolar couplings for several solutes dissolved in this material as a function of the angle Theta between the non-spinning sample tube and the static magnetic field are consistent with the value of P(2)(cosTheta)=(3cos(2)Theta-1)/2 . The variable-angle correlation spectrum for these solutes is shown to separate residual quadrupolar effects from isotropic chemical shifts . Finally, the compatibility of Pluronic F-127 with NMR studies of aqueous biological macromolecules is demonstrated in a measurement of residual dipolar couplings in an 15N-labeled nucleic acid.

Mol Cancer Ther, 2001 Dec, 1(2), 79 - 84
Bivalent disulfide-stabilized fragment variable immunotoxin directed against mesotheliomas and ovarian cancer; Bera TK et al.; We have used protein engineering to generate a stable bivalent fragment variable (Fv) molecule from the antimesothelin antibody SS, in which the VH and VL domains of the Fv are linked to each other by a disulfide bond, and the two Fvs are connected by a flexible 15-amino acid (Gly4-Ser)3 linker . The SS (dsFv)2 molecule is fused to a M(r) 38,000 truncated form of Pseudomonas exotoxin to generate a bivalent, disulfide stabilized, (dsFv)2 immunotoxin . The immunotoxin was expressed in Escherichia coli, refolded in vitro, and purified to approximately 95% purity with a high yield of > 10% . Binding studies demonstrated that the (dsFv)2 molecule has 40 times higher apparent affinity for recombinant mesothelin than a monovalent dsFv molecule . The (dsFv)2 immunotoxin was 4-10-fold more cytotoxic to three mesothelin antigen-positive cell lines than the monovalent dsFv immunotoxin . However, when tested in mice bearing tumor cells expressing mesothelin, the antitumor activity of the bivalent immunotoxin is very similar to the activity of the lower affinity monovalent immunotoxin . Our data indicate that increasing affinity of an antibody fragment does not necessarily lead to higher antitumor activity of an immunotoxin in vivo.

Appl Microbiol Biotechnol, 2002 Dec, 60(4), 475 - 80 Epub 2002 Oct 12.
PAH utilization by Pseudomonas rhodesiae KK1 isolated from a former manufactured-gas plant site; Kahng HY et al.; Pseudomonas rhodesiae KK1 was isolated from a former manufactured-gas plant site, due to its ability to grow rapidly in a mixture of polycyclic aromatic hydrocarbons (PAHs) . Radiorespirometric analysis revealed that strain KK1 was found to be able to mineralize anthracene, naphthalene and phenanthrene . Notably, phenanthrene-grown cells were able to mineralize anthracene much more rapidly than naphthalene-grown cells . Comparative analysis of amino acid sequences from 17 randomly selected dioxygenases capable of hydroxylating unactivated aromatic nuclei indicated that the enzymes for catabolism of PAHs, such as naphthalene and phenanthrene, might exist redundantly in strain KK1 . Northern hybridization for cells grown on naphthalene or phenanthrene, using the putative naphthalene or phenanthrene dioxygenase gene fragment as a probe, suggested that the enzyme for naphthalene catabolism might share some homology in deduced amino acid sequences with phenanthrene dioxygenases . Also, it was found that three lipids (17:0 cyclo, 18:1 omega7c, 19:0 cyclo) increased in response to both naphthalene and phenanthrene, while the shift of other lipids varied from substrate to substrate.

BioDrugs, 2002, 16(6), 433 - 7
Retention of specific yolk IgY in the human oral cavity; Carlander D et al.; INTRODUCTION: The increasing prevalence of antibiotic-resistant bacteria emphasises the need for new treatments that can replace traditional antibiotics . Oral immunotherapy with yolk antibodies from hyperimmunised hens is a new promising treatment strategy, primarily for infections in the mouth and gastrointestinal tract . Several studies show that bacterial and viral infections can be prevented with egg yolk immunoglobulin (IgY) in a dose-dependent manner . Oral treatment could potentially be used against many frequently encountered diseases (e.g . common cold, tonsillitis and caries) . GROUP STUDIED: Healthy volunteers . STUDY DESIGN: We studied the presence of yolk anti-Pseudomonas aeruginosa antibodies in saliva from healthy volunteers over time after 1 or 2 minutes' mouth rinse, performed in the evening, with an aqueous IgY antibody preparation . The test persons rinsed the mouth with 8.0ml phosphate buffered saline before gargling with the antibody preparation 8 and 24 hours later . Statistical analysis was performed with the Mann-Whitney U test . METHODS: The antibody titres in the mouth rinses were tested for their specific activity against P . aeruginosa by ELISA . RESULTS AND CONCLUSION: The next morning there were still active antibodies detected in the saliva from 18 of 19 subjects . After 24 hours, active antibodies could be detected in saliva from only a few of the subjects . A 2-minute mouth rinse resulted in higher mean ELISA absorbance values than a 1-minute rinse.

Biochemistry (Mosc), 2002 Oct, 67(10), 1145 - 51
Effect of interactions between amino acid residues 43 and 61 on thermal stability of bacterial formate dehydrogenases; Fedorchuk VV et al.; NAD+-dependent formate dehydrogenases (EC 1.2.1.2, FDH) of methylotrophic bacteria Pseudomonas sp . 101 (PseFDH) and Mycobacterium vaccae N10 (MycFDH) exhibit high homology . They differ in two amino acid residues only among a total of 400, i.e., Ile35 and Glu61 in MycFDH substitute for Thr35 and Lys61 as in PseFDH . However, the rate constant for MycFDH thermal inactivation in the temperature range of 54-65 degrees C is 4-6-times higher than the corresponding rate constant for the enzyme from Pseudomonas sp . 101 . To clarify the role of these residues in FDH stability the dependence of the apparent rate constant for enzyme inactivation on phosphate concentration was studied . Kinetic and thermodynamic parameters for thermal inactivation were obtained for both recombinant wild-type and mutant forms, i.e., MycFDH Glu61Gln, Glu61Pro, Glu61Lys and PseFDH Lys61Arg . It has been shown that the lower stability of MycFDH compared to that of PseFDH is caused mainly by electrostatic repulsion between Asp43 and Glu61 residues . Replacement of Lys61 with an Arg residue in the PseFDH molecule does not result in an increase in stability.

J Microbiol Methods, 2003 Feb, 52(2), 261 - 6
Highly sensitive detection of Pseudomonas savastanoi pv . savastanoi in asymptomatic olive plants by nested-PCR in a single closed tube; Bertolini E et al.; A nested-polymerase chain reaction (PCR) has been set up to be performed in a single closed tube for the detection of Pseudomonas savastanoi pv . savastanoi . Nested-PCR coupled with dot-blot hybridization was able to detect up to one cell of the target per ml of olive extract, showing the greatest sensitivity compared with all previously reported detection assays . Validation of the developed procedure for diagnosis and epidemiological purposes was achieved by testing ca . 240 asymptomatic plant samples from olive trees . When performing the other previously reported techniques (bacterial isolation and single PCR), P . savastanoi was detected in 50 of the analyzed samples, while with the new developed nested-PCR assay, the bacterium was detected in 82 samples .

Can J Microbiol, 2002 Sep, 48(9), 772 - 86
Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood; Krechel A et al.; To study the effect of microenvironments on potato-associated bacteria, the abundance and diversity of bacteria isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field grown potato was analyzed . Culturable bacteria were obtained after plating on R2A medium . The endophytic populations averaged 10(3) and 10(5) CFU/g (fresh wt.) for the endosphere and endorhiza . respectively, which were lower than those for the ectophytic microenvironments, with 10(5) and 10(7) CFU/g (fresh wt.) for the phyllosphere and rhizosphere, respectively . The composition and richness of bacterial species was microenvironment-dependent . The occurrence and diversity of potato-associated bacteria was additionally monitored by a cultivation-independent approach using terminal restriction fragment length polymorphism analysis of 16S rDNA . The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of microenvironment-specific communities . In an approach to measure the antagonistic potential of potato-associated bacteria, a total of 440 bacteria was screened by dual testing for in vitro antagonism towards the soilborne pathogens Verticillium dahliae and Rhizoctonia solani . The proportion of isolates with antagonistic activity was highest for the rhizosphere (10%), followed by the endorhiza (9%), phyllosphere (6%), and endosphere (5%) . All 33 fungal antagonists were characterized by testing their in vitro antagonistic mechanisms, including their glucanolytic, chitinolytic, pectinolytic, cellulolytic, and proteolytic activity, and by their BOX-PCR fingerprints . In addition, they were screened for their biocontrol activity against Meloidogyne incognita . Overall, nine isolates belonging to Pseudomonas and Streptomyces species were found to control both fungal pathogens and M . incognita and were therefore considered as promising biological control agents.

Int J Cancer, 2003 Jan 1, 103(1), 45 - 52
Tumor regression mechanisms by IL-13 receptor-targeted cancer therapy involve apoptotic pathways; Kawakami M et al.; IL-13 cytotoxin, composed of IL-13 and a truncated form of Pseudomonas exotoxin, targets IL-13R-overexpressing tumor cell lines in vitro and in vivo . To reveal the molecular mechanism of IL-13 cytotoxin-induced cell death in vivo, we demonstrate activation of apoptotic pathways in 2 s.c . growing human SCCHN tumor models in immunodeficient mice after i.t . administration of IL-13 cytotoxin . Treatment of HN12 tumor bearing mice with i.p . or i.t . administration of IL-13 cytotoxin mediated marked regression of established tumors with complete remission . Interestingly, after a single i.t . administration, IL-13 cytotoxin disappeared within 6 hr but accumulation of caspase-3, -8 and -9 and cleavage of procaspase-3 and PARP continued within the tumors for a prolonged period . We further demonstrate that IL-13 cytotoxin also utilizes an alternate pathway of cell death via the release of cytochrome c from mitochondria to the cytosol . Our results indicate that IL-13 cytotoxin induces 2 major pathways of apoptosis, which may play a role in tumor regression . In addition, apoptotic molecules may serve as surrogate molecular markers of tumor response to IL-13R-directed cytotoxin therapy .

Biotechnol Appl Biochem, 2002 Dec, 36(Pt 3), 181 - 6
Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel; Hsu AF et al.; Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts . The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran- T.l . and gran- C.a., respectively), C . antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol-gel matrix (IM PS-30) . All alcoholysis reactions were carried out in solvent-free media employing a one-step addition of the alcohol to the reaction system . Of the lipases studied, IM PS-30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease . The processes catalysed by gran- T.l . and gran- C.a . lipases gave poor conversions to esters, and the SP435-catalysed reactions gave intermediate yields of ethyl and methyl esters . Water activity (a(w)) was an important factor in the methanolysis reactions; reaction media with a(w)<0.5 resulted in the highest conversions to methyl esters . Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS-30 . The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched-chain alcohols.

Appl Environ Microbiol, 2002 Dec, 68(12), 5973 - 80
Arthrobacter aurescens TC1 metabolizes diverse s-triazine ring compounds; Strong LC et al.; Arthrobacter aurescens strain TC1 was isolated without enrichment by plating atrazine-contaminated soil directly onto atrazine-clearing plates . A . aurescens TC1 grew in liquid medium with atrazine as the sole source of nitrogen, carbon, and energy, consuming up to 3,000 mg of atrazine per liter . A . aurescens TC1 is metabolically diverse and grew on a wider range of s-triazine compounds than any bacterium previously characterized . The 23 s-triazine substrates serving as the sole nitrogen source included the herbicides ametryn, atratone, cyanazine, prometryn, and simazine . Moreover, atrazine substrate analogs containing fluorine, mercaptan, and cyano groups in place of the chlorine substituent were also growth substrates . Analogs containing hydrogen, azido, and amino functionalities in place of chlorine were not growth substrates . A . aurescens TC1 also metabolized compounds containing chlorine plus N-ethyl, N-propyl, N-butyl, N-s-butyl, N-isobutyl, or N-t-butyl substituents on the s-triazine ring . Atrazine was metabolized to alkylamines and cyanuric acid, the latter accumulating stoichiometrically . Ethylamine and isopropylamine each served as the source of carbon and nitrogen for growth . PCR experiments identified genes with high sequence identity to atzB and atzC, but not to atzA, from Pseudomonas sp . strain ADP.

Appl Environ Microbiol, 2002 Dec, 68(12), 5882 - 90
Purification and characterization of carbazole 1,9a-dioxygenase, a three-component dioxygenase system of Pseudomonas resinovorans strain CA10; Nam JW et al.; The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd) . Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity . CarAa was found to be trimeric and to have one Rieske type {2Fe-2S} cluster and one mononuclear iron center in each monomer . Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one {2Fe-2S} cluster per monomer protein; His-tagged CarAc, one Rieske type {2Fe-2S} cluster per monomer protein) . Both NADH and NADPH were effective as electron donors for His-tagged CarAd . However, since the k(cat)/K(m) for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd . In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd . Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH . The three purified proteins could reconstitute the CARDO activity in vitro . In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.

Biochemistry, 2002 Dec 3, 41(48), 14430 - 7
Atrazine chlorohydrolase from Pseudomonas sp . strain ADP is a metalloenzyme; Seffernick JL et al.; Atrazine chlorohydrolase (AtzA) from Pseudomonas sp . ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine . Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily . AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion . Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts . Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II) . Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal . Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.

Prikl Biokhim Mikrobiol, 2002 Nov-Dec, 38(6), 653 - 7
Growth phase dependent substrate utilization by Pseudomonas strain PH1; Narde GK et al.; Pseudomonas strain PH1 can utilize nitro-, chloro-, and aminophenols and has been used in this study . The enzymes of the two-pathway viz., phenol, and meta-aminophenol (MAP) were analyzed under different growth conditions . The enzymes responsible for phenol to catechol conversion followed by the ring cleavage enzyme for catechol; and also the enzymes responsible for MAP oxidation and hydroxylation of resorcinol, were studied . Enzyme and respirometric assays were carried out with cells harvested from log phase and stationary phase from medium with different carbon sources and nitrogen levels . It was observed that the first step for utilization of both the substrates requires the same physiological state of the cells; whereas, the subsequent step follows independent approach to intermediates, based on cellular physiology.

Semin Hematol, 2002 Oct, 39(4 Suppl 3), 12 - 9
Monoclonal antibody therapies in leukemias; Tallman MS; Significant advances in the development of monoclonal antibodies (unconjugated) and monoclonal antibodies conjugated to potent toxins or cytotoxic agents (immunoconjugates) have enabled improved targeting of leukemic cells with acceptable toxicities . Gemtuzumab ozogamicin, a calicheamicin-conjugated anti-CD33 monoclonal antibody, has demonstrated substantial efficacy in patients with acute myeloid leukemia (AML) and has induced remissions in patients with favorable-, intermediate-, and poor-risk cytogenetics . The immunoconjugate BL-22, comprised of an anti-CD22 monoclonal antibody fused to a fragment of pseudomonas exotoxin PE38, has produced high response rates in patients with purine analog-resistant hairy cell leukemia . Campath-1H (Wellcome, Beckenham, UK, and Ilex Oncology, San Antonio, TX), an anti-CD52 monoclonal antibody, has demonstrated significant activity in patients with previously untreated, relapsed, or refractory chronic lymphocytic leukemia (CLL), as well as in patients with T-cell prolymphocytic leukemia . The anti-CD20 monoclonal antibody rituximab also is effective in treating CLL and is being evaluated in combination with chemotherapeutic agents (cyclophosphamide) and fludarabine . Monoclonal antibodies may sensitize cells to chemotherapy . The optimal role of targeted therapy with monoclonal antibodies and immunoconjugates in acute and chronic leukemias has not yet been determined, but these novel therapies are beginning to fulfill their promise .

Biochem Biophys Res Commun, 2002 Dec 6, 299(3), 352 - 9
Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation; Ryang SH et al.; We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization . In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR . The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI . The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker . After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs . Therefore, only the tester/tester is selectively amplified in subsequent PCR . This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population . Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency . This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv . tomato (Pst), which elicits a hypersensitive response in Chinese cabbage . After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis . Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment . Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein . Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.

J Basic Microbiol, 2002, 42(6), 434 - 43
Evaluation of carbazole degradation by Pseudomonas rhodesiae strain KK1 isolated from soil contaminated with coal tar; Yoon BJ et al.; In this study, strain KK1 isolated from coal tar-contaminated soil was found to be able to mineralize carbazole as a sole source of carbon by radiorespirometric analysis . KK1 cells pregrown on phenanthrene were able to mineralize carbazole much more rapidly than cells pregrown on naphthalene, suggesting a possible close linkage between the pathways for carbazole and phenanthrene catabolism . Also, Rieske-type iron sulfur center sequence of dioxygenase from KK1 was analyzed to evaluate carbazole catabolism by KK1 . A gene cloned out from KK1 using a universal dioxygenase primer set was found a dioxygenase for initial catabolism of carbazole based on deduced amino acid sequences . Northern hybridization using the putative carbazole dixoygenase gene fragment as a probe provided the information that catabolism of carbazole might be greatly activated in phenanthrene-grown cells . Analysis of PLFAs extracted from KK1 cells exposed to carbazole revealed that lipids 10:0 3OH, 17:0 cyclo, and 18:0 were representatives produced or significantly increased in response to carbazole . Strain KK1 was identified as Pseudomonas species with 94% confidence when BIOLOG system was applied, as Pseudomonas sp . with over 90% confidence by total cellular compositions of fatty acid, and as Pseudomonas rhodesiae with 99% confidence by 16S rRNA sequence . Accordingly, strain KK1 was identified as Pseudomonas rhodesiae based on combination of the data, and designated Pseudomonas rhodesiae KK1 . The phylogenetic tree based on 16S rRNA suggested that strain KK1 was far away in the phylogenetic distance from the strains that can degrade carbazole.

Steroids, 2002 Dec, 67(13-14), 1121 - 7
Use of bioconversion for the preparation of {4-14C}-labeled 7 alpha- and 7 beta-hydroxylated derivatives of dehydroepiandrosterone and epiandrosterone; Chalbot S et al.; The 7 alpha- and 7 beta-hydroxylated derivatives of {4-14C}-dehydroepiandrosterone were prepared with use of the yeast-expressed human cytochrome p4507B1 . Epiandrosterone (EPIA), 5 alpha-androstane-3beta,17 beta-diol, and 5 alpha-androstane-3,17-dione were obtained after incubation of {4-14C}-5 alpha-dihydrotestosterone with Escherichia coli-expressed (3beta,17 beta)-hydroxysteroid dehydrogenase from Pseudomonas testosteroni . The 7 alpha- and 7 beta-hydroxylated derivatives of {4-14C}-EPIA produced were prepared after incubation with mycelium of Rhizopus nigricans . Each labeled steroid was purified by chromatography and identified by crystallization to constant specific activity after isotopic dilution with each authentic steroid carrier . Production yields and radio-purity measurements allowed the use of such procedures for the preparation of the described radio-steroids for studies of metabolism and mode of action.

Mol Plant Microbe Interact, 2002 Oct, 15(10), 1025 - 30
Constitutive activation of jasmonate signaling in an Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum, Pseudomonas syringae, and Myzus persicae; Ellis C et al.; In Arabidopsis spp., the jasmonate (JA) response pathway generally is required for defenses against necrotrophic pathogens and chewing insects, while the salicylic acid (SA) response pathway is generally required for specific, resistance (R) gene-mediated defenses against both biotrophic and necrotrophic pathogens . For example, SA-dependent defenses are required for resistance to the biotrophic fungal pathogen Erysiphe cichoracearum UCSC1 and the bacterial pathogen Pseudomonas syringae pv . maculicola, and also are expressed during response to the green peach aphid Myzus persicae . However, recent evidence indicates that the expression of JA-dependent defenses also may confer resistance to E . cichoracearum . To confirm and to extend this observation, we have compared the disease and pest resistance of wild-type Arabidopsis plants with that of the mutants coil, which is insensitive to JA, and cev1, which has constitutive JA signaling . Measurements of the colonization of these plants by E . cichoracearum, P . syringae pv . maculicola, and M . persicae indicated that activation of the JA signal pathway enhanced resistance, and was associated with the activation of JA-dependent defense genes and the suppression of SA-dependent defense genes . We conclude that JA and SA induce alternative defense pathways that can confer resistance to the same pathogens and pests.

Mol Plant Microbe Interact, 2002 Oct, 15(10), 1014 - 24
A gene in the Pseudomonas syringae pv . tomato Hrp pathogenicity island conserved effector locus, hopPtoA1, contributes to efficient formation of bacterial colonies in planta and is duplicated elsewhere in the genome; Badel JL et al.; The ability of Pseudomonas syringae to grow in planta is thought to be dependent upon the Hrp (type III secretion) system and multiple effector proteins that this system injects into plant cells . ORF5 in the conserved effector locus of the P . syringae pv . tomato DC3000 Hrp pathogenicity island was shown to encode a Hrp-secreted protein and to have a similarly secreted homolog encoded in an effector-rich pathogenicity island located elsewhere in the genome . These putative effector genes were designated hopPtoA1 and hopPtoA2, respectively . DNA gel blot analysis revealed that sequences hybridizing with hopPtoA1 were widespread among P . syringae pathovars, and some strains, like DC3000, appear to have two copies of the gene . uidA transcriptional fusions revealed that expression of hopPtoA1 and hopPtoA2 can be activated by the HrpL alternative sigma factor . hopPtoA1 and hopPtoA1/hopPtoA2 double mutants were not obviously different from wild-type P . syringae pv . tomato DC3000 in their ability to produce symptoms or to increase their total population size in host tomato and Arabidopsis leaves . However, confocal laser-scanning microscopy of GFP (green fluorescent protein)-labeled bacteria in Arabidopsis leaves 2 days after inoculation revealed that the frequency of undeveloped individual colonies was higher in the hopPtoA1 mutant and even higher in the hopPtoA1/hopPtoA2 double mutant . These results suggest that hopPtoA1 and hopPtoA2 contribute redundantly to the formation of P . syringae pv . tomato DC3000 colonies in Arabidopsis leaves.

Curr Microbiol, 2003 Jan, 46(1), 39 - 42
Naphtho{1,2- b}thiophene degradation by Pseudomonas sp . HKT554: involvement of naphthalene dioxygenase; Matsui T et al.; Pseudomonas sp . strain HKT554 degrades naphtho{1,2- b}thiophene and two other isomers, naphtho{2,1- b}thiophene and naphtho{2,3- b}thiophene, by cometabolism, in the absence of any specific inducer, at similar degradation rates . A mutant of strain HKT554, deficient in dibenzothiophene degradation, was generated by using a recently developed transposition system . Sequence analysis of the mutant revealed that the knocked out gene was almost identical to naphthalene dioxygenase (EC 1.14.12.12) . The mutant, HKT554M, degraded neither the naphthothiophene isomers nor dibenzothiophene, suggesting that the naphthalene dioxygenase is responsible for the initial catabolic reactions onto naphthothiophenes and dibenzothiophene.

Chem Commun (Camb), 2002 Oct 21, (20), 2304 - 5
Hammett analysis of a C-C hydrolase-catalysed reaction using synthetic 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates; Speare DM et al.; A Hammett plot (rho = -0.71) has been measured for C-C hydrolase enzyme BphD from Pseudomonas LB400, using six 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acids synthesised by a Heck coupling strategy.

Clin Cancer Res, 2002 Nov, 8(11), 3503 - 11
Interleukin 4 receptor on human lung cancer: a molecular target for cytotoxin therapy; Kawakami M et al.; Previous studies have demonstrated that human lung tumor cell lines express interleukin 4 (IL-4) receptors, and IL-4 can mediate modest to moderate antiproliferative activity in vitro and in vivo in animal models of human lung tumors . On the basis of these studies, IL-4 was tested in clinical trials; however, it showed little antitumor activity in lung cancer patients . In the present study, we examined the expression of IL-4 receptors (IL-4Rs) in lung tumor samples and normal lung tissues and tested whether an IL-4R targeted agent will have better antitumor activity in vitro and in vivo compared with IL-4 . IL-4R expression was tested by immunohistochemistry in 54 lung tumor samples and normal lung tissues in a tissue array, by reverse-transcription PCR and Northern blot analyses in lung tumor cell lines . Cytotoxic activity of IL-4 cytotoxin {IL-4(38-37)-PE38KDEL}, composed of a circular permuted IL-4 and a mutated form of Pseudomonas exotoxin (PE38KDEL) was tested by protein synthesis inhibition and clonogenic assays in seven lung tumor cell lines . Antitumor activity of IL-4 cytotoxin was tested in vitro and in immunodeficient animal models of human lung tumors . We observed that IL-4Rs are expressed at higher levels in situ in lung tumor samples compared with normal lung tissues and IL-4 cytotoxin is highly and specifically cytotoxic to lung tumor cell lines in vitro . Intratumoral and i.p . administration of IL-4 cytotoxin to immunodeficient mice with s.c . established human lung H358 non-small cell lung cancer tumors mediated considerable antitumor activity in a dose-dependent manner with the higher dose producing durable complete responses . On the other hand, H460 non-small cell lung cancer tumors expressing low levels of IL-4R did not respond to IL-4 cytotoxin therapy . Because IL-4 cytotoxin mediates its antitumor activity through IL-4R, and a variety of lung tumors expressed high levels of IL-4R, we propose testing the safety of this agent in patients with lung cancer.

Microbiology, 2002 Nov, 148(Pt 11), 3617 - 29
Molecular analysis of the soluble butane monooxygenase from 'Pseudomonas butanovora'; Sluis MK et al.; 'Pseudomonas butanovora' is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO) . In vitro oxidation of ethylene (an alternative substrate for sBMO) was reconstituted in the soluble portion of cell extracts and was NADH-dependent . Butane monooxygenase was separated into three components which were obligately required for substrate oxidation . The N-terminal sequences of the peptides associated with butane monooxygenase led to the cloning and sequencing of the 5797 nucleotide bmo gene cluster . Comparisons of the deduced amino acid sequences with other multicomponent monooxygenases suggest that sBMO is a multimeric hydroxylase with 61, 45 and 19 kDa subunits encoded by bmoXYZ, a 40 kDa oxidoreductase encoded by bmoC, and a 15 kDa regulatory protein encoded by bmoB . A sixth structural gene (bmoD) encodes a 9.6 kDa protein with similarity exclusively to mmoD (orfY), a putative metal centre assembly protein of the soluble methane monooxygenases . Insertional inactivation of bmoX resulted in a mutant 'P . butanovora' strain incapable of growth with butane . A putative promoter element characteristic of promoters associated with sigma(54)-dependent transcription initiation was located upstream of the bmo genes . Expression of all six genes was detected in butane-induced cells . Butane monooxygenase from 'P . butanovora' aligns most closely with non-haem carboxylate-bridged diiron monooxygenases and, moreover, contains the characteristic iron-binding motif . The structural and mechanistic implications of the high sequence identity (up to 64%) between the peptides of butane monooxygenase and methane monooxygenases are discussed.

Cell Prolif, 2002 Dec, 35(6), 343 - 61
A mathematical model of the impact of infused targeted cytotoxic agents on brain tumours: implications for detection, design and delivery; Wein LM et al.; Motivated by the recent development of highly specific agents for brain tumours, we develop a mathematical model of the spatio-temporal dynamics of a brain tumour that receives an infusion of a highly specific cytotoxic agent (e.g . IL-4-PE, a cytotoxin comprised of IL-4 and a mutated form of Pseudomonas exotoxin) . We derive an approximate but accurate mathematical formula for the tumour cure probability in terms of the tumour characteristics (size at time of detection, proliferation rate, diffusion coefficient), drug design (killing rate, loss rate and convection constants for tumour and tissue), and drug delivery (infusion rate, infusion duration) . Our results suggest that high specificity is necessary but not sufficient to cure malignant gliomas; a nondispersed spatial profile of pretreatment tumour cells and/or good drug penetration are also required . The most important levers to improve tumour cure appear to be earlier detection, higher infusion rate, lower drug clearance rate and better convection into tumour, but not tissue . In contrast, the tumour cure probability is less sensitive to a longer infusion duration and enhancements in drug potency and drug specificity.

J Bacteriol, 2002 Dec, 184(23), 6746 - 9
Low-temperature-induced changes in composition and fluidity of lipopolysaccharides in the antarctic psychrotrophic bacterium Pseudomonas syringae; Kumar GS et al.; The Antarctic psychrotrophic bacterium Pseudomonas syringae was more sensitive to polymyxin B at a lower (4 degrees C) temperature of growth than at a higher (22 degrees C) temperature . The amount of hydroxy fatty acids in the lipopolysaccharides (LPS) also increased at the lower temperature . These changes correlated with the increase in fluidity of the hydrophobic phase of lipopolysaccharide aggregates in vitro.

Biomacromolecules, 2002 Nov-Dec, 3(6), 1320 - 6
Change of surface structure of poly(3-hydroxybutyrate) film upon enzymatic hydrolysis by PHB depolymerase; Yoshie N et al.; The change in the surface structure of poly{(R)-3-hydroxybutyrate} {PHB} films upon the enzymatic hydrolysis was analyzed by attenuated total reflection infrared {ATR/IR} spectrometry . As enzymes, PHB depolymerases isolated from Ralstonia pickettii T1 and Pseudomonas stutzeri were used . By curve decomposition of the carbonyl stretching band of ATR/IR spectra, the change in the surface crystallinity of PHB films by exposure to buffer containing 0, 1, and 4 microg of PHB depolymerases was estimated . It has been widely believed that the enzymatic hydrolysis first occurs in the amorphous phase, followed by the degradation in the crystalline phase, and extracellular PHB depolymerase can degrade only polymer chains in the surface layer of the film . Therefore, the surface crystallinity had been expected to increase upon the enzymatic degradation . However, the results were contrary to this expectation . The surface crystallinity was decreased by the enzymatic attack . Because ATR/IR spectrometry is sensitive to a small change in molecular structure of the sample surface, the decrease in the crystallinity shown by ATR/IR experiments probably does not indicate the complete loss of regularity of the crystalline phase . Because the chains at crystalline surface are more mobile than those inside the crystals, the C=O band for crystalline surface may appear at a position similar to those of the amorphous or interfacial phase in ATR/IR spectra of PHB . Only the chains inside the crystals may contribute to the C=O band of the crystalline phase . Thus, we rather suppose that the decrease in the crystalline peak of the ATR/IR spectra reflects the change in chain mobility or the increase of crystalline surface area by cracking of lamellas at the surface layers of PHB films or both.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2001, 66(2a), 241 - 7
Effects of nutritional factors on production of tabtoxin, a phytotoxin, by Pseudomonas syringae pv . tabaci; Dehbi F et al.; Pseudomonas syringae pv . tabaci, the causal agent of the wildfire of tobacco, produces the phytotoxin tabtoxin . The effects of carbon, nitrogen sources and amino acids on growth and tabtoxin production by pv . tabaci, were examined by varying the components of a defined basal medium, which contained the following nutrients per liter: sucrose (10 g), KNO3 (5 g), MgSO(4).7H2O (0.2 g), CaCl(2).2H2O (0.11 g), FeSO(4).7H2O (20 mg), NaH2PO(4).2H2O (0.9 g) and H2PO(4).3HO (1 g) . Bacterial growth was determined by cell density, and tabtoxin production was measured by the E . coli bioassay technique . Both growth and quantity of tabtoxin synthesized were significantly affected by carbon source, nitrogen source and amino acid supplements . Sorbitol, xylose and sucrose proved to be the best carbon sources for tabtoxin production . Specific toxin production was very low using glucose as a single carbohydrate source, although bacterial growth was well supported by glucose . Amount and type of nitrogen sources (NH4Cl or KNO3) affected the growth of pv . tabaci and quantities of tabtoxin produced . Nitrate was the best of these two forms of nitrogen for production of tabtoxin . Adding threonine to pv . tabaci grown in batch culture decreased the amount of tabtoxin production . Similar results were obtained with lysine, whereas, serine had no effects on quantities of tabtoxin production . The results of the present study were to formulate a medium which allowed for enhanced tabtoxin production by Pseudomonas syringae pv . tabaci.

Eur J Biochem, 2002 Nov, 269(22), 5689 - 99
Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex; Cafaro V et al.; This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization . Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined . The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity . Tomo F component is an NADH oxidoreductase . The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one {2Fe-2S} cluster, and exhibits a typical flavodoxin absorption spectrum . Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence . We have shown that this depends on minor errors in the gene sequence that we have corrected . C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one {2Fe-2S} cluster . The cluster is very sensitive to oxygen damage . Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified . Finally, experimental evidence is reported which strongly support a model for the electron transfer . Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.

Rev Argent Microbiol, 2002 Jul-Sep, 34(3), 138 - 49
{Analysis of aromatic hydrocarbon catabolic genes in strains isolated from soil in Patagonia}; Vacca GS et al.; Seven strains belonging to genus Pseudomonas were isolated from an enrichment with hydrocarbon mixtures . Tests for enzyme activities showed that five strains used predominantly the catabolic meta-pathway for aromatic hydrocarbon degradation . Furthermore, the xylE gene which encodes a catechol 2,3-dioxygenase was amplified by PCR, and in two strains the nahAc gene, a key enzyme for naphthalene catabolism, was also found . The xylE gene might be a good marker to identify aromatic hydrocarbon degrading bacteria in soils from Patagonia.

Pediatr Dev Pathol, 2003 Jan-Feb, 6(1), 88 - 93 Epub 2002 Nov 06.
Cystic fibrosis and Chiari type I malformation: autopsy study of two infants with a rare association; Rakheja D et al.; Cystic fibrosis (CF), an epithelial cell transport disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is not generally associated with malformations of the central nervous system (CNS) . This report describes two African-American children who died at less than 2 years of age with known Chiari I malformations and were found, unexpectedly at autopsy, to have the classic pancreatic and respiratory changes of CF . Both patients had suffered from failure to thrive that had been attributed to their CNS malformations . One child also had recurrent pneumonia and died with Pseudomonas sepsis . Mutational analysis for > 70 common CFTR mutations identified a single delta F508 mutation in one patient and a single 3120+1G to A mutation in the other . Their second CFTR mutations were not identified . The association of CF with Chiari I malformation is not likely to be purely coincidental, as the probability of such an occurrence in African-Americans is greater than one in 7,500,000 patients . It is possible that the CFTR gene may play a previously unrecognized role in CNS development . Alternatively, this CNS abnormality may be acquired due to the metabolic and electrolyte imbalances that characteristically occur in CF.

Plant J, 2002 Nov, 32(3), 299 - 315
Comprehensive transcript profiling of Pto- and Prf-mediated host defense responses to infection by Pseudomonas syringae pv . tomato; Mysore KS et al.; The disease resistance gene Pto encodes a serine/threonine protein kinase that confers resistance in tomato to Pseudomonas syringae pv . tomato strains that express the effector protein AvrPto . Pto-mediated resistance to bacterial speck disease also requires Prf, a protein with leucine-rich repeats and a putative nucleotide-binding site, although the role of Prf in the defense pathway is not known . We used GeneCalling, an open-architecture, mRNA-profiling technology, to identify genes that are either induced or suppressed in leaves 4 h after bacterial infection in the Pto- and Prf-mediated tomato-Pseudomonas(avrPto) interaction . Over 135 000 individual cDNA fragments representing an estimated 90% of the transcripts expressed in tomato leaves were examined and 432 differentially expressed genes were identified . The genes encode over 25 classes of proteins including 11 types of transcription factors and many signal transduction components . Differential expression of 91% of the genes required both Pto and Prf . Interestingly, differential expression of 32 genes did not require Pto but was dependent on Prf . Thus, our data support a role for Prf early in the Pto pathway and indicate that Prf can also function as an independent host recognition determinant of bacterial infection . Comprehensive expression profiling of the Pto-mediated defense response allows the development of many new hypotheses about the molecular basis of resistance to bacterial speck disease.

Bioorg Khim, 2002 Sep-Oct, 28(5), 434 - 9
{Hydrophobic formate dehydrogenase from Pseudomonas sp . 101 in the system of reverse micelles of aerosol OT in octane}; Trofimova DN et al.; NAD(+)-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2-10) . The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane . Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer . The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree . Thus, the modified enzyme mainly functions in the form of monomer and octamer . The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Mar, 22(3), 206 - 7
Purification and renaturation of recombiant human interleukin-2-pseudomonas exotoxin (IL2-PE66(4GLU)) fusion protein; Hu ZM et al.; OBJECTIVE: To evaluate the effect of a novel approach for purification and renaturation of recombinant human interleukin-2-pseudomonas exotoxin (IL2-PE66(4Glu)) fusion protein . METHODS: A novel purification method established in our laboratory was adopted for the purification of the inclusion body, and after renaturation, recombinant human IL2-PE66(4Glu) fusion protein was purified by DEAE-Sepharose FF ion-exchange chromatography . RESULTS: The purity of the fusion protein that retain its biological activity was as high as 95%, and a recovery rate over 80% of the refolded IL2-PE66(4Glu) fusion protein was achieved . CONCLUSION: The purification and refolding method for inclusion body adopted in this study is simple and practical, which lays the foundation for a large-scale production of the fusion protein.

Cancer Immunol Immunother, 2002 Nov, 51(10), 565 - 73 Epub 2002 Sep 13.
A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells; Epel M et al.; Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR) . Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity . Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53 . An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide . In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex . To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38) . The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide . Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner . These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex . Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy.

Eur J Biochem, 2002 Oct, 269(20), 4985 - 93
The beta-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions; De Vries RP et al.; The Aspergillus nigerbeta-1,4-endogalactanase encoding gene (galA) was cloned and characterized . The expression of galA in A . niger was only detected in the presence of sugar beet pectin, d-galacturonic acid and l-arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes . The corresponding enzyme, endogalactanase A (GALA), contains both active site residues identified previously for the Pseudomonas fluorescensbeta-1,4-endogalactanase . The galA gene was overexpressed to facilitate purification of GALA . The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5 . Incubations of arabinogalactans of potato, onion and soy with GALA resulted initially in the release of d-galactotriose and d-galactotetraose, whereas prolonged incubation resulted in d-galactose and d-galactobiose, predominantly . MALDI-TOF analysis revealed the release of l-arabinose substituted d-galacto-oligosaccharides from soy arabinogalactan . This is the first report of the ability of a beta-1,4-endogalactanase to release substituted d-galacto-oligosaccharides . GALA was not active towards d-galacto-oligosaccharides that were substituted with d-glucose at the reducing end.

BMC Plant Biol . 2002 Oct 15;2(1):9.
Two pathways act in an additive rather than obligatorily synergistic fashion to induce systemic acquired resistance and PR gene expression; Zhang C et al.; BACKGROUND: Local infection with necrotizing pathogens induces whole plant immunity to secondary challenge . Pathogenesis-related genes are induced in parallel with this systemic acquired resistance response and thought to be co-regulated . The hypothesis of co-regulation has been challenged by induction of Arabidopsis PR-1 but not systemic acquired resistance in npr1 mutant plants responding to Pseudomonas syringae carrying the avirulence gene avrRpt2 . However, experiments with ndr1 mutant plants have revealed major differences between avirulence genes . The ndr1-1 mutation prevents hypersensitive cell death, systemic acquired resistance and PR-1 induction elicited by bacteria carrying avrRpt2 . This mutation does not prevent these responses to bacteria carrying avrB . RESULTS: Systemic acquired resistance, PR-1 induction and PR-5 induction were assessed in comparisons of npr1-2 and ndr1-1 mutant plants, double mutant plants, and wild-type plants . Systemic acquired resistance was displayed by all four plant lines in response to Pseudomonas syringae bacteria carrying avrB . PR-1 induction was partially impaired by either single mutation in response to either bacterial strain, but only fully impaired in the double mutant in response to avrRpt2 . PR-5 induction was not fully impaired in any of the mutants in response to either avirulence gene . CONCLUSION: Two pathways act additively, rather than in an obligatorily synergistic fashion, to induce systemic acquired resistance, PR-1 and PR-5 . One of these pathways is NPR1-independent and depends on signals associated with hypersensitive cell death . The other pathway is dependent on salicylic acid accumulation and acts through NPR1 . At least two other pathways also contribute additively to PR-5 induction.

Can J Microbiol, 2002 Aug, 48(8), 739 - 48
Physiological and genetic characterization of fluorescent Pseudomonas associated with Cantharellus cibarius; Rangel-Castro JI et al.; Fluorescent Pseudomonas spp . isolated from fruiting bodies (FB) of Cantharellus cibarius were characterized physiologically and genetically and were compared with fluorescent Pseudomonas from forest soil and with sequences from the GenBank database . Pseudomonas spp . from FB differed physiologically from isolates from soil lacking FB and had some similarities with the strains obtained from soil underneath the FB . Analyses of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) patterns and partial sequencing analysis of the 16S-rDNA region indicated that the bacteria isolated from these environments were different . However, there was no specific Pseudomonas genotype restricted to the FB environment . Utilization of the reported fungal exudates trehalose and mannitol may explain how millions of bacteria survive in the C . cibarius FB without deteriorating the fungal mycelium . The importance of the metabolic characterization of bacteria and the possible mechanisms involved in the association with C cibarius are discussed . Our study showed that standard processes for bacterial identification, e.g., Biolog and 16S-rDNA are insufficient until databases for different ecosystems are created.

Biochem Biophys Res Commun, 2002 Oct 18, 298(1), 46 - 53
A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation; Koike M et al.; A novel cDNA clone, Tad1, was isolated from crown tissue of winter wheat after differential screening of cold acclimation-induced genes . The Tad1 cDNA encoded a 23kDa polypeptide with a potential N-terminal signal sequence . The putative mature sequence showed striking similarity to plant defensins or gamma-thionins, representing low molecular size antipathogenic polypeptides . High levels of Tad1 mRNA accumulation occurred within one day of cold acclimation in crown tissue and the level was maintained throughout 14 days of cold acclimation . Similar rapid induction was observed in young seedlings treated with low temperature but not with exogenous abscisic acid . In contrast to defensins from other plant species, neither salicylic acid nor methyl jasmonate induced expression of Tad1 . The recombinant mature form of TAD1 polypeptide inhibited the growth of the phytopathogenic bacteria, Pseudomonas cichorii; however, no antifreeze activity was detected . Collectively, these data suggested that Tad1 is induced in cold-acclimated winter wheat independent of major defense signaling(s) and is involved in low temperature-induced resistance to pathogens during winter hardening.

Trends Microbiol, 2002 Oct, 10(10), 462 - 9
Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors; Collmer A et al.; Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells . Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete . A pre-closure draft sequence of Pseudomonas syringae pv . tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain . These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P . syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.

Structure (Camb), 2002 Oct, 10(10), 1292 - 5
The ABCDs of periplasmic copper trafficking; Puig S et al.; The structure of the CopC protein of Pseudomonas syringae pathovar tomato provides fascinating clues, not only to its role in the periplasmic space in copper resistance, but also to features important for copper trafficking and homeostasis that may be conserved in a variety of biological systems.

Plant Physiol, 2002 Oct, 130(2), 549 - 60
Hydrogen peroxide activates cell death and defense gene expression in birch; Pellinen RI et al.; The function of hydrogen peroxide (H(2)O(2)) as a signal molecule regulating gene expression and cell death induced by external stresses was studied in birch (Betula pendula) . Ozone (O(3)), Pseudomonas syringae pv syringae (Pss), and wounding all induced cell death of various extents in birch leaves . This was temporally preceded and closely accompanied by H(2)O(2) accumulation at, and especially surrounding, the lesion sites . O(3) and Pss, along with an artificial H(2)O(2) producing system glucose (Glc)/Glc oxidase, elicited elevated mRNA levels corresponding to genes encoding reactive oxygen species detoxifying enzymes, Pal, Ypr10, and mitochondrial phosphate translocator 1 . In addition to the regulation of gene expression, Glc/Glc oxidase also induced endogenous H(2)O(2) production in birch leaves, accompanied by cell death that resembled O(3) and Pss damage . Wound-induced gene expression differed from that induced by O(3) and Pss . Thus, it appears that at least two separate defense pathways can be activated in birch leaves by stress factors, even though the early H(2)O(2) accumulation response is common among them all.

Clin Cancer Res, 2002 Oct, 8(10), 3092 - 9
A phase I trial of the single-chain immunotoxin SGN-10 (BR96 sFv-PE40) in patients with advanced solid tumors; Posey JA et al.; PURPOSE: Our purpose in the study was to establish the maximum tolerated dose and toxicity profile of SGN-10 (or BR96 sFv-PE40), a single-chain immunotoxin . SGN-10 is composed of the fused gene products encoding the translocating and ADP-ribosylating domains of Pseudomonas exotoxin (PE40) and the variable heavy (V(H)) and variable light (V(L)) regions of BR96 monoclonal antibody . This antibody is specific for a Lewis(Y) (Le(Y))-related carbohydrate antigen expressed on multiple carcinomas . Experimental Design: A total of 46 patients with Le(Y)-positive metastatic carcinoma were enrolled in a Phase I dose-escalation study in cohorts of three to six patients who received SGN-10 at doses ranging from 0.024 to 0.962 mg/m(2), administered on days 1, 4, 8, and 11, followed by 2 weeks of rest and a second cycle of therapy . Pharmacokinetics and human antibody response to SGN-10 were also determined . RESULTS: The maximum tolerated dose of SGN-10 was 0.641 mg/m(2) with gastrointestinal dose-limiting toxicity . Pharmacokinetic studies performed in eight patients at the 0.641-mg/m(2) dose revealed a t({1/2}) of 2.5 +/- 0.3 h and a C(max) of 389 +/- 112 ng/ml . Pharmacodynamic analyses demonstrated a rapid clearance of the drug by day 11 associated with an antitoxin human antitoxin antibody (HATA) response in most patients . Signs consistent with a modest vascular leak syndrome, specifically, transient hypoalbuminemia, were observed in patients treated with doses of > or =0.384 mg/m(2) . No complete or partial tumor responses were observed at an 8-week evaluation, although 31% of patients had stable disease . CONCLUSIONS: The maximal tolerated dose of SGN-10 given twice weekly for 2 weeks is 0.641 mg/m(2) with gastrointestinal dose-limiting toxicity . The immunogenicity of the toxin moiety limits the ability of SGN-10 to circulate by day 11 of therapy . Studies are ongoing to evaluate strategies to ameliorate toxicities and to inhibit the development of the anti-SGN-10 immune response.

Biochemistry, 2002 Oct 15, 41(41), 12488 - 97
The three-dimensional structure of the gallium complex of azoverdin, a siderophore of Azomonas macrocytogenes ATCC 12334, determined by NMR using residual dipolar coupling constants; Wasielewski E et al.; In iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore . Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer-membrane receptor . This compound is related to the pyoverdins, the peptidic siderophores of Pseudomonas, but differs in the site on the chromophore at which the peptide is covalently linked . This feature identifies azoverdin as a member of a new class of pyoverdins: the isopyoverdins . We report the three-dimensional structure of azoverdin-Ga(III) in solution . The use of orientational constraints obtained from the measurement of residual dipolar couplings using samples dissolved in a liquid crystalline medium allowed us to define the absolute configuration of the metal complex, which is Delta . The structure is characterized by a U-shape adopted by the peptide chain, with the N(delta)-acetyl-N(delta)-hydroxyornithine side chains adopting extended conformations in order to chelate the gallium ion . This conformation leaves a large open space permitting access to the gallium ion . The structural consequences of the particular isopyoverdin chemical structure are discussed in the context of the three-dimensional structures of other pyoverdins.

Am J Pathol, 2002 Oct, 161(4), 1283 - 97
Interleukin-13 fusion cytotoxin arrests Schistosoma mansoni egg-induced pulmonary granuloma formation in mice; Jakubzick C et al.; Schistosoma mansoni egg-induced lung pathology requires the actions of interleukin (IL)-4 and IL-13 . Because receptors for IL-4 and IL-13 share chains, we examined the effect of a fusion protein comprised of IL-13 and Pseudomonas exotoxin (IL13-PE) on the development of pulmonary granulomas in mice . At day 8 after an intravenous injection of live S . mansoni eggs, whole lung samples from IL13-PE-treated mice exhibited significantly lower IL-4 and IL-13 gene expression, smaller granulomas, decreased collagen levels, and increased IL-13 receptor alpha2 gene expression compared to controls . The therapeutic effects of IL13-PE were also observed at day 16 despite the termination of IL13-PE treatment at day 8 . These studies demonstrate that targeting IL-4- and IL-13- responsive cells with IL13-PE effectively arrests S . mansoni egg granuloma formation.

Mol Microbiol, 2002 Oct, 46(1), 223 - 34
Modulation of pPS10 host range by DnaA; Maestro B et al.; Narrow-host-range plasmid pPS10, originally found in Pseudomonas savastanoi, is unable to replicate in other strains such as Escherichia coli . Here, we report that the establishment of pPS10 in E . coli can be achieved by a triple mutation in the dnaA gene of E . coli (dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant) . As the E . coli strain used contained double amber suppressor mutations (supE, supF), the amber codon in dnaA403 can be translated into glutamine or tyrosine . Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon in E . coli . The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity . To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon in E . coli and provides support for the hypothesis that interactions between RepA and DnaA modulate the establishment of pPS10 in that bacteria and probably in other species.

Bioresour Technol, 2002 Dec, 85(3), 313 - 5
Impact of fly ash and phosphate solubilising bacteria on soybean productivity; Gaind S et al.; Fly ash was characterized for the leaching potential of some major and minor constituents and then added to soil at 20, 40, 60 and 80 t/ha with N and P fertilizer to evaluate its effect on nutrient uptake and soybean yield singly as well as in combination with an efficient phosphate solubilizer Pseudomonas striata . The application of fly ash at 40 t/ha in conjunction with P . striata inoculation improved the bean yield and P uptake by grain . The available phosphorus of soil also showed an upward trend . The fly ash did not exert any detrimental effect on the population of inoculated bacteria . However, the uptake of trace elements did not improve significantly.

Nucleic Acids Res, 2002 Oct 1, 30(19), 4138 - 44
RNA recognition site of PP7 coat protein; Lim F et al.; The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity . Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas . Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta . Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e . SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu . However, another structural class possessing only some of these features is capable of binding almost as tightly.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1749 - 58
Phenotypic and genomic evidence for the revision of Pseudomonas corrugata and proposal of Pseudomonas mediterranea sp . nov; Catara V et al.; To re-examine the taxonomic status of Pseudomonas corrugata, 27 strains of this species were studied using a polyphasic approach . Numerical analysis of phenotypic data revealed two phena, A (including the P . corrugata type strain) and B, which could be clearly differentiated by the assimilation of mesotartrate, 2-ketogluconate and histamine . The mean DNA reassociation values with labelled DNA of P . corrugata type strain CFBP 2431T (phenon A) and strain CFBP 5447T (phenon B) were high for strains belonging to the same phenon (96.9 and 98.5%, respectively), whereas the DNA relatedness between the two phena was assessed as being close to 70%, which represents the value that is accepted for the definition of a bacterial species . Phena A and B were also differentiated by means of DNA profiles generated by heteroduplex mobility assay of PCR products of 16S rDNA hypervariable region 2, HaeIII restriction of the amplified internal transcribed spacer, REP- and BOX-PCR profiles, and by PCR with two pairs of specific primers . A comparison of the 16S rRNA sequences of strains CFBP 5447T and CFBP 5458 from phenon B with the available sequences of Pseudomonas species showed that these strains formed a cluster distinct from the P . corrugata type strain . Thus, a new species, Pseudomonas mediterranea, is proposed for strains of phenon B . The type strain is strain CFBP 5447T (= ICMP 14184T); its G+C content is 60.2 mol%.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1559 - 67
Pseudomonas indica sp . nov., a novel butane-utilizing species; Pandey KK et al.; The taxonomic position of two butane-utilizing bacteria was studied using a polyphasic approach . Biochemical and physiological characteristics indicated these to be members of the genus Pseudomonas, showing more similarity to Pseudomonas mendocina than to any other species . The major fatty acids found in these two strains also pointed to their similarity to P . mendocina . On the other hand, DNA-DNA hybridization studies with seven related Pseudomonas species belonging to the gamma-Proteobacteria and the deltaTm values of reassociated molecules clearly showed that these two strains do not belong to any of the seven species tested . The 16S rRNA gene was sequenced and compared with the sequences available in the GenBank database . Phylogenetic analysis using the region covering positions 31-1488 (Escherichia coli numbering) confirmed these observations and placed these two strains as members of the authentic Pseudomonas, but not in any existing species of the genus . On the basis of biochemical characteristics, fatty acid profiles, DNA-DNA reassociation and deltaTm values, as well as 16S rRNA gene sequence analyses, these two isolates were shown to belong to one species but to have characteristics distinct from those of validly described species of Pseudomonas (sensu stricto) . These strains, therefore, should be recognized as a novel species, for which the name Pseudomonas indica sp . nov . is proposed . The type strain is strain IMT37T (= MTCC 3713T = DSM 14015T).

Lett Appl Microbiol, 2002, 35(4), 276 - 80
PCR assays for specific and sensitive detection of Pseudomonas tolaasii, the cause of brown blotch disease of mushrooms; Lee HI et al.; AIMS: The present study describes PCR assays to detect specifically Pseudomonas tolaasii from various samples . METHODS AND RESULTS: Two sets of PCR primers were developed to amplify genes required for tolaasin production . Only a PCR product of 449 bp or 249 bp was produced in PCR reactions with the Pt-1A/Pt-1D1 or Pt-PM/Pt-QM primer sets, respectively, and DNA and cells of Ps . tolaasii . Nested and immunocapture-nested PCR could detect to 3 cells of Ps . tolaasii and amplify the Ps . tolaasii-specific DNA from a sample containing 10 000 times more other bacterial cells than Ps . tolaasii, respectively . CONCLUSIONS: The PCR assays are simple, rapid and reliable methods for detection and identification of Ps . tolaasii . SIGNIFICANCE AND IMPACT OF THE STUDY: The protocols can effectively distinguish Ps . tolaasii from other bacteria and detect Ps . tolaasii from various samples for studying ecology of the bacterium and preventing the use of contaminated water or spawn or medium in mushroom cultivation.

Biochemistry, 2002 Oct 8, 41(40), 11946 - 53
Characterization of subunit-specific interactions in a double-stranded RNA virus: Raman difference spectroscopy of the phi6 procapsid; Benevides JM et al.; The icosahedral core of a double-stranded (ds) RNA virus hosts RNA-dependent polymerase activity and provides the molecular machinery for RNA packaging . The stringent requirements of dsRNA metabolism may explain the similarities observed in core architecture among a broad spectrum of dsRNA viruses, from the mammalian rotaviruses to the Pseudomonas bacteriophage phi6 . Although the structure of the assembled core has been described in atomic detail for Reoviridae (blue tongue virus and reovirus), the molecular mechanism of assembly has not been characterized in terms of conformational changes and key interactions of protein constituents . In the present study, we address such questions through the application of Raman spectroscopy to an in vitro core assembly system--the procapsid of phi6 . The phi6 procapsid, which comprises multiple copies of viral proteins P1 (copy number 120), P2 (12), P4 (72), and P7 (60), represents a precursor of the core that is devoid of RNA . Raman signatures of the procapsid, its purified recombinant core protein components, and purified sub-assemblies lacking either one or two of the protein components have been obtained and interpreted . The major procapsid protein (P1), which forms the skeletal frame of the core, is an elongated and monomeric molecule of high alpha-helical content . The fold of the core RNA polymerase (P2) is also mostly alpha-helical . On the other hand, the folds of both the procapsid accessory protein (P7) and RNA-packaging ATPase (P4) are of the alpha/beta type . Raman difference spectra show that conformational changes occur upon interaction of P1 with either P4 or P7 in the procapsid . These changes involve substantial ordering of the polypeptide backbone . Conversely, conformations of procapsid subunits are not significantly affected by interactions with P2 . An assembly model is proposed in which P1 induces alpha-helix in P4 during formation of the nucleation complex . Subsequently, the partially disordered P7 subunit is folded within the context of the procapsid shell.

J Ind Microbiol Biotechnol, 2002 Oct, 29(4), 185 - 8
Isolation of a mutant strain of Pseudomonas sp ATCC 31461 exhibiting elevated polysaccharide production; West TP; A mutant strain of the bacterium Pseudomonas sp . ATCC 31461 that exhibited elevated production of the polysaccharide gellan on glucose or corn syrup as a carbon source was isolated . Gellan production by the mutant strain was about twofold higher than its parent strain on glucose or corn syrup after 48 h of growth, and about 1.4-fold higher after 72 h . An increase in biomass production was not correlated with enhanced gellan synthesis by the mutant strain . The increased gellan production by the mutant strain on either carbon source resulted in an increase in its culture medium viscosity and the viscosity of the isolated polysaccharide produced by glucose-grown cells . No differences in the glucuronic acid content of the polysaccharides produced by the mutant and parent strains were observed.

Planta, 2002 Oct, 215(6), 997 - 1005 Epub 2002 Jun 20.
OsBIMK1, a rice MAP kinase gene involved in disease resistance responses; Song F et al.; The activation of mitogen-activated protein kinases (MAPKs) has been previously implicated in signal transduction during plant responses to pathogen attack as well as to various environmental stresses . We have isolated from rice a new MAPK cDNA, OsBIMK1 ( O ryza s ativa L . BTH-induced MAPK 1), which encodes a 369-amino-acid protein with moderate to high nucleotide sequence similarity to previously reported plant MAPK genes . OsBIMK1 contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY . We analyzed in detail the expression of OsBIMK1 upon treatment with various chemical and biological inducers of resistance responses in rice and in both incompatible and compatible interactions between rice and Magnaporthe grisea . Expression of OsBIMK1 was activated rapidly upon treatment with benzothiadiazole (BTH) as well as with dichloroisonicotinic acid, probenazole, jasmonic acid and its methyl ester, Pseudomonas syringae pv . syringae, or wounding . Expression of OsBIMK1 was induced rapidly during the first 36 h after inoculation with M . grisea in BTH-treated rice seedlings and in an incompatible interaction between M . grisea and a blast-resistant rice genotype . BTH treatment induced a systemic activation of OsBIMK1 expression . These results suggest that OsBIMK1 plays an important role in rice disease resistance.

Plant Cell Physiol, 2002 Sep, 43(9), 1049 - 53
A pathogen-responsive cDNA from potato encodes a protein with homology to a phosphate starvation-induced phosphatase; Petters J et al.; Infiltration of potato leaves with the phytopathogenic bacteria Pseudomonas syringae pv . maculicola induces local and systemic defense gene expression as well as increased resistance against subsequent pathogen attacks . By cDNA-AFLP a gene was identified that is activated locally in potato leaves in response to bacterial infiltration and after infection with Phytophthora infestans, the causal agent of late blight disease . The encoded protein has high homology to a phosphate starvation-induced acid phosphatase from tomato . Possibly, decreased phosphate availability after pathogen infection acts as a signal for the activation of the potato phosphatase gene.

Syst Appl Microbiol, 2002 Aug, 25(2), 220 - 7
Specific PCR amplification for the genus Pseudomonas targeting the 3' half of 16S rDNA and the whole 16S-23S rDNA spacer; Locatelli L et al.; A PCR protocol was developed for the selective amplification of a segment of the ribosomal RNA operon in Pseudomonas strains . Two specific conserved sequences suitable for PCR priming were identified in the middle of the 16S rDNA and at the very beginning of the 23S rDNA respectively . As a result, amplified region includes the 3' half of the 16S rDNA with the whole 16S-23S rRNA Internal Transcripted Spacer (ITS1) sequence . The specificity of the primer set was checked on sequence databases and validated on collection strains and on one hundred soil bacterial isolates . Our results showed that both collection, soil-inhabiting Pseudomonas and some Pseudomonas-related Azotobacter DNAs could be amplified . This specific PCR for the detection of Pseudomonas strains was in good agreement with colony hybridisation using a Pseudomonas-specific probe . The targeted segment is relevant for a characterisation at the species (16S rDNA) as well as at the infraspecific (ITS1) levels . This PCR-based approach offers promising potential for the characterisation of environmental Pseudomonas populations.

Laryngoscope, 2002 Sep, 112(9), 1619 - 22
Evolving resistant pseudomonas to ciprofloxacin in malignant otitis externa; Berenholz L et al.; OBJECTIVE: To determine whether there has been an increase in ciprofloxacin-resistant pseudomonas malignant otitis externa, and if this has increased the morbidity of the disease . STUDY DESIGN: Retrospective . SETTING: Tertiary referral center . PATIENTS: Twenty-eight patients over 13 years . RESULTS: The records of a total of 28 patients who were admitted between 1988 and 2001 with the diagnosis of malignant otitis externa were reviewed . Seven patients had ciprofloxacin-resistant pseudomonas on their hospital culture and sensitivity test . Five of the 7 resistant cases appeared in the last 3 years, as opposed to 2 of the 7 who appeared in the 10 years before that period . In our series, there is a significant trend developing over time of pseudomonas resistant to treatment with ciprofloxacin . No increased morbidity or mortality was found in the ciprofloxacin-resistant pseudomonas group compared with the remaining patients who were sensitive to ciprofloxacin . CONCLUSIONS: In our series, resistance to ciprofloxacin in patients with malignant otitis externa is increasing over time . This may have an impact on the relatively successful outpatient treatment of these patients in the past decade . A return to inpatient or outpatient intravenous treatment with third-generation cephalosporins/antipseudomonal penicillins and more frequent debridement will be required in these patients.

FEMS Microbiol Lett, 2002 Sep 10, 214(2), 171 - 6
Induction of carbofuran oxidation to 4-hydroxycarbofuran by Pseudomonas sp . 50432; Chaudhry GR et al.; Pseudomonas sp . 50432 biotransformed a highly toxic pesticide, carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) to 7-phenol (2,3-dihydro-2,2-dimethyl-7-hydroxy benzofuran) and several unknown metabolites . One of the unknown metabolites identified by gas chromatography/mass spectroscopy was 4-hydroxycarbofuran (2,3-dihydro-2,2-dimethyl-4-hydroxybenzofuran-7-yl methylcarbamate) . It had a mass (237) similar to 3-hydroxycarbofuran and 5-hydroxycarbofuran but different fragmentation patterns . This is the first report in which an inducible oxidative enzyme, hydroxylase, mediated the conversion of carbofuran to 4-hydroxycarbofuran . A second constitutively synthesized enzyme hyrolase transformed carbofuran to 7-phenol.

J Biochem Mol Biol, 2002 Jul 31, 35(4), 432 - 6
Chloroplast-type ferredoxin involved in reactivation of catechol 2,3-dioxygenase from Pseudomonas sp . S 47; Park DW et al.; Pseudomonas sp . S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway . XylTE products catalyze the dioxygenation of the aromatics . The xylT of the strain S-47 is located just upstream of the xylE gene . XylT is a typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81 . The chloroplast-type ferredoxin of Pseudomonas sp . S-47 exhibited a 98% identity with that of P . putida mt-2 (TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms . We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XylT for the reactivation of the catechol 2,3-dioxygenase (XylE) that is oxidized with hydrogen peroxide . The pRES1 that was treated with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 minutes after incubation, but the pRES101 showed no recovery . That means that the typical chloroplast-type ferredoxin (XylT) of Pseudomonas sp . S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.

Plant Cell, 1993 Jan, 5(1), 57 - 63
Suppression of Bean Defense Responses by Pseudomonas syringae; Jakobek JL et al.; We have developed a model system to examine suppression of defense responses in bean by the compatible bacterium Pseudomonas syringae pv phaseolicola . Previously, we have shown that there is a general mechanism for the induction of the bean defense genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and chitinase (CHT) by incompatible, compatible, and nonpathogenic bacteria . Here, we show that bean plants infiltrated with isolates of P . s . phaseolicola failed to produce transcripts for PAL, CHS, or CHI up to 120 hr after infiltration and CHT transcript accumulation was significantly delayed when compared to the incompatible P . syringae strains . Infiltration of bean plants with 108 cells per mL of P . s . phaseolicola NPS3121 8 hr prior to infiltration with an equal concentration of incompatible P . s . pv tabaci Pt11528 significantly reduced the typical profile of defense transcript accumulation when compared to plants infiltrated with Pt11528 alone . A corresponding suppression of phytoalexin accumulation was also observed . NPS3121 also suppressed PAL, CHS, CHI, and CHT transcript accumulation and phytoalexin production induced by Escherichia coli DH5{alpha} or the elicitor glutathione . Heat-killed NPS3121 cells or cells treated with protein synthesis inhibitors lost the suppressor activity . Taken together, these experiments suggest that NPS3121 has an active mechanism to suppress the accumulation of defense transcripts and phytoalexin biosynthesis in bean.

Plant Cell, 1993 Jan, 5(1), 49 - 56
Generalized Induction of Defense Responses in Bean Is Not Correlated with the Induction of the Hypersensitive Reaction; Jakobek JL et al.; Transcripts for phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, and chitinase accumulated in common bean after infiltration with the Pseudomonas syringae pv tabaci Hrp- mutant Pt11528::Hrp1, even though a hypersensitive reaction did not occur . The temporal pattern of this transcript accumulation was similar to that seen after infiltration with wild-type P . s . tabaci Pt11528, which resulted in a hypersensitive reaction . Escherichia coli DH5{alpha}, P . fluorescens Pf101, heat-killed Pt11528 cells, and Pt11528 cells treated with protein synthesis inhibitors also induced accumulation of defense transcripts but not a hypersensitive reaction . In contrast, these transcripts were not detected in plants infiltrated with water or P.s . pv phaseolicola NPS3121, a compatible pathogen that causes halo blight . Phytoalexins were produced in bean after infiltration with Pt11528, Pt11528::Hrp1, Pt11528 cells treated with neomycin, or Pf101, but not in plants infiltrated with NPS3121 or water . These results suggest that there are unique biochemical events associated with the expression of a hypersensitive reaction which are distinct from other plant defense responses such as phytoalexin biosynthesis . In addition, our results support the hypothesis that there is a general, nonspecific mechanism for the induction of defense transcripts and phytoalexins by pathogenic and saprophytic bacteria that is distinct from the more specific mechanism associated with the induction of the hypersensitive reaction.

Plant Cell, 1994 May, 6(5), 751 - 759
Arabidopsis Mutants Selected for Resistance to the Phytotoxin Coronatine Are Male Sterile, Insensitive to Methyl Jasmonate, and Resistant to a Bacterial Pathogen; Feys B et al.; The phytotoxin coronatine and the plant growth regulator methyl jasmonate (MeJA) caused similar growth-inhibitory effects on Arabidopsis seedlings . To test whether these two compounds have similar action, 14 independent coi1 (coronatine-insensitive) mutants of Arabidopsis were selected . The mutants segregated as single recessive Mendelian markers, and all were alleles at the coi1 locus . All coi1 mutants were also insensitive to MeJA and were male sterile . Both coronatine and MeJA inhibited root growth, stimulated anthocyanin accumulation, and increased the level of two proteins of ~31 and ~29 kD detected in SDS-polyacrylamide gels of wild-type Arabidopsis but caused none of these effects in the coi1 mutant . Coronatine and MeJA also induced the systemic appearance of proteinase inhibitor activity in tomato . The male-sterile flowers of the coi1 mutant produced abnormal pollen and had reduced level of an ~31-kD protein, which was abundant in the wild-type flowers . A coronatine-producing strain of Pseudomonas syringae grew in leaves of wild-type Arabidopsis to a population more than 100 times greater than it reached in the coi1 mutant . We conclude that coronatine mimics the action of MeJA and that coi1 controls a step in MeJA perception/response and in flower development.

Plant Cell, 1994 Dec, 6(12), 1703 - 1712
Function of Oxidative Cross-Linking of Cell Wall Structural Proteins in Plant Disease Resistance; Brisson LF et al.; Elicitation of soybean cells causes a rapid insolubilization of two cell wall structural proteins, p33 and p100 . Likewise, a short elicitation of 30 min rendered cell walls more refractory to enzyme digestion as assayed by the yield of protoplasts released . This effect could be ascribed to protein cross-linking because of its insensitivity to inhibitors of transcription (actinomycin D) and translation (cycloheximide) and its induction by exogenous H2O2 . Moreover, the induced loss of protoplasts could be prevented by preincubation with DTT, which also blocks peroxidase-mediated oxidative cross-linking . The operation of protein insolubilization in plant defense was also demonstrated by its occurrence in the incompatible interaction but not in the compatible interaction between soybean and Pseudomonas syringae pv glycinea . Likewise, protein insolubilization was observed in bean during non-host hypersensitive resistance to the tobacco pathogen P . s . pv tabaci mediated by the hypersensitive resistance and pathogenicity (Hrp) gene cluster . Our data strongly suggest that rapid protein insolubilization leads to a strengthened cell wall, and this mechanism functions as a rapid defense in the initial stages of the hypersensitive response prior to deployment of transcription-dependent defenses.

Plant Cell, 1994 Nov, 6(11), 1583 - 1592
Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance; Cao H et al.; Systemic acquired resistance (SAR) is a general defense response in plants that is characterized by the expression of pathogenesis-related (PR) genes . SAR can be induced after a hypersensitive response to an avirulent pathogen or by treatment with either salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) . To dissect the signal transduction pathway of SAR, we isolated an Arabidopsis mutant that lacks the expression of an SA-, INA-, and pathogen-responsive chimeric reporter gene composed of the 5{prime} untranslated region of an Arabidopsis PR gene, {beta}-1,3-glucanase (BGL2), and the coding region of {beta}-glucuronidase (GUS) . This mutant, npr1 (nonexpresser of PR genes), carries a single recessive mutation that abolishes the SAR-responsive expression of other PR genes as well . While SA-, INA-, or avirulent pathogen-induced SAR protects wild-type plants from Pseudomonas syringae infection, the mutant cannot be protected by pretreatment with these inducers . The insensitivity of npr1 to SA, INA, and avirulent pathogens in SAR induction indicates that these inducers share a common signal transduction pathway . Moreover, in npr1, the localized expression of PR genes induced by a virulent Pseudomonas pathogen is disrupted, and the lesion formed is less confined . These results suggest a role for PR genes in preventing the proximal spread of pathogens in addition to their suggested role in SAR.

Biosens Bioelectron, 2002 Oct, 17(10), 867 - 73
Amperometric biosensors for detection of phenol using chemically modified electrodes containing immobilized bacteria; Skladal P et al.; Eight strains of Pseudomonas were studied for development of phenol sensor . The immobilization of cells was performed by absorbing them on the working part of mediator-modified screen-printed electrodes (SPEs) . Only three Pseudomonas strains were able to transfer electrons resulting from specific oxidation of phenol to the electrode by means of mediators; ferrocene, duroquinone and dimethyferrocene were successfully used with the strains 394 (p20), 74-III and 83-IV (working names), respectively . The lower limits for detection of phenol were 1 micro M for the strain 74-III and 10 micro M for the strain 83-IV and 394 (p20) . Calibrations were obtained as the dependencies of logarithm of current changes (log deltaI) on logarithm of concentration (logC), log delta I vs . logC . Among all substrates tested (phenol, catechol, hydroquinone, ethanol, methanol, propanol, isopropanol, isobutanol, isoamylalcohol, acetate, glucose, xylose, vanillin, 2,4,6-trichlorphenol, 2,3,6-trichlorphenol, 4-hydroxy-3-methoxybenzoic acid, coumarin, pentafluorophenol), bacterial sensor demonstrated a good selectivity with respect to phenol and lower responses to catechol and hydroquinone (10-times lower) . The dependence of signals on operating conditions was studied . The biosensor should be used during the day of preparation . The operational stability was satisfactory to perform up to 10 consecutive measurements . Low cost and very simple manufacturing procedure allow for bacterial sensor to be applied as disposable devices.

Plant Cell, 1995 Oct, 7(10), 1529 - 1536
Expression of the Tomato Pto Gene in Tobacco Enhances Resistance to Pseudomonas syringae pv tabaci Expressing avrPto; Thilmony RL et al.; The Pto gene encodes a serine-threonine kinase that confers resistance in tomato to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto . We examined the ability of Pto to function in tobacco, a species that is sexually incompatible with tomato . Evidence that a heterologous Pto-like signal transduction pathway is present in tobacco was suggested by the fact that tobacco line Wisconsin-38 exhibits a hypersensitive response after infection with P . syringae pv tabaci expressing avrPto . We introduced a Pto transgene into cultivar Wisconsin-38 and assessed the ability of transformed plants to further inhibit growth of the P . s . tabaci strain expressing avrPto . The Pto-transformed tobacco plants exhibited a significant increase in resistance to the avirulent P . s . tabaci strain compared with wild-type tobacco as indicated by (1) more rapid development of a hypersensitive resistance response at high inoculum concentrations (108 colony-forming units per mL); (2) lessened severity of disease symptoms at moderate inoculum concentrations (106 and 107 colony-forming units per mL); and (3) reduced growth of avirulent P . s . tabaci in inoculated leaves . The results indicate that essential components of a Pto-mediated signal transduction pathway are conserved in tobacco and should prompt examination of resistance gene function across even broader taxonomic distances.

Plant Cell, 1996 Feb, 8(2), 251 - 257
Interference between Two Specific Pathogen Recognition Events Mediated by Distinct Plant Disease Resistance Genes; Ritter C et al.; We demonstrate that the interaction of the avirulence gene avrRpt2 and the cognate resistance gene RPS2 interferes with the interaction of avrRpm1-RPM1 in Arabidopsis . Interference is mediated outside of the bacterial pathogen Pseudomonas syringae, presumably at the level of recognition of avr-dependent signals, yet does not require the wild-type RPS2 product . A numerical excess of P . syringae expressing avrRpm1 can overcome this interference in mixed inoculations . The interference of avrRpt2-RPS2 engagement with RPM1-dependent functions is mirrored by transcriptional activation of genes preferentially expressed during RPM1- or RPS2-mediated disease resistance reactions . This demonstration of interference between two plant disease resistance genes suggests that their products compete for a common element(s) in a signal transduction pathway leading to disease resistance.

J Laryngol Otol, 2002 Jul, 116(7), 556 - 8
Masked pseudomonal skull base osteomyelitis presenting with a bilateral Xth cranial nerve palsy; Rowlands RG et al.; Skull base osteomyelitis classically presents as a complication of severe external otitis, middle ear, mastoid or sinus infection and can lead to multiple lower cranial nerve palsies when the jugular foramen is involved as a consequence of widespread involvement of the skull base . Bilateral skull base osteomyelitis is a recognized phenomenon, but has not previously been reported secondary to pseudomonal infection in the absence of a clinically obvious focus of infection . We report the case of a 77-year-old diabetic patient who presented with dysphonia and dysphagia and had a bilateral Xth cranial nerve palsy . No focus of infection was evident on presentation . Subsequent radiological investigation confirmed the diagnosis of bilateral skull base osteomyelitis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(1), 70 - 76
The Purification and Renaturation of the Interleukin-2-Pseudomonas Exotoxin Fusion Protein (IL-2-PE); Gao JM et al.; The IL-2-PE fusion protein expressed in E . coli forms the inclusion body which can be isolated easily . The purity of the fusion protein was 90%-95% after washing the inclusion body with PBS containing 4 M urea and 0.5% Triton X-100 followed by chromatography on Sephacryl S-300 and DEAE-Sepharose FF . Then, parameters relative to the renaturation, including the concentrations of GSSG and L-Arg, the initiation concentrations of the protein, pH, temperature and Reaction time etc, were systematically analysed . An optimum condition for IL-2-PE renaturation was proposed.

Curr Microbiol, 2002 Nov, 45(5), 323 - 7
Development of heavy metal-resistant mutants of phosphate solubilizing Pseudomonas sp . NBRI 4014 and their characterization; Gupta A et al.; Pseudomonas sp . NBRI 4014 is a potent phosphorus solubilizer (284 microg/ml) . It also produced significant levels of siderophore (143.87 microg/ml) and IAA (5.6 microg/ml) . Siderotyping indicated it was P . aeruginosa siderovar 1 . Cadmium (180 microM), nickel (420 microM), and chromium (370 microM) resistant mutants were developed and characterized for their PGPR properties . Mutants were stable under non-selective pressure . In cases of nickel and cadmium, there were reductions of the siderophore levels . However, they were able to promote root and shoot elongation in soybeans ( Glycine max PK 564) at a significant level (p < 0.05) in the presence of metals unfamiliar to the wild type . The persistence and stability of mutants were evident in rhizospheric soil, thus their exploitation for polluted/contaminated sites was supported.

Plant Physiol, 1995 Nov, 109(3), 1107 - 1114
Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants; Meuwly P et al.; Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens . Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more {14C}Phe into {14C}SA than mock-inoculated controls . The identity of SA was confirmed by gas chromatography-mass spectrometry . No reduction in specific activity of {14C}SA was observed for either free or bound SA between control and infected plants after feeding {14C}Phe . A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of {14C}Phe into {14C}SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce {14C}SA from {14C}benzoic acid . Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation . Uninfected tissues from an infected plant synthesized de novo more SA than did controls . This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance.

Plant Physiol, 1995 Aug, 108(4), 1379 - 1385
Systemic Responses in Arabidopsis thaliana Infected and Challenged with Pseudomonas syringae pv syringae; Summermatter K et al.; Attack of plants by necrotizing pathogens leads to acquired resistance to the same or other pathogens in tissues adjacent to or remotely located from the site of initial attack . We have used Arabidopsis thaliana inoculated with the incompatible pathogen Pseudomonas syringae pv syringae on the lower leaves to test the induction of systemic reactions . When plants were challenged with Pseudomonas syringae pv syringae in the upper leaves, bacterial titers remained stable in those preinfected on the lower leaves . However, there was a distinct decrease in symptoms that correlated with a local and systemic increase in salicylic acid (SA) and in chitinase activity . Peroxidase activity only increased at the site of infection . No changes in catalase activity were observed, either at the local or at the systemic level . No inhibition of catalase could be detected in tissue in which the endogenous levels of SA were elevated either naturally (after infection) or artificially (after feeding SA to the roots) . The activity of catalase in homogenates of A . thaliana leaves could not be inhibited in vitro by SA . SA accumulation was induced by H2O2 in leaves, suggesting a link between H2O2 from the oxidative burst commonly observed during the hypersensitive reaction and the induction of a putative signaling molecule leading to system acquired resistance.

Plant Physiol, 1995 Jun, 108(2), 633 - 639
Salicylic Acid in Rice (Biosynthesis, Conjugation, and Possible Role); Silverman P et al.; Salicylic acid (SA) is a natural inducer of disease resistance in some dicotyledonous plants . Rice seedlings (Oryza sativa L.) had the highest levels of SA among all plants tested for SA content (between 0.01 and 37.19 {mu}g/g fresh weight) . The second leaf of rice seedlings had slightly lower SA levels than any younger leaves . To investigate the role of SA in rice disease resistance, we examined the levels of SA in rice (cv M-201) after inoculation with bacterial and fungal pathogens . SA levels did not increase after inoculation with either the avirulent pathogen Pseudomonas syringae D20 or with the rice pathogens Magnaporthe grisea, the causal agent of rice blast, and Rhizoctonia solani, the causal agent of sheath blight . However, leaf SA levels in 28 rice varieties showed a correlation with generalized blast resistance, indicating that SA may play a role as a constitutive defense compound . Biosynthesis and metabolism of SA in rice was studied and compared to that of tobacco . Rice shoots converted {14C}cinnamic acid to SA and the lignin precursors p-coumaric and ferulic acids, whereas {14C}benzoic acid was readily converted to SA . The data suggest that in rice, as in tobacco, SA is synthesized from cinnamic acid via benzoic acid . In rice shoots, SA is largely present as a free acid; however, exogenously supplied SA was converted to {beta}-O-D-glucosylSA by an SA-inducible glucosyltransferase (SA-GTase) . A 7-fold induction of SA-GTase activity was observed after 6 h of feeding 1 mM SA . Both rice roots and shoots showed similar patterns of SA-GTase induction by SA, with maximal induction after feeding with 1 mM SA.

Plant Physiol, 1995 Jun, 108(2), 503 - 516
Hrp Mutant of Pseudomonas syringae pv phaseolicola Induces Cell Wall Alterations but Not Membrane Damage Leading to the Hypersensitive Reaction in Lettuce; Bestwick CS et al.; Both wild-type (S21-WT) and hrpD- (S21-533) strains of Pseudomonas syringae pv phaseolicola induced the formation of large paramural papillae in lettuce (Lactuca sativa) mesophyll cells adjacent to bacterial colonies . Localized alterations to the plant cell wall included deposition of hydroxyproline-rich glycoproteins, phe-nolics, and callose, and were associated with proliferation of the endoplasmic reticulum and multivesicular bodies . Tissue collapse during the hypersensitive reaction caused by S21-WT was associated with electrolyte leakage and rapid accumulation of the phy-toalexin lettucenin A, both of which followed membrane damage indicated by the failure of mesophyll cells to plasmolyze . A few cells lost the ability to plasmolyze after inoculation with S21-533, and low levels of lettucenin A were recorded, but neither leakage of electrolytes nor tissue collapse were detected . Dysfunction of the plasma membrane in cells adjacent to colonies of S21-WT led to extensive vacuolation of the cytoplasm, organelle disruption, and cytoplasmic collapse{mdash}changes unlike those occurring in cells undergoing apoptosis . Strain S21-533 remained viable within symptomless tissue, whereas cells of S21-WT were killed as a consequence of the hypersensitive reaction . Our observations emphasize the subtle coordination of the plant's response occurring at the subcellular level.

Plant Physiol, 1995 May, 108(1), 353 - 359
A Noninvasive Technique for Monitoring Peroxidative and H2O2-Scavenging Activities during Interactions between Bacterial Plant Pathogens and Suspension Cells; Baker CJ et al.; Stimulation of active oxygen metabolism occurs during the early stages of interactions involving bacteria and plant cell suspensions . Although many cellular processes are known to affect active oxygen metabolism in plants, it is not known which of these factors affect active oxygen levels during plant-bacteria interactions . Extracellular peroxidases have been shown to participate in both the production and utilization of active oxygen species such as H2O2 and superoxide . Catalase and other scavenging mechanisms also affect the overall level of active oxygen . In this study the luminol-dependent chemiluminescent reaction previously used to measure H2O2 levels in suspension cells was modified to allow the assay of both peroxidase and H2O2-scavenging activity . The early stages of the interactions between tobacco (Nicotiana tabacum) and Pseudomonas syringae pv syringae, as well as between soybean (Glycine max) and P . syringae pv glycinea, were investigated . This method of monitoring peroxidase and H2O2-scavenging activity proved to be rapid, sensitive, and nonintrusive, allowing the processing of multiple samples using intact cells or cell-free preparations . The results from the study demonstrate that the scavenging activities can be significant and must be considered when studying active oxygen production in biological interactions.

Plant Physiol, 1995 Feb, 107(2), 603 - 612
Analysis of Sweet cherry (Prunus avium L.) Leaves for Plant Signal Molecules That Activate the syrB Gene Required for Synthesis of the Phytotoxin, Syringomycin, by Pseudomonas syringae pv syringae; Mo YY et al.; An important aspect of the interaction of Pseudomonas syringae pv syringae with plant hosts is the perception of plant signal molecules that regulate expression of genes, such as syrB, required for synthesis of the phytotoxin, syringomycin . In this study, the leaves of sweet cherry (Prunus avium L.) were analyzed to determine the nature of the syrB-inducing activity associated with tissues of a susceptible host . Crude leaf extracts yielded high amounts of total signal activity of more than 12,000 units g-1 (fresh weight) based on activation of a syrB-lacZ fusion in strain B3AR132 . The signal activity was fractionated by C18 reversed-phase high-performance liquid chromatography and found to be composed of phenolic glycosides, which were resolved in three regions of the high-performance liquid chromatography profile, and sugars, which eluted with the void volume . Two flavonol glycosides, quercetin 3-rutinosyl-4{prime}-glucoside and kaempferol 3-rutinosyl-4{prime}-glucoside, and a flavanone glucoside, dihydrowogonin 7-glucoside, were identified . The flavonoid glycosides displayed similar specific signal activities and were comparable in signal activity to arbutin, a phenyl {beta}-glucoside, giving rise to between 120 and 160 units of {beta}-galactosidase activity at 10 {mu}M . Although D-fructose exhibits intrinsic low level syrB-inducing signal activity, D-fructose enhanced by about 10-fold the signal activities of the flavonoid glycosides at low concentrations (e.g . 10 {mu}M) . This demonstrates that flavonoid glycosides, which represent a new class of phenolic plant signals sensed by P . s . syringae, are in sufficient quantities in the leaves of P . avium to activate phytotoxin synthesis.

J Org Chem, 2002 Sep 20, 67(19), 6845 - 7
Ionic liquid-coated enzyme for biocatalysis in organic solvent; Lee JK et al.; Ionic liquid-coated enzyme (ILCE) is described as a useful catalyst for biocatalysis in organic solvent . An ionic liquid, {PPMIM}-{PF(6)} (1, {PPMIM} = 1-(3'-phenylpropyl)-3-methylimidazolium), which is solid at room temperature and becomes liquid above 53 degrees C, was synthesized in two steps from N-methylimidazole . The coating of enzyme was done by simply mixing commercially available enzyme with 1 in the liquid phase above 53 degrees C and then allowing the mixture to cool . A representative ILCE, prepared with a lipase from Pseudomonas cepacia, showed markedly enhanced enantioselectivity without losing any significant activity.

Plant Physiol, 2002 Sep, 130(1), 120 - 7
A strobilurin fungicide enhances the resistance of tobacco against tobacco mosaic virus and Pseudomonas syringae pv tabaci; Herms S et al.; The strobilurin class of fungicides comprises a variety of synthetic plant-protecting compounds with broad-spectrum antifungal activity . In the present study, we demonstrate that a strobilurin fungicide, F 500 (Pyraclostrobin), enhances the resistance of tobacco (Nicotiana tabacum cv Xanthi nc) against infection by either tobacco mosaic virus (TMV) or the wildfire pathogen Pseudomonas syringae pv tabaci . F 500 was also active at enhancing TMV resistance in NahG transgenic tobacco plants unable to accumulate significant amounts of the endogenous inducer of enhanced disease resistance, salicylic acid (SA) . This finding suggests that F 500 enhances TMV resistance in tobacco either by acting downstream of SA in the SA signaling mechanism or by functioning independently of SA . The latter assumption is the more likely because in infiltrated leaves, F 500 did not cause the accumulation of SA-inducible pathogenesis-related (PR)-1 proteins that often are used as conventional molecular markers for SA-induced disease resistance . However, accumulation of PR-1 proteins and the associated activation of the PR-1 genes were elicited upon TMV infection of tobacco leaves and both these responses were induced more rapidly in F 500-pretreated plants than in the water-pretreated controls . Taken together, our results suggest that F 500, in addition to exerting direct antifungal activity, may also protect plants by priming them for potentiated activation of subsequently pathogen-induced cellular defense responses.

Plant Physiol, 1996 Apr, 110(4), 1257 - 1266
A Complex Array of Proteins Related to the Multimeric Leucine Aminopeptidase of Tomato; Gu YQ et al.; Leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding, pathogen infection, and insect infestation (V . Pautot, F.M . Holzer, B . Reisch, L.L . Walling {1993} Proc Natl Acad Sci USA 90: 9906-9910) . Polyclonal antibodies to a glutathione S-transferase-LAP fusion protein and affinity-purified antibodies recognizing LAP antigenic determinants detected four classes of polypeptides in tomato (Lycopersicon esculentum) leaves . All four classes had multiple polypeptides in two-dimensional polyacrylamide gel electrophoresis immunoblots . Although antigenically related to the wound-induced tomato LAP proteins, the 77- and 66-kD LAP-like proteins accumulated in both healthy and wounded leaves . Two classes of 55-kD polypeptides with distinctive isoelectric points were designated as plant LAPs; only the acidic LAP proteins accumulated to high levels after mechanical wounding or Pseudomonas syringae pv tomato infection of tomato leaves . The temporal accumulation of LAP mRNAs was correlated with the increase in acidic LAP protein subunits . A slow-migrating LAP activity was detected using a native gel assay after wounding . The molecular mass of the native wound-induced LAP enzyme was 353 kD . The 55-kD acidic LAP proteins were associated with induced LAP activity, whereas the neutral LAPs and the LAP-like proteins were not associated with this exopeptidase . A second, fast-migrating aminopeptidase was detected in both healthy and wounded tomato leaves . Cell fractionation experiments revealed that wound-induced LAP is a soluble enzyme.

Plant Physiol, 1996 Mar, 110(3), 759 - 763
The Active Oxygen Response of Cell Suspensions to Incompatible Bacteria Is Not Sufficient to Cause Hypersensitive Cell Death; Glazener JA et al.; The inoculation of tobacco (Nicotiana tabacum L.) suspension cells with bacterial pathogens that elicit the hypersensitive response (HR) in leaves has been shown to elicit production of active oxygen . This response occurs in two phases, the second of which occurs 1 to 3 h after bacterial addition and is unique to HR-causing interactions . The relationship between the phase II active oxygen response and the HR was characterized using Pseudomonas syringae pv syringae and P . fluorescens (pHIR11), which contains a cosmid clone of the hrp/hrm region from P . syringae pv syringae . TnphoA mutations in complementation groups II through XIII of the hrp cluster blocked the phase II active oxygen response, whereas mutations in the group I hrmA locus did not affect phase II . Despite the normal active oxygen response, bacteria with mutations in the hrmA region did not cause the HR in intact tobacco leaves nor did they induce hypersensitive cell death in cell suspensions . The data indicate that the bacteria do not require the hrmA region to elicit active oxygen production, but a full and intact hrp/hrm region is required to elicit hypersensitive cell death . Therefore, the phase II active oxygen response does not directly cause hypersensitive cell death nor is the response itself sufficient to trigger the HR.

Plant Physiol, 1997 Sep, 115(1), 291 - 298
Salicylic Acid Is Needed in Hypersensitive Cell Death in Soybean but Does Not Act as a Catalase Inhibitor; Tenhaken R et al.; The function of salicylic acid (SA) in hypersensitive cell death was studied in a soybean (Glycine max)-Pseudomonas syringae pv glycinea system . The infection of cell cultures with bacteria leads to a hypersensitive reaction (HR), which is dependent on an appropriate avirulence gene and on low concentrations of SA . The requirement for SA is essential for a process shortly before the onset of the HR-caused cell death 5 to 6 h after infection with bacteria . SA given 10 to 12 h after infection or preincubation cannot rescue the completion of the cell death program . SA does not inhibit catalase or ascorbate peroxidase in soybean . In addition, the in vivo capacity of the cell culture for the rapid metabolism of H2O2 is not altered by SA . This clearly shows that SA is needed for the HR-caused cell death for a reaction downstream of the oxidative burst . Lipid peroxides accumulate during the HR, but the loss of membrane control precedes the generation of lipid peroxides . The accumulation of lipid peroxides in the HR can be prevented by lipid antioxidants . Nevertheless, cell death kinetics remain unaltered in the presence of antioxidants . It is concluded that lipid peroxides are a consequence of cell death, but not the primary cause of it.

Plant Physiol, 1997 Sep, 115(1), 61 - 70
Gene-Expression Patterns and Levels of Jasmonic Acid in Rice Treated with the Resistance Inducer 2,6-Dichloroisonicotinic Acid; Schweizer P et al.; Acquired disease resistance can be induced in rice (Oryza sativa) by a number of synthetic or natural compounds, but the molecular mechanisms behind the phenomenon are poorly understood . One of the synthetic inducers of resistance, 2,6-dichloroisonicotinic acid (INA), efficiently protected rice leaves from infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr . A comparison of gene-expression patterns in plants treated with INA versus plants inoculated with the compatible pathogen M . grisea or the incompatible pathogen Pseudomonas syringae pv syringae revealed only a marginal overlap: 6 gene products, including pathogenesis-related proteins (PR1-PR9), accumulated in both INA-treated and pathogen-attacked leaves, whereas 26 other gene products accumulated only in INA-treated or only in pathogen-attacked leaves . Lipoxygenase enzyme activity and levels of nonconjugated jasmonic acid (JA) were enhanced in leaves of plants treated with a high dose of INA (100 ppm) . Exogenously applied JA enhanced the gene induction and plant protection caused by lower doses of INA (0.1 to 10 ppm) that by themselves did not give rise to enhanced levels of endogenous (-)-JA . These data suggest that INA, aside from activating a pathogen-induced signaling pathway, also induces events that are not related to pathogenesis . JA acts as an enhancer of both types of INA-induced reactions in rice.

Plant Physiol, 1997 Feb, 113(2), 327 - 334
Observations of Ice Nucleation and Propagation in Plants Using Infrared Video Thermography; Wisniewski M et al.; We evaluated the use of infrared (IR) video thermography to observe directly ice nucleation and propagation in plants . An imaging radiometer with an HgCdTe long-wave (8-12 {mu}m) detector was utilized to image the thermal response of plants during freezing . IR images were analyzed in real time and recorded on videotape . Information on the videotape was subsequently accessed and analyzed utilizing IR image analysis software . Freezing of water droplets as small as 0.5 {mu}L was clearly detectable with the radiometer . Additionally, a comparison of temperature tracking data collected by the radiometer with data collected with thermocouples showed close correspondence . Monitoring of an array of plant species under different freezing conditions revealed that ice nucleation and propagation are readily observable by thermal imaging . In many instances, the ice nucleation-active bacterium Pseudomonas syringae placed on test plants could be seen to initiate freezing of the whole plant . Apparent ice nucleation by intrinsic nucleators, despite the presence of ice nucleation-active bacteria, was also evident in some species . Floral bud tissues of peach (Prunus persica) could be seen to supercool below the temperature of stem tissues, and ice nucleation at the site of insertion of the thermocouple was frequently observed . Rates of propagation of ice in different tissues were also easily measured by thermal imaging . This study demonstrates that IR thermography is an excellent method for studying ice nucleation and propagation in plants.

J Dairy Res, 2002 May, 69(2), 227 - 41
Spoilage patterns of skim and whole milks; Deeth HC et al.; The reason for the reported difference in spoilage behaviour of skim and whole pasteurised milks was investigated . The rates of growth of psychrotrophic bacteria were not significantly different in the two milks and the bacterial types, all pseudomonads, present at spoilage were also similar . However, when representative spoilage organisms were cultured into freshly pasteurised skim and whole milks, skim milks exhibited predominantly bitter flavours while whole milk showed mostly sour flavours . The different spoilage behaviours can be largely explained by greater proteolvsis in skim milk than in whole milk, caused by higher production of protease and greater susceptibility of the protein to protease attack . In addition, lipolysis in whole milk, caused by the substantial quantities of lipase produced by spoilage pseudomonads in this milk, also contributes to the different flavours produced during cold storage of these milk types.

Biochemistry, 2002 Sep 17, 41(37), 11152 - 60
Kinetic, Raman, NMR, and site-directed mutagenesis studies of the Pseudomonas sp . strain CBS3 4-hydroxybenzoyl-CoA thioesterase active site; Zhuang Z et al.; 4-Hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase catalyzes the hydrolysis of 4-HBA-CoA to 4-hydroxybenzoate and CoA . X-ray crystallographic analysis of the liganded enzyme has shown that the benzoyl thioester and pantetheine moieties of the substrate ligand are bound in a narrow crevice while the nucleotide moiety rests on the protein surface (Thoden, J . B., Holden, H . M., Zhuang, Z . and Dunaway-Mariano, D . (2002) X-ray Crystallographic Analyses of Inhibitor and Substrate Complexes of Wild-type and Mutant 4-Hydroxybenzoyl-CoA Thioesterase, J . Biol . Chem., in press) . Asp17 is positioned in the crevice, close to the substrate thioester C=O, which in turn interacts with the positive pole of an alpha-helix macrodipole . In this paper we report the results from spectral, mutagenesis, and kinetic studies which show (1) that substrate activation involves restricted thioester C=O conformational freedom and a modest enhancement of C=O bond polarization, (2) that the nucleotide unit of the substrate is bound through interaction with the protein surface, and (3) that Asp17 contributes a rate factor of 10(4), consistent with its proposed role of general base or nucleophile.

J Bacteriol, 2002 Oct, 184(19), 5376 - 84
Purification, substrate range, and metal center of AtzC: the N-isopropylammelide aminohydrolase involved in bacterial atrazine metabolism; Shapir N et al.; N-Isopropylammelide isopropylaminohydrolase, AtzC, the third enzyme in the atrazine degradation pathway in Pseudomonas sp . strain ADP, catalyzes the stoichiometric hydrolysis of N-isopropylammelide to cyanuric acid and isopropylamine . The atzC gene was cloned downstream of the tac promoter and expressed in Escherichia coli, where the expressed enzyme comprised 36% of the soluble protein . AtzC was purified to homogeneity by ammonium sulfate precipitation and phenyl column chromatography . It has a subunit size of 44,938 kDa and a holoenzyme molecular weight of 174,000 . The K(m) and k(cat) values for AtzC with N-isopropylammelide were 406 micro M and 13.3 s(-1), respectively . AtzC hydrolyzed other N-substituted amino dihydroxy-s-triazines, and those with linear N-alkyl groups had higher k(cat) values than those with branched alkyl groups . Native AtzC contained 0.50 eq of Zn per subunit . The activity of metal-depleted AtzC was restored with Zn(II), Fe(II), Mn(II), Co(II), and Ni(II) salts . Cobalt-substituted AtzC had a visible absorbance band at 540 nm (Delta epsilon = 84 M(-1) cm(-1)) and exhibited an axial electron paramagnetic resonance (EPR) signal with the following effective values: g((x)) = 5.18, g((y)) = 3.93, and g((z)) = 2.24 . Incubating cobalt-AtzC with the competitive inhibitor 5-azacytosine altered the effective EPR signal values to g((x)) = 5.11, g((y)) = 4.02, and g((z)) = 2.25 and increased the microwave power at half saturation at 10 K from 31 to 103 mW . Under the growth conditions examined, our data suggest that AtzC has a catalytically essential, five-coordinate Zn(II) metal center in the active site and specifically catalyzes the hydrolysis of intermediates generated during the metabolism of s-triazine herbicides.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(4), 337 - 342
The Influence of Different Physicochemical Parameters of the Solvent On The Activity and Selectivity of Lipase; Gao XG et al.; The asymmetric esterification of octanoic acid with racemic 2-octanol catalyzed by a lipase from Pseudomonas sp . was investigated in several typical solvents . It was found that the catalytic activity and the enantioselectivity of the enzyme were governed by different physicochemical parameters of the solvent employed . While the former depended on the hydrophobicity (lgP) of the solvent, by contrast the latter was a function of the dielectric constant and the dipole moment . A mechanistic model for the binding site of the enzyme was postulated to rationalize this phenomenon based on the results of the kinetic studies of the reaction in some representative solvents.

Microbiology, 2002 Sep, 148(Pt 9), 2687 - 95
Cloning and expression of the phosphotriesterase gene hocA from Pseudomonas monteilii C11; Horne I et al.; The cloning of a gene encoding the novel phosphotriesterase from Pseudomonas monteilii C11, which enabled it to use the organophosphate (OP) coroxon as its sole phosphorus source, is described . The gene, called hocA (hydrolysis of coroxon) consists of 501 bp and encodes a protein of 19 kDa . This protein had no sequence similarity to any proteins in the SWISS-PROT/GenBank databases . When a spectinomycin-resistance cassette was placed in this gene, phosphotriesterase activity was abolished and P . monteilii C11 could no longer grow with coroxon as the sole phosphorus source . Overexpression and purification of HocA as a maltose-binding protein fusion produced a protein having a broad substrate specificity across oxon and thion OPs . Michaelis-Menten kinetics were observed with the oxon OPs, but not with the thion OPs . End-product inhibition was observed for coroxon-hydrolytic activity . Increased expression of hocA was observed from an integrative hocA-lacZ fusion when cultures were grown in the absence of phosphate, suggesting that it might be part of the Pho regulon, but the phosphate-regulated promoter was not cloned in this study . This is believed to be the first study in which a gene required for an organism to grow with OP pesticides as a phosphorus source has been isolated.

Biochim Biophys Acta, 2002 Sep 5, 1584(1), 55 - 64
Oxylipin profiling in pathogen-infected potato leaves; Gobel C et al.; Plants respond to pathogen attack with a multicomponent defense response . Synthesis of oxylipins via the lipoxygenase (LOX) pathway appears to be an important factor for establishment of resistance in a number of pathosystems . In potato cells, pathogen-derived elicitors preferentially stimulate the 9-LOX-dependent metabolism of polyunsaturated fatty acids (PUFAs) . Here we show by oxylipin profiling that potato plants react to pathogen infection with increases in the amounts of the 9-LOX-derived 9,10,11- and 9,12,13-trihydroxy derivatives of linolenic acid (LnA), the divinyl ethers colnelenic acid (CnA) and colneleic acid (CA) as well as 9-hydroxy linolenic acid . Accumulation of these compounds is faster and more pronounced during the interaction of potato with the phytopathogenic bacterium Pseudomonas syringae pv . maculicola, which does not lead to disease, compared to the infection of potato with Phytophthora infestans, the causal agent of late blight disease . Jasmonic acid (JA), a 13-LOX-derived oxylipin, accumulates in potato leaves after infiltration with P . syringae pv . maculicola, but not after infection with P . infestans.

J Basic Microbiol, 2002, 42(4), 260 - 7
Purification and properties of phenylalanyl aminopeptidase synthesised by Pseudomonas sp; Jankiewicz U et al.; Intracellular aminopeptidase synthesized by a soil strain of Pseudomonas sp . was purified 323-fold using the following procedure: saturation with ammonium sulfate, separation by preparative electrophoresis, anion-exchange chromatography and gel filtration chromatography . Molecular weight of the enzyme determined according to the latter method was 57 kDa . Aminopeptidase showed a high substrate specificity and affinity to Phe-beta-naphtylamide (Phe-beta-NA) as a substrate . A considerable inhibition of the enzymatic activity by iodoacetamide and p-chloromercuribenzoate (p-CMB) led to the conclusion that it was a cysteine aminopeptidase . Hydrosulphide compounds markedly stabilised the enzyme . Ethylenediaminetetra-acetic acid (EDTA), a metalloenzyme inhibitor, caused a double increase in the phenylalanyl aminopeptidase activity.( )Mg(2+) ions activated the enzyme to a negligible extent, whereas Co(2+), Cu(2+), Cd(2+) and Pb(2+) ions contributed to its inhibition . The highest enzymatic activity was observed at 37 degrees C and pH 7.0.

Biotechnol Bioeng, 2002 Oct 5, 80(1), 33 - 41
Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase; Panke S et al.; Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp . strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99% . This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion . The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5 . At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited . The reactor had an estimated power input in the order of 5 W . L(-1), which is close to values typically obtained with commercially operating units . The product could be easily purified by fractional distillation to a purity in excess of 97% . The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products .

Appl Environ Microbiol, 2002 Sep, 68(9), 4509 - 16
Lipopeptide production in Pseudomonas sp . strain DSS73 is regulated by components of sugar beet seed exudate via the Gac two-component regulatory system; Koch B et al.; Pseudomonas sp . strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi . By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8 . The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase . DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS . Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds . This growth phenotype could be complemented by purified amphisin . Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase . This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase . Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus . Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.

Appl Environ Microbiol, 2002 Sep, 68(9), 4416 - 24
Cloning and characterization of the ferulic acid catabolic genes of Sphingomonas paucimobilis SYK-6; Masai E et al.; Sphingomonas paucimobilis SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway . We obtained a Tn5 mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid . A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated . The 5.2-kb BamHI-EcoRI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on Escherichia coli host cells . A sequencing analysis revealed the genes ferB and ferA in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively . The deduced amino acid sequence of ferB showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of Pseudomonas and Amycolatopsis ferulic acid degraders . On the other hand, the deduced amino acid sequence of ferA showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders . However, the deduced amino acid sequence of ferA did show 31% identity with pimeloyl-CoA synthetase of Pseudomonas mendocina 35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L . B . Sanchez, M . Y . Galperin, and M . Muller, J . Biol . Chem . 275:5794-5803, 2000) . On the basis of the enzyme activity of E . coli carrying each of these genes, ferA and ferB were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively . p-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities . We found a ferB homolog, ferB2, upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW) involved in the degradation of 5,5'-dehydrodivanillic acid . The deduced amino acid sequence of ferB2 showed 49% identity with ferB, and its gene product showed feruloyl-CoA hydratase/lyase activity with a substrate specificity similar to that of FerB . Insertional inactivation of each fer gene in S . paucimobilis SYK-6 suggested that the ferA gene is essential and that ferB and ferB2 genes are involved in ferulic acid degradation.

Appl Environ Microbiol, 2002 Sep, 68(9), 4383 - 9
Assessment of the importance of similarity in carbon source utilization profiles between the biological control agent and the pathogen in biological control of bacterial speck of tomato; Ji P et al.; Bacterial speck of tomato, caused by Pseudomonas syringae pv . tomato, was used to determine whether similarity in carbon source utilization between a preemptive biological control agent and the pathogen was significant in determining the ability of the bacterium to suppress disease . Similarity in carbon source utilization was quantified as the ratio of the number of tomato carbon sources utilized in vitro by the biological control agent to the number of tomato carbon sources utilized in vitro by the target pathogen (the niche overlap index {NOI}) . Suppression of the disease was quantified as the percent reduction in disease severity compared to the pathogen-only control when nonpathogenic bacteria were applied to foliage 48 h prior to the pathogen . In the collection of 36 nonpathogenic bacterial strains, there was a significant (P < 0.01), but weak (r(2) = 0.25), correlation between reduction in disease severity and similarity in carbon source utilization, suggesting that similarity in carbon source use was significant in determining ability to suppress disease . The relationship was investigated further using catabolic mutants of P . syringae strain TLP2, an effective biological control agent of speck . Catabolic mutants exhibited lower levels of similarity (NOI = 0.07 to 0.90) than did wild-type TLP2 (NOI = 0.93) . With these catabolic mutants there was a significant (P < 0.01), and stronger (r(2) = 0.42), correlation between reduction in disease severity and similarity in carbon source utilization . This suggests that similarity in carbon source utilization was a more important component of biological control ability for the catabolic mutants than for the nonpathogenic bacteria . Together, these studies indicate that suppression of bacterial speck of tomato was correlated with nutritional similarity between the pathogenic and nonpathogenic bacteria and suggest that preemptive utilization of carbon sources was probably involved in the biological control of the disease by both the naturally occurring nonpathogenic bacteria and the catabolic mutants.

Appl Environ Microbiol, 2002 Sep, 68(9), 4315 - 21
Biotransformation of eugenol to ferulic acid by a recombinant strain of Ralstonia eutropha H16; Overhage J et al.; The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp . strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette . This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16 . A recombinant strain of R . eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase . Cells of R . eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid . A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.

J Biol Chem, 2002 Nov 1, 277(44), 42121 - 7 Epub 2002 Aug 26.
Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit; Otten LG et al.; Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins . The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77 . The gene fragment encoding the beta-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR . Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source . In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis . Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266 . Five mutants were further characterized by determination of their Michaelis-Menten parameters . Strikingly, mutant SY-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA . In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA . These results are discussed in the light of a structural model of SY-77 glutaryl acylase.

Biochem Soc Trans, 2002 Aug, 30(4), 653 - 8
Cytochrome cbb(3) oxidase and bacterial microaerobic metabolism; Pitcher RS et al.; Cytochrome cbb(3) oxidase is a member of the haem-copper oxidase superfamily . It is characterized by its high oxygen affinity, while retaining the ability to pump protons . These attributes are central to its proposed role in bacterial microaerobic metabolism . Recent spectroscopic characterization of both the cytochrome cbb(3) oxidase complex from Pseudomonas stutzeri and the dihaem ccoP subunit expressed separately in Escherichia coli has revealed the presence of a low-spin His/His co-ordinated c-type cytochrome . The low midpoint reduction potential of this haem (E(m)<+100 mV), together with its unexpected ability to bind CO in the reduced state at the expense of the distal histidine ligand, raises questions about the role of the ccoP subunit in the delivery of electrons to the active site.

J Antibiot (Tokyo), 2002 Jun, 55(6), 543 - 51
Argyrins, immunosuppressive cyclic peptides from myxobacteria . I . Production, isolation, physico-chemical and biological properties; Sasse F et al.; A group of cyclic peptides consisting of 8 amino acid residues, named argyrins A to H, were isolated from the culture broth of strains of the myxobacterium Archangium gephyra . Argyrin B was found to be a potent inhibitor of T cell independent antibody formation by murine B cells and strongly inhibited the two way murine mixed lymphocyte reaction . All argyrins had slight antibiotic activity, especially against Pseudomonas sp., and inhibited growth of mammalian cell cultures . The growth inhibition was often incomplete and varied highly with different cell lines.

Curr Cancer Drug Targets, 2002 Mar, 2(1), 19 - 36
Diphtheria fusion protein therapy of chemoresistant malignancies; Frankel AE et al.; Patients with widespread cancer respond initially to combination chemotherapy, immunotherapy, and/or radiotherapy, but most relapse with chemoresistant disease . Novel methods of killing resistant neoplastic stem cells are needed . One such approach is therapy with targeted toxins composed of tumor cell selective ligands covalently linked to group I peptide toxins (group II and III peptide toxins act on the cell surface) . The targeted toxin is delivered to the cell by a tumor selective ligand . Once bound, the ligand-receptor complex is internalized . The catalytic domain escapes to the cytosol . The toxin then enzymatically modifies a critical cell function (protein synthesis, p21 Rho activity, protein kinase signaling, cyclic AMP signaling or others) . The irreversibly damaged cells fails to divide and, eventually, undergoes lysis or programmed cell death . Targeted peptide toxins used to date in the treatment of chemotherapy refractory cancers include ricin toxin, Pseudomonas exotoxin, pokeweed antiviral protein, saporin, gelonin and diphtheria toxin . In this review, we have focused on the applications of genetically engineered diphtheria toxin for cancer therapy.

Anticancer Drugs, 2002 Aug, 13(7), 693 - 9
Cytokine receptor as a sensitizer for targeted cancer therapy; Kawakami K et al.; Introducing a cytokine receptor as a sensitizer into cancer cells offers a unique opportunity for receptor-targeted cancer therapy . It has been shown that transfection of the tumor necrosis factor (TNF) receptor gene in cancer cells or exposing cancer cells to certain reagents which increase expression of TNF receptors results in enhancement of the cytotoxic effect of TNF . In addition, the literature suggests that Fas/CD95-mediated apoptotic tumor cell killing is augmented by gene transfer of Fas into cancer cells or treatment of cells with agents like cisplatin and interferon (IFN)-gamma . In contrast to these approaches, we have discovered a new approach to cancer therapy; wherein introduction of a cytokine receptor chain into cancer cells sensitizes them to receptor-directed cytotoxins . We have demonstrated that when interleukin (IL)-13 receptor (IL-13R) alpha2 chain, one of the two known IL-13 binding proteins, is introduced into cancer cells that do not express this chain the cells acquire extreme sensitivity to a chimeric fusion cytotoxin composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE) . Cells that do not express this chain or express low levels show limited sensitivity to IL13-PE . Acquisition of sensitivity to IL13-PE was observed both in vitro and in vivo when IL-13R alpha2-transfected human tumor cells were implanted in immunodeficient animals followed by systemic or regional IL13-PE therapy . Our third generation experiments suggest that this approach is feasible for clinical situations as intratumor administration of plasmid carrying the IL-13R alpha2 chain gene sensitized these tumors to systemic or regional IL13-PE therapy . This unique approach comprising gene transfer of cytokine receptor chain and receptor-targeted cytotoxin administration represents a novel strategy for cancer therapy .

Biochemistry, 2002 Aug 27, 41(34), 10680 - 91
Biochemical, Mössbauer, and EPR studies of the diiron cluster of phenol hydroxylase from Pseudomonas sp . strain CF 600; Cadieux E et al.; Phenol hydroxylase of Pseudomonas sp . strain CF600 comprises three components: DmpP is an FAD- and {2Fe-2S}-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase . Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM:1 DmpLNO . Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex . Mossbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites . Cluster form I, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and delta = 0.48 mm/s . Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged . Reduction of the samples with dithionite yields the diferrous forms of the clusters . Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity . The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6 . Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species . Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H(2)O(2) generation . In addition, Mossbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.

J Chem Ecol, 2002 Jun, 28(6), 1131 - 59
Antagonism between jasmonate- and salicylate-mediated induced plant resistance: effects of concentration and timing of elicitation on defense-related proteins, herbivore, and pathogen performance in tomato; Thaler JS et al.; The jasmonate (JA) and salicylate (SA) signaling pathways in plants provide resistance to herbivorous insects and pathogens . It is known that these pathways interact, sometimes resulting in antagonism between the pathways . We tested how the timing and concentration of elicitation of each pathway influenced the interaction between the jasmonate and salicylate pathways measured in terms of five biochemical responses and biological resistance to caterpillars and bacteria . The salicylate pathway had a stronger effect on the jasmonate pathway than did the reverse . The negative signal interaction was generated by two distinct paths in the plant . A negative interaction in the biochemical expression of the two pathways was most consistent in the simultaneous elicitation experiments compared to when the elicitors were temporally separated by two days . Herbivore bioassays with Spodoptera exigua also consistently reflected an interaction between the two pathways in the simultaneous elicitation experiments . The negative signal interaction reducing biological resistance to the herbivore was also demonstrated in some temporally separated treatment combinations where attenuation of the biochemical response was not evident . Concentration of the elicitors had an effect on the pathway interaction with consistent biochemical and biological antagonism in the high concentration experiments and inconsistent antagonism in the low concentration experiments . The bacterial pathogen, Pseudomonas syringae pv . tomato (Pst), consistently showed reduced lesion development on plants with SA responses activated and, in some experiments, on JA-elicited plants . Resistance to Pst was not reduced or enhanced in dual-elicited plants . Thus, signal interaction is most consistent when elicitors are applied at the same time or when applied at high doses . Signal interaction affected the herbivore S . exigua, but not the pathogen Pst.

J Appl Physiol, 2002 Sep, 93(3), 957 - 65
Delayed rectifier potassium channels contribute to the depressed pulmonary artery contractility in pneumonia; Yaghi A et al.; We investigated the role of K(+) channels in the attenuated pulmonary artery (PA) contractility characteristic of acute Pseudomonas pneumonia . Contractility of PA rings from the lungs of control or pneumonia rats was assessed in vitro by obtaining cumulative concentration-response curves to the contractile agonists KCl, phenylephrine, or PGF(2 alpha) on PA rings before and after treatment with K(+) channel blockers . In rings from pneumonia rats, paxilline (10 microM), tetraethylammonium (2 mM) (blockers of large-conductance Ca(2+)-activated K(+) channels), and glybenclamide (ATP-sensitive K(+) channel blocker, 80 microM) had no significant effect on the attenuated contractile responses to KCl, phenylephrine, and PGF(2 alpha) . However, 4-aminopyridine (2 mM), a blocker of voltage-gated K(+) channels (delayed rectifier K(+) channel) reversed this depressed contractility . Therefore, large-conductance Ca(2+)-activated K(+) and ATP-sensitive K(+) channels do not contribute to the attenuated PA contractility observed in this model of acute pneumonia . In contrast, 4-aminopyridine enhances contraction in PA rings from pneumonia lungs, consistent with involvement of a voltage-gated K(+) channel in the depressed PA contractility in acute pneumonia . Unraveling the precise mechanism of attenuated contractility in pneumonia could lead to innovative therapies for the pulmonary vascular abnormalities associated with this disease.

Mol Plant Microbe Interact, 2002 Aug, 15(8), 808 - 16
Differential expression of genes encoding Arabidopsis phospholipases after challenge with virulent or avirulent Pseudomonas isolates; de Torres Zabela M et al.; Phospholipase D (PLD; EC 3.1.4.4) has been linked to a number of cellular processes, including Tran membrane signaling and membrane degradation . Four PLD genes (alpha, beta, gamma1, and gamma2) have been cloned from Arabidopsis thalami . They encode isoforms with distinct regulatory and catalytic properties but little is known about their physiological roles . Using cDNA amplified fragment length polymorphism display and RNA blot analysis, we identified Arabidopsis PLDgamma1 and a gene encoding a lysophospholipase (EC 3.1.1.5), lysoPL1, to be differentially expressed during host response to virulent and avirulent pathogen challenge . Examination of the expression pattern of phospholipase genes induced in response to pathogen challenge was undertaken using the lysoPL1 and gene-specific probes corresponding to the PLD isoforms a, beta, and gamma1 . Each mRNA class exhibited different temporal patterns of expression after infiltration of leaves with Pseudomonas syringae pv . tomato with or without avrRpm1 . PLDalpha was rapidly induced and remained constitutively elevated regardless of treatment . PLDbeta was transiently induced upon pathogen challenge . However, mRNA for the lysoPL1 and PLDgamma1 genes showed enhanced and sustained elevation during an incompatible interaction, in both ndr1 and overexpressing NahG genetic backgrounds . Further evidence for differential engagement of these PLD mRNA during defense responses, other than gene-for-gene interactions, was demonstrated by their response to salicylic acid treatment or wounding . Our results indicate that genes encoding lysoPL1, PLDgamma1, and PLDbeta are induced during early responses to pathogen challenge and, additionally, PLDyl and lysoPL1 are specifically upregulated during gene-for-gene interactions, leading to the hypersensitive response . We discuss the possible role of these genes in plant-pathogen interactions.

J Exp Bot, 2002 Sep, 53(376), 1887 - 90
In vitro freezing in microtitre plates applied to tobacco plants transformed with the inaZ gene of Pseudomonas syringae; Anastassopoulos E et al.; High throughput assays have been developed to measure the ice nucleation activity of transgenic tobacco, Nicotiana tabacum L . cv . Petit Havana SR1 plants expressing the ice nucleation gene, inaZ, from Pseudomonas syringae at a young seedling stage, as well as in leaf tissue . Both assays are carried out in 96-well microtitre plates . The first assay involves direct seeding in vitro, one seed per microtitre plate well containing Murashige-Skoog agar . When seedlings reach the two-leaf stage, they are exposed to freezing temperatures by floating the plates on a circulating alcohol bath set at temperatures colder than -9 degrees C . The second assay involves placing small leaf discs individually in microtitre plate wells containing sterile distilled water . The assays complement each other, give highly reproducible results, are technically simple and enable the detection of freezing events in large numbers of plants . The utility and limitations of these assays are discussed.

Arch Biochem Biophys, 2002 Sep 1, 405(1), 95 - 103
Biochemical observations on medium-chain-length polyhydroxyalkanoate biosynthesis and accumulation in Pseudomonas mendocina; Kroumova AB et al.; Certain Pseudomonads are capable of accumulating high levels of medium-chain-length polyhydroxyalkanates (PHAmcl) when grown with carbohydrates as the main carbon source . 3-OH acyl components of PHAmcl are derived from fatty acid synthase (FAS) and these components are accessed by action of 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase (transacylase) . However, little is known with regard to the time courses of 3-OH acyl component occurrence and of transacylase activity during PHAmcl induction . Also, little is known with regard to the coupling mechanism between FAS and PHAmcl synthesis or whether the FAS pathway itself is specialized in PHAmcl-producing cells . Our results with regard to the time course of formation of 3-OH acids, 3-OH acyl-ACPs, and PHAmcl are consistent with the view that transacylase provides the key link between FAS and PHAmcl synthase . They also suggest that FAS specialization is not a feature of the mechanism . Further, we observed the formation of a 3-OH 10:0 homopolymer early in the induction phase followed by later formation of a mixed polymer containing 3-OH 8:0 and 3-OH 12:0 in addition to 3-OH 10:0 . Early occurrence of 3-OH 10:0-CoA transacylase activity was coincident with homopolymer formation.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11270 - 4 Epub 2002 Aug 09.
Genetic variation for disease resistance and tolerance among Arabidopsis thaliana accessions; Kover PX et al.; Pathogens can be an important selective agent in plant evolution because they can severely reduce plant fitness and growth . However, the role of pathogen selection on plant evolution depends on the extent of genetic variation for resistance traits and their covariance with host fitness . Although it is usually assumed that resistance traits will covary with plant fitness, this assumption has not been tested rigorously in plant-pathogen interactions . Many plant species are tolerant to herbivores, decoupling the relationship between resistance and fitness . Tolerance to pathogens can reduce selection for resistance and alter the effect of pathogens on plant evolution . In this study, we measured three components of Arabidopsis thaliana resistance (pathogen growth, disease symptoms, and host fitness) to the bacteria Pseudomonas syringae and investigated their covariation to determine the relative importance of resistance and tolerance . We observed extensive quantitative variation in the severity of disease symptoms, the bacterial population size, and the effect of infection on host fitness among 19 accessions of A . thaliana infected with P . syringae . The severity of disease symptoms was strongly and positively correlated with bacterial population size . Although the average fitness of infected plants was smaller than noninfected plants, we found no correlation between the bacterial growth or symptoms expressed by different accessions of A . thaliana and their relative fitness after infection . These results indicate that the accessions studied vary in tolerance to P . syringae, reducing the strength of selection on resistance traits, and that symptoms and bacterial growth are not good predictors of host fitness.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(4), 393 - 396
Secretory Expression of GL-7-ACA Acylase in E . coli; Chen JF et al.; The gene of beta-subunit of GL-7-ACA acylase {7beta-(4-carboxybutanamido) cephalosporanic acid acylase} was cloned into pTrc99B, an IPTG inducible pasmid, to form the recombinant called pTrc-CA1B . Another recombinant plasmid pTrcCA1S was obtained by cloning the gene encoding alpha-subunit of GL-7-ACA acylase, the signal peptide and the expression elements from Pseudomonas sp . into pTrcCA1B . Then, recombinant plasmid pKKCA1S was constructed by cloning the gene encoding the signal peptide, expression elements and GL-7-ACA acylase into the vector pKK235 . pTrcCA1S and pKKCA1S were allowed to transform TG1 . These two plamids were able to transfer the expression product into the periplasmic space of the host bacteria . As a result, in the whole cell of TG1/pTrcCAIS, the specific activity of GL-7-ACA acylase was 23.9 u/g cell, 8 fold higher than that of TG1/pMR24 . And in the whole cell of TG1/pKKCA1S, the specific activity of acylase was 18.3 u/g cell, 6 fold higher than that of TG1/pMR24.

J Am Chem Soc, 2002 Aug 14, 124(32), 9378 - 9
Resonance Raman detection of a ferrous five-coordinate nitrosylheme b(3) complex in cytochrome cbb(3) oxidase from Pseudomonas stutzeri; Pinakoulaki E et al.; Understanding the chemical nature of the nitric oxide (NO) moiety of nitrosylheme copper oxidases is crucial for elucidation of the NO activation process . In the present work, direct resonance Raman spectroscopic observation of both the Fe(2+)-NO and the N-O stretching modes unambiguously establishes the vibrational characteristics of the NO-bound heme moiety in cytochrome cbb(3) from Pseudomonas stutzeri . Addition of NO to fully reduced enzyme causes the rupture of the proximal His-heme b(3) bond resulting in the formation of a five-coordinate heme b(3)(2+)-NO species with nu(Fe-NO) and nu(NO) at 524 and 1679 cm(-1), respectively . The frequencies of the nitrosyl species we detect are very similar to those obtained in other model- and protein heme-NO complexes . To account for this observation, we propose a model describing the oxidation and ligand-binding states in fully reduced cytochrome cbb(3) upon addition of NO.

J Biol Chem, 2002 Oct 11, 277(41), 38262 - 71 Epub 2002 Aug 02.
Isolation and biochemical characterization of hypophosphite/2-oxoglutarate dioxygenase . A novel phosphorus-oxidizing enzyme from Psuedomonas stutzeri WM88; White AK et al.; The htxA gene is required for the oxidation of hypophosphite in Pseudomonas stutzeri WM88 (Metcalf, W . W., and Wolfe, R . S . (1998) J . Bacteriol . 180, 5547-5558) . Amino acid sequence comparisons suggest that hypophosphite:2-oxoglutarate dioxygenase (HtxA) is a novel member of the 2-oxoglutarate-dependent dioxygenase enzyme family . To provide experimental support for this hypothesis, HtxA was overproduced in Escherichia coli and purified to apparent homogeneity . Recombinant HtxA is identical to the native enzyme based on amino terminus sequencing and mass spectral analysis, and it catalyzes the oxidation of hypophosphite to phosphite in a process strictly dependent on 2-oxoglutarate, ferrous ions, and oxygen . Succinate and phosphite are stoichiometrically produced, indicating a strict coupling of the reaction . Size exclusion analysis suggests that HtxA is active as a homodimer, and maximal activity is observed at pH 7.0 and at 27 degrees C . The apparent K(m) values for hypophosphite and 2-oxoglutarate were 0.58 +/- 0.04 mm and 10.6 +/- 1.4 microm, respectively . V(max) and k(cat) values were determined to be 10.9 +/- 0.30 micromol min(-1) mg(-1) and 355 min(-1), respectively . 2-Oxoadipate and pyruvate substitute poorly for 2-oxoglutarate as a cosubstrate . The highest specific activity is observed with hypophosphite as substrate, but HtxA is also able to oxidize formate and arsenite at significant rates . The substrate analog inhibitors, formate and nitrate, significantly reduce HtxA activity.

Environ Microbiol, 2002 Aug, 4(8), 465 - 76
Identification of complex composition, strong strain diversity and directional selection in local Pseudomonas stutzeri populations from marine sediment and soils; Sikorski J et al.; Members of Pseudomonas stutzeri have been isolated world-wide from various habitats including aquatic and terrestrial ecosystems . The global population has a clonal structure, is of exceptionally high genetic diversity and has been grouped into eight genomovars . We have analysed four local populations (n = 89-125) from three geographically separated habitats (two from a marine sediment and two from different soils) by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), restriction fragment length polymorphism (RFLP) of the rpoB gene and 16S rDNA sequences in order to quantify the influence of evolutionary forces on closely related groups of proliferating cells in situ . All populations consisted of a complex structure of genomic subgroups with variable numbers of members . The analyses revealed that the two populations from marine sediment were rather similar . At least three of the populations were influenced by migrational input as concluded from the presence of members from different genomovars . All populations showed very high strain diversity suggesting strong influence of mutability . Neutrality tests indicated that two or possibly three of the populations were shaped by directional selection . Thus, the local populations of P . stutzeri reflect already the high genetic diversity of the global population and are influenced, to different extents, by migration, mutation and directional selection.

Leuk Lymphoma, 2002 Apr, 43(4), 885 - 8
Augmentation of the activity of an immunotoxin, anti-Tac(Fv)-PE40KDEL, in T cell lines infected with human T cell leukemia virus type-I; Ohno N et al.; Therapy with an immunotoxin, anti-Tac(Fv)-PE38, which is a conjugate of the variable domains of an anti-Tac monoclonal antibody and Pseudomonas exotoxin, was reported to be useful for adult T cell leukemia (ATL) patients but a considerable amount of the immunotoxin is needed for the therapy and some side effects were also observed . We have previously demonstrated that an immunotoxin, anti-Tac(Fv)-PE40KDEL, showed strong cytotoxic effects on malignant cells from ATL patients . Therefore, we searched for agents that enhance the effects of the immunotoxin . PAK-200, FK-506, quinidine, cepharanthine and cyclosporine A (CsA) augmented the ability of the immunotoxin to inhibit protein synthesis in two human T cell leukemia virus type-I infected T cell lines, KUT-1 and KUT-2 . CsA was the most potent agent in both the cell lines . Augmentation of the cytotoxic effect of the immunotoxin by these agents, especially CsA, may be useful in the immunotoxin therapy of ATL.

Ukr Biokhim Zh, 2002 Mar-Apr, 74(2), 115 - 9
{Inhibitory effect of lead ions on cells of some strains of Pseudomonas species bacteria}; Gruzina TG et al.; The inhibition effect of ionic lead on membrane ATPase activity, transmembrane potential (delta psi) and permeability level of the Pb-sensitive P . fluorescens B894 and Pb-resistant P . fluorescens B4252 bacteria cells have been studied . It have been shown that decreasing ATPase activity and transmembrane potential values and the increasing of permeability by lead are higher for Pb-sensitive strain then for Pb-resistant . It is suggested that mechanism of the ionic lead toxic effect deals with plasma membrane biochemical parameters (ATPase activity, value of delta psi) alterations and interruption of it barrier function.

Biotechnol Appl Biochem, 2002 Aug, 36(Pt 1), 63 - 70
Purification and biochemical characterization of the carbamate hydrolase from Pseudomonas sp . 50432; Chaudhry GR et al.; A soluble carbamate hydrolase that had a wide specificity was purified 2032-fold from Pseudomonas sp . 50432 . This was achieved using a combination of anion-exchange, gel-filtration and hydrophobic-interaction- chromatography techniques . Carbamate hydrolase cleaved the ester linkage of the N-methylcarbamates . The native enzyme was a monomer with a molecular mass of 88 kDa . The optimum pH and temperature of the enzyme activity were 8.5 and 37 degrees C respectively . The tested cations or EDTA did not affect the enzyme activity . However, 2-mercaptoethanol reversibly inhibited the enzyme activity . The enzyme showed the K(m) values of 16 and 12 microM for carbofuran and carbaryl respectively . The purified enzyme did not hydrolyse o-nitrophenyl dimethylcarbamate but hydrolysed several N-methylcarbamates and 1-naphthyl acetate.

Biotechnol Appl Biochem, 2002 Aug, 36(Pt 1), 47 - 55
Characterization and physicochemical properties of a lipase from Pseudomonas mendocina 3121-1; Surinenaite B et al.; The lipase from Pseudomonas mendocina 3121-1 was found to be homogeneous with a molecular mass of 30 kDa by SDS/PAGE . It is composed of two identical subunits . A molecular mass of 62 kDa was determined by gel chromatography on a Toyopearl HW-55F column . Some physicochemical properties of the lipase were investigated using p-nitrophenyl butyrate (p-NPB), Tween 80 solution and Sigma olive-oil emulsion as substrates . The optimum temperature was determined to be 52 degrees C with p-NPB, in the range 50-60 degrees C with Tween 80 and in the range 50-65 degrees C with olive-oil emulsion . The optimum pH was determined to be in the pH range 7.2-7.5, both with Tween and the emulsion, but was unusually alkaline (pH 9.5) with p-NPB . The enzyme was activated for p-NPB hydrolysis by thermal treatment up to 60 min at 60 degrees C, pH 7.0-8.2, but was rapidly inactivated at 70-80 degrees C and at pH 7.0 . The lipase was shown to be more thermolabile at 60 degrees C with respect to other two substrates . Using the emulsified substrate, no activity was obtained after preincubating the enzyme for 30 min at 70 degrees C . The enzyme was found to be pH-tolerant when stored at 20 degrees C, pH 6.3-10.3 (100 mM Briton-Robson buffer) as the half-life (t(1/2)) was more than 240 h when p-NPB was used as the substrate . By contrast, the pH-stability range was more narrow (pH 8.0-10.5) with olive-oil emulsion . The effect of various metal ions and EDTA depended on the nature of the substrate.

Appl Environ Microbiol, 2002 Aug, 68(8), 3948 - 55
Molecular cloning and characterization of the gene coding for the aerobic azoreductase from Xenophilus azovorans KF46F; Blumel S et al.; The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp . strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4'-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy . The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases . A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase . The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared . The results suggested that whole cells of the recombinant E . coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity . The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E . coli strain was demonstrated.

Biochem J, 2002 Nov 1, 367(Pt 3), 841 - 7
Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae; Serov AE et al.; A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD(+) to NADP(+) has been constructed by introducing two single-point mutations, Asp(196)-->Ala (D196A) and Tyr(197)-->Arg (Y197R) . The mutagenesis was based on the results of homology modelling of a NAD(+)-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template . The resulting model structure suggested that Asp(196) and Tyr(197) mediate the absolute coenzyme specificity of SceFDH for NAD(+).

J Bacteriol, 2002 Aug, 184(16), 4343 - 50
Roles for the two 1-butanol dehydrogenases of Pseudomonas butanovora in butane and 1-butanol metabolism; Vangnai AS et al.; Pseudomonas butanovora grown on butane or 1-butanol expresses two 1-butanol dehydrogenases, a quinoprotein (BOH) and a quinohemoprotein (BDH) . BOH exhibited high affinity towards 1-butanol (K(m) = 1.7 +/- 0.2 microM) . BOH also oxidized butyraldehyde and 2-butanol (K(m) = 369 +/- 85 microM and K(m) = 662 +/- 98 microM, respectively) . The mRNA induction profiles of BOH and BDH at three different levels of 1-butanol, a nontoxic level (0.1 mM), a growth-supporting level (2 mM), and a toxic level (40 mM), were similar . When cells were grown in citrate-containing medium in the presence of different levels of 1-butanol, wild-type P . butanovora could tolerate higher levels of 1-butanol than the P . butanovora boh::tet strain and the P . butanovora bdh::kan strain . A model is proposed in which the electrons from 1-butanol oxidation follow a branched electron transport chain . BOH may be coupled to ubiquinone, with the electrons being transported to a cyanide-sensitive terminal oxidase . In contrast, electrons from BDH may be transferred to a terminal oxidase that is less sensitive to cyanide . The former pathway may function primarily in energy generation, while the latter may be more important in the detoxification of 1-butanol.

Med Microbiol Immunol (Berl), 2002 May, 191(1), 17 - 24
Virolytic action of lipoprotein lipase on hepatitis C virus in human sera; Thomssen R et al.; In most sera of hepatitis C virus (HCV)-infected patients beta-lipoproteins are bound to HCV RNA-carrying material, most often simultaneously with immunoglobulins (IgG, IgM) and sometimes additionally with high-density lipoproteins, forming complexes of low density (1.04-1.06 g/ml) . To separate HCV particles from bound material, we tried to destroy the lipoprotein enzymatically by incubating HCV-positive human sera with lipoprotein lipase derived from Pseudomonas spp . (LPL-Ps) . After this treatment, titers of HCV RNA in human sera (determined by a simple semiquantitative reverse transcription-PCR assay) were strongly reduced, regardless of whether primers of the NTR or NS5 region were used . Inactivation of HCV RNA could be inhibited by the addition of RNAguard to the serum-enzyme mixture . The lytic effect of the LPL-Ps preparation could be inhibited by tetrahydrolipstatin . Hence LPL-Ps seems to disrupt the HCV particle structure, including the putative core of the virus, and makes HCV RNA sensitive for RNase present in the reaction mixture . HBV was not destroyed by LPL-Ps . Porcine pancreatic lipase had no effect on HCV . The implications of these observations for the structure and biology of HCV and for its stability and inactivation in human sera are discussed.

Acta Crystallogr D Biol Crystallogr, 2002 Aug, 58(Pt 8), 1350 - 2 Epub 2002 Jul 20.
Crystallization and preliminary crystallographic analysis of the terminal oxygenase component of carbazole 1,9a-dioxygenase of Pseudomonas resinovorans strain CA10; Nam JW et al.; The terminal oxygenase component (CarAa) of carbazole 1,9a-dioxygenase from Pseudomonas resinovorans strain CA10 was crystallized at 293 K using the sitting-drop vapour-diffusion method under the following conditions: 0.1 M sodium citrate pH 5.6 in the presence of 0.5 M ammonium sulfate and 1.0 M lithium sulfate . By using additive reagents with the crystallizing condition, improved diffraction was obtained from the crystals . Preliminary X-ray diffraction analysis indicated that CarAa crystals are hexagonal and belong to space group P6(2) or P6(4), with unit-cell parameters a = b = 244.5, c = 65.7 A, alpha = beta = 90.0, gamma = 120.0 degrees . Diffraction data were collected to 3.0 A resolution . The V(M) value is 2.16 A(3) Da(-1), which indicates a solvent content of 43.0% . This is the first report of crystallization of the terminal oxygenase component of an angular-type dioxygenase.

Biophys Chem, 2002 Jul 10, 98(1-2), 27 - 34
Intramolecular electron transfer in cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri; kinetics and thermodynamics; Farver O et al.; Cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri catalyzes the one electron reduction of nitrite to nitric oxide . It is a homodimer, each monomer containing one heme-c and one heme-d(1), the former being the electron uptake site while the latter is the nitrite reduction site . Hence, internal electron transfer between these sites is an inherent element in the catalytic cycle of this enzyme . We have investigated the internal electron transfer reaction employing pulse radiolytically produced N-methyl nicotinamide radicals as reductant which reacts solely with the heme-c in an essentially diffusion controlled process . Following this initial step, the reduction equivalent is equilibrating between the c and d(1) heme sites in a unimolecular process (k=23 s(-1), 298 K, pH 7.0) and an equilibrium constant of 1.0 . The temperature dependence of this internal electron transfer process has been determined over a 277-313 K temperature range and yielded both equilibrium standard enthalpy and entropy changes as well as activation parameters of the specific rate constants . The significance of these parameters obtained at low degree of reduction of the enzyme is discussed and compared with earlier studies on cd(1) nitrite reductases from other sources.

Mol Microbiol, 2002 Jul, 45(2), 397 - 409
Lon protease functions as a negative regulator of type III protein secretion in Pseudomonas syringae; Bretz J et al.; The central conserved region of the Pseudomonas syringae hrp pathogenicity island encodes a type III protein secretion system (TTSS) that is required for pathogenicity in plants . Expression of the hrp TTSS is controlled by the alternative sigma factor, HrpL, whose expression, in turn, is positively controlled by two truncated enhancer binding proteins, HrpR and HrpS . Although a number of environmental conditions are known to modulate hrp TTSS expression, such as stringent conditions and pathogenesis, the mechanism by which the activities of these transcriptional factors are modulated had not been established . In this study, HrpR and HrpS were found to be constitutively expressed under conditions in which the hrpL promoter was inactive . To identify a postulated negative regulator of hrpL expression, transposome (Tz) mutagenesis was used to isolate hrp constitutive mutants . P . syringae Pss61 and DC3000 hrp constitutive mutants were identified that carried lon::Tz insertions and exhibited increased cell length and UV sensitivity typical of Delta lon mutants . The P . syringae Lon protease retained structural features of its homologues found in other bacteria and was capable of complementing an Escherichia coli Delta lon mutant . P . syringae lon::Tz mutants exhibited enhanced expression of the hrpL promoter, suggesting an effect on HrpR and/or HrpS . HrpR was observed to be unstable in wild-type P . syringae strains grown in non-inductive media . However, the apparent half-life increased more than 10-fold in the P . syringae lon::Tz mutants or upon transfer to an inductive medium . The P . syringae lon mutants elicited rapidly developing plant responses and were shown to hypersecrete effector proteins, such as AvrPto . These results indicate that expression of the hrp regulon and type III secretion are negatively regulated by Lon-mediated degradation of HrpR.

Plant Cell, 2002 Jul, 14(7), 1469 - 82
Arabidopsis SON1 is an F-box protein that regulates a novel induced defense response independent of both salicylic acid and systemic acquired resistance; Kim HS et al.; One of several induced defense responses in plants is systemic acquired resistance (SAR), which is regulated by salicylic acid and in Arabidopsis by the NIM1/NPR1 protein . To identify additional components of the SAR pathway or other genes that regulate SAR-independent resistance, we performed genetic suppressor screens of mutagenized nim1-1 seedlings, which are highly susceptible to infection by Peronospora parasitica . We isolated the son1 (suppressor of nim1-1) mutant, which shows full restoration of pathogen resistance without the induction of SAR-associated genes and expresses resistance when combined with a salicylate hydroxylase (nahG) transgene . These features indicate that son1-mediated resistance is distinct from SAR . Resistance is effective against both the virulent oomycete Peronospora and the bacterial pathogen Pseudomonas syringae pv tomato strain DC3000 . We cloned SON1 and found it to encode a novel protein containing an F-box motif, an element found within the specificity determinant in the E3 ubiquitin-ligase complex . We propose the existence of a novel defense response that is independent of SAR and negatively regulated in Arabidopsis by SON1 through the ubiquitin-proteosome pathway.

J Biol Chem, 2002 Sep 13, 277(37), 34383 - 90 Epub 2002 Jul 15.
Increased affinity and stability of an anti-HIV-1 envelope immunotoxin by structure-based mutagenesis; McHugh L et al.; HIV-infected cells are selectively killed by an immunotoxin in which a truncated form of Pseudomonas exotoxin A is joined to the variable region of a broadly neutralizing antibody (3B3) that recognizes the viral envelope glycoprotein (Env) . To improve the efficacy of this molecule, we used three-dimensional structural information and phage selection data to design 23 single and multiple point mutations in the antibody variable region sequences that contact Env . Substituting an aromatic residue for an aspartate in the third complementarity-determining region of V(H) increased the potency of the immunotoxin by approximately 10-fold in a cell-killing assay . Detailed analysis of one such mutant, N31H/Q100eY, revealed both a higher affinity for monomeric and cell surface Env and an increased stability against aggregation compared with the starting immunotoxin . Conversion to a disulfide-linked two-chain format further stabilized the protein . N31H/Q100eY retained the ability to bind to Env from multiple viral isolates, to inhibit Env-mediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells . Such site-directed mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans.

Biochem J, 2002 Oct 15, 367(Pt 2), 483 - 9
Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp; Hopper DJ et al.; The gene encoding the enzyme lupanine hydroxylase was isolated by PCR using chromosomal DNA from a lupanine-utilizing Pseudomonas sp . as template and primers based on the sequences of the N- and C-termini of the purified protein . The derived sequence for the mature gene product gave a protein with an M (r) of 72256, in good agreement with the value found by SDS/PAGE of the pure enzyme, and contained the sequences of several peptides obtained after endoproteinase Lys-C digestion of the pure enzyme . The gene, under the transcriptional control of a phoA promotor and with the Escherichia coli alkaline phosphatase signal sequence, was expressed in E . coli containing a plasmid expressing the genes for cytochrome c maturation proteins constitutively . Haem-containing inactive protein in inclusion bodies was renatured and reactivated with pyrroloquinoline quinone (PQQ) and Ca(2+) to give active enzyme . The lupanine hydroxylase (luh) gene coded for a protein with a cleavable 26-residue signal sequence at its N-terminus, required for the transport of the enzyme to its periplasmic location . Analysis of the protein sequence showed that it contains two domains, a large PQQ-binding N-terminal domain and a smaller cytochrome c C-terminal domain . Comparison of the derived sequence with those of other proteins showed considerable similarity with other quino(haemo)proteins, including alcohol dehydrogenases from a variety of bacteria . The PQQ-binding domain sequence contains W motifs, characteristic of the eight-bladed "propeller" structure of methanol dehydrogenase, but lacks the unusual disulphide ring structure formed from two adjacent cysteines seen in this enzyme . The C-terminus shares some similarity with bacterial cytochrome c and includes the haem-binding consensus sequence CXXCH.

Mol Plant Microbe Interact, 2002 Jul, 15(7), 654 - 61
Overexpression of Pto induces a salicylate-independent cell death but inhibits necrotic lesions caused by salicylate-deficiency in tomato plants; Li J et al.; Tomato plants overexpressing the disease resistance gene Pto (35S::Pto) exhibit spontaneous cell death, accumulation of salicylic acid (SA), elevated expression of pathogenesis-related genes, and enhanced resistance to a broad range of pathogens . Because salicylate plays an important role in the cell death and defense activation in many lesion mimic mutants, we investigated the interaction of SA-mediated processes and the 35S::Pto-mediated defense pathway by introducing the nahG transgene that encodes salicylate hydroxylase . Here, we show that SA is not required for the 35S::Pto-activated microscopic cell death and plays a minor role in defense gene activation and general disease resistance in 35S::Pto plants . In contrast, temperature greatly affects the spontaneous cell death and general resistance in 35S::Pto plants, and high temperature inhibits the cell death . The NahG tomato plants develop spontaneous, unconstrained necrotic lesions on leaves . These lesions also are initiated by the inoculation of a virulent strain of Pseudomonas syringae pv . tomato . However, the NahG-dependent necrotic lesions are inhibited in the NahG/35S::Pto plants . This inhibition is most pronounced under conditions favoring the 35S::Pto-mediated spontaneous cell death development . These results indicate that the signaling pathways activated by Pto overexpression suppress the cellular damage that is caused by SA depletion . We also found that ethylene is dispensable for the 35S::Pto-mediated general defense.

Bioresour Technol, 2002 Sep, 84(3), 207 - 11
Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulfate precipitation technique; Kanwar L et al.; Among the various lipidic and non-lipidic substances, normal alkanes within the chain lengths of C-12 to C-20 served as the best carbon substrates for the production of extracellular lipase by Pseudomonas species G6 . Maximum lipase production of 25 U/ml of the culture broth was obtained by using n-hexadecane as the sole carbon substrate . The optimum pH of 8 and temperature of 34 + 1 degrees C were demonstrated for the production of lipase in n-hexadecane substrate . The optimum concentration of iron, which played a critical role on the lipase production, was found to be 0.25 mg/l . Lipase production could be enhanced to nearly 2.4-fold by using tributyrin at a concentration of 0.05% (v/v) in the culture medium . High recovery of the lipase protein (83%) from the culture broth was achieved by treating the culture supernatant with Silicone 21 Defoamer followed by ammonium sulfate (60% saturation) fractionation.

Clin Cancer Res, 2002 Jul, 8(7), 2345 - 55
Novel anti-CD30 recombinant immunotoxins containing disulfide-stabilized Fv fragments; Nagata S et al.; PURPOSE: To develop a novel targeting reagent to CD30 expressed on Hodgkin'sdisease and anaplastic large cell lymphoma, we made a panel of recombinant immunotoxins specific for CD30 using Fvs of newly produced anti-CD30 monoclonal antibodies (MAbs) and a M(r) 38,000 truncated mutant of Pseudomonas exotoxin . EXPERIMENTAL DESIGN: A group of MAbs against CD30 was produced and characterized for their reactivity and epitopes . Recombinant immunotoxins were made using the Fv genes cloned from the hybridomas . Their cytotoxic activities were examined on various CD30-positive cell lines . RESULTS: Six MAbs were produced . All reacted with recombinant soluble CD30 and to a CD30-Fc fusion protein, and bound to native CD30 expressed on Hodgkin's lymphoma-derived cell lines . The epitopes of the six MAbs were classified into two groups by a mutual competition assay for the binding to CD30 on cells . Sequencing the cDNAs revealed that all of the variable chains are unique except one valiable light that is shared by two MAbs . We made four disulfide stabilized Fv-based recombinant immunotoxins, in which the valiable heavy, which is genetically fused with truncated mutant of Pseudomonas exotoxin, forms a disulfide bond with the valiable light . The purified immunotoxins bound to recombinant soluble CD30 immobilized on a biosensor chip with K(d)s of 4-400 nM . Fluorescence-activated cell sorter analysis confirmed their specific binding . In vitro cytotoxicity tests showed that the immunotoxins specifically kill a variety of CD30-positive lymphoma cell lines as well as CD30-transfected A431 cells . The IC(50) ranged from 0.3 to 100 ng/ml . CONCLUSIONS: Four anti-CD30 disulfide stabilized Fv immunotoxins were successfully produced . Two of these showed good cytotoxic activity to various CD30-positive cell lines . These newly produced immunotoxins should be additionally evaluated for the treatment of CD30-positive lymphomas.

Chirality, 2002 Jul, 14(7), 558 - 61
Enzyme immobilization utilizing a porous ceramics support for chiral synthesis; Kamori M et al.; A novel porous ceramics support, named "Toyonite," for the immobilization of enzymes was prepared from the minerals of kaolinite under acidic conditions . Modification of the porous surface of Toyonite with two different organic coating agents gave Toyonite 200-M (TN-M), and Toyonite 200-A (TN-A), possessing methacryloyloxy and amino groups on the respective surfaces . Compared with other solid supports, TN-M and TN-A supports exhibited high selectivity for lipase PS (Pseudomonas cepacia, Amano) and glucoamylase (Gluczyme AF 6, Amano) proteins, respectively . The activities of both the transesterification of rac-1 with TN-M PS lipase and the hydrolysis of starch with TN-A glucoamylase were greater than those of similar reactions with these two enzymes immobilized on other solid supports . Further, TN-M PS lipase showed higher reactivity toward synthetic substrates, including aromatic and aliphatic secondary alcohols, than the free enzyme powder .

J Bacteriol, 2002 Aug, 184(15), 4124 - 33
Evidence for temporal regulation of the two Pseudomonas cellulosa xylanases belonging to glycoside hydrolase family 11; Emami K et al.; Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium . Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized . The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively . In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan . Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound . xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or beta-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes . Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media . Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes . Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone . A transposon mutant of P . cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P . cellulosa . Based on the data presented here, we propose a model for xylan degradation by P . cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.

J Biol Chem, 2002 Sep 13, 277(37), 34161 - 7 Epub 2002 Jul 08.
Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase; Meyer A et al.; Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC ) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpA(ind)) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole . The wild-type protein does not catalyze these reactions . HbpA(ind) contains amino acid substitutions D222V and V368A . The activity for indole hydroxylation was increased 18-fold in this variant . Concomitantly, the K(d) value for indole decreased from 1.5 mm to 78 microm . Investigation of the major reaction products of HbpA(ind) with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate . Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin . The activity of the HbpA(ind) mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme . In HbpA(ind), there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V . The position of Asp(222) in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp(222) is involved in substrate activation in HbpA.

Plant J, 2002 Jul, 31(1), 87 - 95
Phytochrome signalling modulates the SA-perceptive pathway in Arabidopsis; Genoud T et al.; The interaction of phytochrome signalling with the SA signal transduction pathway has been investigated in Arabidopsis using single and multiple mutants affected in light perception (phyA and phyB deficient) and light-signal processing (psi2, phytochrome signalling) . The induction of PR1 by SA and functional analogues has been found to strictly correlate with the activity of the signalling pathway controlled by both phyA and phyB photoreceptors . In darkness as well as dim light, and independently of a carbohydrate source, SA-induced PR gene expression as well as the hypersensitive response to pathogens (HR) are strongly reduced . Moreover, the initiation of HR also exhibits a strict dependence upon both the presence and the amplitude of a phytochrome-elicited signal . The growth of an incompatible strain of bacterial a pathogen (Pseudomonas syringae pv . tomato) was enhanced in phyA-phyB and decreased in psi2 mutants . While functional chloroplasts were found necessary for the development of an HR, the induction of PRs was strictly dependent on light, but independent of functional chloroplasts . Taken together, these data demonstrate that the light-induced signalling pathway interacts with the pathogen/SA-mediated signal transduction route . These results are summarized in a formalism that allows qualitative computer simulation.

Biomacromolecules, 2002 Jul-Aug, 3(4), 787 - 92
Isolation and characterization of polyhydroxyalkanoates inclusions and their associated proteins in Pseudomonas sp . 61-3; Matsumoto K et al.; Two types of polyester inclusions of poly(3-hydroxybutyrate) {P(3HB)} and poly(3HB-co-3-hydroxyalkanoates) {P(3HB-co-3HA)} were isolated from crude extract of Pseudomonas sp . 61-3 . Proteins associated with each inclusion were separated by SDS-PAGE . PHA synthase 1 (PhaC1(Ps)), PhaF(Ps), and PhaI(Ps) were identified from P(3HB-co-3HA) inclusions by N-terminal amino acid sequences analyses, as well as PHB synthase (PhbC(Ps)) and 24-kDa unknown protein were identified from P(3HB) inclusions . The structural genes of PhaF(Ps) and PhaI(Ps) were located downstream of the pha locus . The relative PHA/PHB synthase activities of each inclusion were measured for various 3-hydroxyacyl-coenzyme As of 4-12 carbon atoms . Direct atomic force microscopy observation of P(3HB) and P(3HB-co-3HA) inclusions demonstrated that the two types of inclusions had different morphologies.

Cancer Res, 2002 Jul 1, 62(13), 3575 - 80
Targeting interleukin-4 receptors for effective pancreatic cancer therapy; Kawakami K et al.; We demonstrate that pancreatic cancer tissues express receptors for interleukin (IL)-4 in situ at high density . Using the approach of selective receptor targeting, we have tested the efficacy of a recombinant cytotoxin IL4-Pseudomonas exotoxin A, which is composed of a targeting moiety (IL-4) and a mutated form of Pseudomonas exotoxin . Our results demonstrate that this molecule exerts vigorous antitumor activity against human pancreatic tumors implanted s.c . in immunodeficient animals . Sixty percent of animals treated with intratumoral injections of IL4-Pseudomonas exotoxin A experienced complete disappearance of established tumors . Animals with pancreatic tumors implanted orthotopically exhibited prolonged survival that was significantly greater by comparison with untreated animals . Thus, IL-4 receptor-targeted cytotoxin represents a potent agent that may provide an effective therapy for pancreatic cancer.

Tsitologiia, 2002, 44(3), 296 - 304
{Activity of toxins produced by Pseudomonas syringae pv . syringae in model and cell membranes}; Gur'nev FA et al.; We studied effects of toxins produced by a bacterium Pseudomonas syringae pv . syringae on the conductance of bilayer lipid membranes (BLM) . The used toxins were as follows: syringopeptin 22A (SP22A), syringomycin E (SPE), syringostatin A (SSA), syringotoxin B (STB), and methylated syringomycin E (CH3-SRE) . All toxins demonstrated channel-forming activity . The threshold sequence for toxin activity was SP22A > SRE approximately equal to SSA > STB > CH3-SRE, and this sequence was independent of lipid membrane composition, and NaCl concentration (pH 6) in the membrane bathing solution (in the range of 0.1-1.0 M) . This sequence correlated with relative bioactivities of toxins . In addition, SRE demonstrated a more potent antifungal activity than CH3-SRE . These findings suggest that ion channel formation may underlie the bioactivities of the above toxins . The properties of single ion channels formed by the toxins in BLMs were found to be similar, which points to the similarity in the channel structures . In negatively charged membranes, bathed with diluted electrolyte solutions (0.1 M NaCl), the channels were seen to open with positive transmembrane potentials (V) (from the side of toxin addition), and close with negative potentials . In uncharged membranes the opposite response to a voltage sign was observed . Increasing the NaCl concentration up to 1 M unified the voltage sensitivity of channels in charged and uncharged membranes: channels opened with negative V, and closed with positive V . With all systems, the voltage current curves of single channels were similarly superlinear in the applied voltage and asymmetric in its sign . It was found that the single channel conductance of STB and SSA was higher than that of other toxin channels . All the toxins formed at least two types of ion channels that were multiple by a factor of either 6 or 4 in their conductance . The results are discussed in terms of the structural features of toxin molecules.

Folia Microbiol (Praha), 2002, 47(3), 230 - 4
Regulation of the activity of intracellular alanylaminopeptidase synthesized by Pseudomonas sp; Jankiewicz U et al.; Activity of purified alanylaminopeptidase of Pseudomonas sp . measured in the presence of the alanine derivative of 2-naphthoic acid (NA-Ala) is inhibited by 1,10-phenanthroline, EDTA, bestatin and amastatin; this finding supports the conclusion that this enzyme is a metallo-aminopeptidase . A decrease of its activity in the presence of iodoacetamide and its activation by thiols points to the significant role of -SH groups in the regulation of its activity . Co2+, Ca2+ and Mg2+ ions increased the enzyme activity while Zn2+, Cd2+ and Pb2+ markedly inhibited the enzyme even at low concentrations . A high thermal stability of alanylaminopeptidase depended on the presence of 1 mmol/L Co2+ and of 1 mmol/L L-cysteine in the incubation mixture.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1097 - 104
Identification, cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp . strain ON-4a; Ohmachi T et al.; The newly isolated strain Pseudomonas sp . ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine . A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli . Transformants expressing cysteine-forming activity were selected by growth of an E . coli mutant defective in the cysB gene . A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed . Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively . E . coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa . From the analyses of crude extracts of E . coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.

Appl Environ Microbiol, 2002 Jul, 68(7), 3308 - 14
Survival of GacS/GacA mutants of the biological control bacterium Pseudomonas aureofaciens 30-84 in the wheat rhizosphere; Chancey ST et al.; GacS/GacA comprises a two-component regulatory system that controls the expression of secondary metabolites required for the control of plant diseases in many pseudomonads . High mutation frequencies of gacS and gacA have been observed in liquid culture . We examined whether gacS/gacA mutants could competitively displace the wild-type populations on roots and thus pose a threat to the efficacy of biological control . The survival of a gac mutant alone and in competition with the wild type on roots was examined in the biological control strain Pseudomonas aureofaciens 30-84 . In this bacterium, GacS/GacA controls the expression of phenazine antibiotics that are inhibitory to plant pathogenic fungi and enhance the competitive survival of the bacterium . Wheat seedlings were inoculated with strain 30-84, and bacteria were recovered from roots after 21 days in sterile or nonsterile soil to check for the presence of gacS or gacA mutants . Although no mutants were detected in the inoculum, gacS/gacA mutants were recovered from 29 out of 31 roots and comprised up to 36% of the total bacterial populations . Southern hybridization analysis of the recovered gacA mutants did not indicate a conserved mutational mechanism . Replacement series analysis on roots utilizing strain 30-84 and a gacA mutant (30-84.gacA) or a gacS mutant (30-84.A2) demonstrated that although the mutant population partially displaced the wild type in sterile soil, it did not do so in natural soil . In fact, in natural soil final rhizosphere populations of wild-type strain 30-84 starting from mixtures were at least 1.5 times larger than would be predicted from their inoculation ratio and generally were greater than or equal to the population of wild type alone despite lower inoculation rates . These results indicate that although gacS/gacA mutants survive in natural rhizosphere populations, they do not displace wild-type populations . Better survival of wild-type populations in mixtures with mutants suggests that mutants arising de novo or introduced within the inoculum may be beneficial for the survival of wild-type populations in the rhizosphere.

FEMS Microbiol Lett, 2002 Jun 18, 212(1), 71 - 5
Polyhydroxyalkanoate biosynthesis in Pseudomonas pseudoalcaligenes YS1; Hang X et al.; Pseudomonas pseudoalcaligenes strain YS1 isolated from oil contaminated soil was able to produce polyhydroxybutyrate blended with medium-chain-length polyhydroxyalkanoates (mcl PHA) . PHA synthesis genes were cloned from this strain . A fadB (gene for fatty acid degradation) deleted mutant Escherichia coli KM32B (FADB::Tet) was constructed to express the cloned PHA synthesis gene phaC1(Pp) or phaC2(Pp) . The fadB deleted mutant KM32B harboring phaC1(Pp) or phaC2(Pp) showed mcl PHA accumulation while the intact E . coli KM32 did not . The results demonstrated that P . pseudoalcaligenes YS1 possessed at least two PHA synthesis pathways; one of them was responsible for production of mcl PHA.

J Org Chem, 2002 Jun 28, 67(13), 4513 - 9
Building blocks for the solution phase synthesis of oligonucleotides: regioselective hydrolysis of 3',5'-Di-O-levulinylnucleosides using an enzymatic approach; Garcia J et al.; A short and convenient synthesis of 3'- and 5'-O-levulinyl-2'-deoxynucleosides has been developed from the corresponding 3',5'-di-O-levulinyl derivatives by regioselective enzymatic hydrolysis, avoiding several tedious chemical protection/deprotection steps . Thus, Candida antartica lipase B (CAL-B) was found to selectively hydrolyze the 5'-levulinate esters, furnishing 3'-O-levulinyl-2'-deoxynucleosides 3 in >80% isolated yields . On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) and Candida antarctica lipase A (CAL-A) exhibit the opposite selectivity toward the hydrolysis at the 3'-position, affording 5'-O-levulinyl derivatives 4 in >70% yields . A similar hydrolysis procedure was successfully extended to the synthesis of 3'- and 5'-O-levulinyl-protected 2'-O-alkylribonucleosides 7 and 8 . This work demonstrates for the first time application of commercial CAL-B and PSL-C toward regioselective hydrolysis of levulinyl esters with excellent selectivity and yields . It is noteworthy that protected cytidine and adenosine base derivatives were not adequate substrates for the enzymatic hydrolysis with CAL-B, whereas PSL-C was able to accommodate protected bases during selective hydrolysis . In addition, we report an improved synthesis of dilevulinyl esters using a polymer-bound carbodiimide as a replacement for dicyclohexylcarbodiimide (DCC), thus considerably simplifying the workup for esterification reactions.

J Ind Microbiol Biotechnol, 2002 Mar, 28(3), 147 - 53
The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans; Ashby RD et al.; Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV) . Acetone fractionation verified the presence of PHB/mcl-PHA mixtures . The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms . In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) . The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179 degrees C, enthalpies of fusion (AHf) of 20 cal/g and glasstransition temperatures (Tg) of 3-4 degrees C . This was due to the large PHB content in the polymer mixtures . On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature . In both cases, the Tm values were 161 degrees C, deltaHf values were 7 cal/g and Tg values were - 3 degrees C.

Rev Med Liege, 2002 Apr, 57(4), 233 - 5
{Green nail syndrome or chloronychia}; Maes M et al.; "Green nails" or chloronychia is an infection mostly caused by Pseudomonas ueruginosa but also by other bacterial or fungal contamination . The clinical appearance consists in a typical triad: green discoloration of the nail plate associated with proximal chronic paronychia and disto-lateral onycholysis . Exposition to moist environment, microtraumatisms, oaychotillomania and associated nail diseases such as psoriasis may promote infection by Pseudomonas . Treatment consists in cutting of the detached nail plate, brushing the nail bed with a 2% sodium hypochlorite solution twice daily and eviction of the repeated immersions by wearing cotton and latex gloves.

Genetics, 2002 Jun, 161(2), 803 - 11
Role of salicylic acid and NIM1/NPR1 in race-specific resistance in arabidopsis; Rairdan GJ et al.; Salicylic acid (SA) and the NIM1/NPR1 protein have both been demonstrated to be required for systemic acquired resistance (SAR) and implicated in expression of race-specific resistance . In this work, we analyzed the role that each of these molecules play in the resistance response triggered by members of two subclasses of resistance (R) genes, members of which recognize unrelated pathogens . We tested the ability of TIR and coiled-coil-class (also known as leucine-zipper-class) R genes to confer resistance to Pseudomonas syringae pv . tomato or Peronospora parasitica in SA-depleted (NahG) and nim1/npr1 plants . We found that all of the P . syringae pv . tomato-specific R genes tested were dependent upon SA accumulation, while none showed strong dependence upon NIM1/NPR1 activity . A similar SA dependence was observed for the P . parasitica TIR and CC-class R genes RPP5 and RPP8, respectively . However, the P . parasitica-specific R genes differed in their requirement for NIM1/NPR1, with just RPP5 depending upon NIM1/NPR1 activity for effectiveness . These data are consistent with the hypothesis that at least in Arabidopsis, SA accumulation is necessary for the majority of R-gene-triggered resistance, while the role of NIM1/NPR in race-specific resistance is limited to resistance to P . parasitica mediated by TIR-class R genes.

J Biol Chem, 2002 Aug 30, 277(35), 31474 - 83 Epub 2002 Jun 17.
Molecular and spectroscopic analysis of the cytochrome cbb(3) oxidase from Pseudomonas stutzeri; Pitcher RS et al.; Cytochrome cbb(3) oxidase, a member of the heme-copper oxidase superfamily, is characterized by its high affinity for oxygen while retaining the ability to pump protons . These attributes are central to its proposed role in the microaerobic metabolism of proteobacteria . We have completed the first detailed spectroscopic characterization of a cytochrome cbb(3) oxidase, the enzyme purified from Pseudomonas stutzeri . A combination of UV-visible and magnetic CD spectroscopies clearly identified four low-spin hemes and the high-spin heme of the active site . This heme complement is in good agreement with our analysis of the primary sequence of the ccoNOPQ operon and biochemical analysis of the complex . Near-IR magnetic CD spectroscopy revealed the unexpected presence of a low-spin bishistidine-coordinated c-type heme in the complex . This was shown to be one of two c-type hemes in the CcoP subunit by separately expressing the subunit in Escherichia coli . Separate expression of CcoP also allowed us to unambiguously assign each of the signals associated with low-spin ferric hemes present in the X-band EPR spectrum of the oxidized enzyme . This work both underpins future mechanistic studies on this distinctive class of bacterial oxidases and raises questions concerning the role of CcoP in electron delivery to the catalytic subunit.

Plant Physiol, 2002 Jun, 129(2), 706 - 16
Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor; Chen C et al.; AtWRKY18 is a pathogen- and salicylic acid-induced Arabidopsis transcription factor containing the plant-specific WRKY zinc finger DNA-binding motif . In the present study, we have transformed Arabidopsis plants with AtWRKY18 under control of the cauliflower mosaic virus 35S promoter . Surprisingly, transgenic plants expressing high levels of AtWRKY18 were stunted in growth . When expressed at moderate levels, AtWRKY18 potentiated developmentally regulated defense responses in transgenic plants without causing substantial negative effects on plant growth . As they grew from seedling to mature stages, transgenic AtWRKY18 plant showed marked increase in the expression of pathogenesis-related genes and resistance to the bacterial pathogen Pseudomonas syringae, whereas wild-type plants exhibited little enhancement in these defense responses . Potentiation of developmentally regulated defense responses by AtWRKY18 was not associated with enhanced biosynthesis of salicylic acid but required the disease resistance regulatory protein NPR1/NIM1 . Thus, AtWRKY18 can positively modulate defense-related gene expression and disease resistance . To study the regulated expression of AtWRKY18, we have identified a cluster of WRKY binding sites in the promoter of the gene and demonstrated that they acted as negative regulatory elements for the inducible expression of AtWRKY18 . These negative cis-acting elements may prevent overexpression of AtWRKY18 during the activation of plant defense responses that could be detrimental to plant growth as inferred from the transgenic plants ectopically expressing the transgene.

Bioorg Med Chem Lett, 2002 Jul 8, 12(13), 1735 - 8
Efficient chemoenzymatic synthesis of (S)- and (R)-5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide: key intermediate for Src-SH2 inhibitor; Kamal A et al.; A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described . Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot . Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.

Mol Microbiol, 2002 Jun, 44(6), 1469 - 81
The ShcA protein is a molecular chaperone that assists in the secretion of the HopPsyA effector from the type III (Hrp) protein secretion system of Pseudomonas syringae; van Dijk K et al.; Pseudomonas syringae uses a type III protein secretion system encoded by the Hrp pathogenicity island (Pai) to translocate effector proteins into plant cells . One of these effector proteins is HopPsyA . A small open reading frame (ORF), named shcA, precedes the hopPsyA gene in the Hrp Pai of P . s . syringae 61 . The predicted amino acid sequence of shcA shares general characteristics with chaperones used in type III protein secretion systems of animal pathogens . A functionally non-polar deletion of shcA in P . s . syringae 61 resulted in the loss of detectable HopPsyA in supernatant fractions, consistent with ShcA acting as a chaperone for HopPsyA . Cosmid pHIR11 carries a functional set of type III genes from P . s . syringae 61 and confers upon saprophytes the ability to secrete HopPsyA in culture and to elicit a HopPsyA-dependent hypersensitive response (HR) on tobacco . P . fluorescens carrying a pHIR11 derivative lacking shcA failed to secrete HopPsyA in culture, but maintained the ability to secrete another type III-secreted protein, HrpZ . This pHIR11 derivative was also greatly reduced in its ability to elicit an HR, indicating that the ability to translocate HopPsyA into plant cells was compromised . Using affinity chromatography, we showed that ShcA binds directly to HopPsyA and that the ShcA binding site must reside within the first 166 amino acids of HopPsyA . Thus, ShcA represents the first demonstrated chaperone used in a type III secretion system of a bacterial plant pathogen . We searched known P . syringae type III-related genes for neighbouring ORFs that shared the general characteristics of type III chaperones and identified five additional candidate type III chaperones . Therefore, it is likely that chaperones are as prevalent in bacterial plant pathogen type III systems as they are in their animal pathogenic counterparts.

Cell, 2002 May 31, 109(5), 589 - 98
Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity; Kim YJ et al.; The Pto serine/threonine kinase of tomato confers resistance to speck disease by recognizing strains of Pseudomonas syringae that express the protein AvrPto . Pto and AvrPto physically interact, and this interaction is required for activation of host resistance . We identified a second Pseudomonas protein, AvrPtoB, that interacts specifically with Pto and is widely distributed among plant pathogens . AvrPtoB is delivered into the plant cell by the bacterial type III secretion system, and it elicits Pto-specific defenses . AvrPtoB has little overall sequence similarity with AvrPto . However, AvrPto amino acids, which are required for interaction with Pto, are present in AvrPtoB and required for its interaction with Pto . Thus, two distinct bacterial effectors activate plant immunity by interacting with the same host protein kinase through a similar structural mechanism.

Dev Cell, 2002 Jun, 2(6), 807 - 17
An evolutionarily conserved mediator of plant disease resistance gene function is required for normal Arabidopsis development; Holt BF 3rd et al.; Plants recognize many pathogens through the action of a diverse family of proteins called disease resistance (R) genes . The Arabidopsis R gene RPM1 encodes resistance to specific Pseudomonas syringae strains . We describe an RPM1-interacting protein that is an ortholog of TIP49a, previously shown to interact with the TATA binding protein (TBP) complex and to modulate c-myc- and beta-catenin-mediated signaling in animals . Reduction of Arabidopsis TIP49a (AtTIP49a) mRNA levels results in measurable increases of two R-dependent responses without constitutively activating defense responses, suggesting that AtTIP49a can act as a negative regulator of at least some R functions . Further, AtTIP49a is essential for both sporophyte and female gametophyte viability . Thus, regulators of R function overlap with essential modulators of plant development.

Cryobiology, 2002 Feb, 44(1), 14 - 23
Inhibition of bacterial ice nucleation by polyglycerol polymers; Wowk B et al.; The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae . PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P . syringae 31A in water . Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 microL volume . The mean freezing temperature was lowered from -6.8 degrees C (control) to -8.0,-9.4,-12.5, and -13.4 degrees C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2 degrees C) . PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven . The activity of PGL therefore seems to be specific against bacterial ice nucleating protein . PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin . However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency . The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification . (c) 2002 Elsevier Science (USA).

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 9004 - 9 Epub 2002 Jun 11.
Local circuit neurons in the striatum regulate neural and behavioral responses to dopaminergic stimulation; Saka E et al.; Interneurons are critical for shaping neuronal circuit activity in many parts of the central nervous system . To study interneuron function in the basal ganglia, we tested and characterized an NK-1 receptor-based method for targeted ablation of specific classes of interneuron in the striatum . Our findings demonstrate that the neurotoxin SP-PE35, a substance P-Pseudomonas exotoxin conjugate, selectively targets striatal cholinergic and nitric oxide synthase/somatostatinergic interneurons when injected locally into the striatum . The effects of this selective cell targeting encompassed alterations in both behavioral and neural responses to dopaminergic stimulation, including altered patterns of early-gene response in striosomes and matrix . We conclude that NK-1-bearing local circuit neurons of the striatum regulate the differential responses of striatal projection neurons to dopamine-mediated signaling.

Clin Cancer Res, 2002 Jun, 8(6), 1948 - 56
Heterogeneity in interleukin-13 receptor expression and subunit structure in squamous cell carcinoma of head and neck: differential sensitivity to chimeric fusion proteins comprised of interleukin-13 and a mutated form of Pseudomonas exotoxin; Joshi BH et al.; Squamous cell carcinoma of the head and neck (SCCHN) is characterized by a high proliferation index and marked propensity for local invasion resulting in poor prognosis for these patients . To develop tumor-targeted novel therapeutic agents, here we demonstrate that SCCHN cell lines express receptors for an immune regulatory cytokine, interleukin (IL) 13 . By reverse transcription-PCR (RT-PCR), we found that 16 SCCHN cell lines express equally strong RT-PCR positive bands for mRNA of IL-13Ralpha1 and IL-4Ralpha chains . However, only three cell lines, HN12, YCUM911, and KCCT873, expressed a strong band for transcripts for IL-13Ralpha2 chain and five cell lines, YCUL891, KCCTC871, KCCL871, KCCTCM901, and RPMI 2650 expressed faint bands . Transcripts for IL-2Rgamma(c) chain were absent in all of the cell lines tested . Indirect immunofluorescence analysis for four different receptor chains confirmed RT-PCR results and showed pronounced expression of IL-13Ralpha2 protein in three high IL-13R expressing cell lines . All of the cell lines were equally positive for IL-13Ralpha1 and IL-4Ralpha chains . Receptor-binding studies demonstrated that IL-13Ralpha2-positive cell lines expressed a high density of IL-13 receptors . Using two chimeric proteins composed of IL-13 and mutated forms of Pseudomonas exotoxin (IL-13-PE38 or IL-13-PE38QQR), we found that these two fusion toxins were highly and equally cytotoxic to IL-13Ralpha2-positive SCCHN, whereas IL-13Ralpha2-negative cell lines showed low or no sensitivity to IL-13 toxins . To additionally substantiate the critical role of the IL-13Ralpha2 chain in IL-13R-mediated cytotoxicity, two head and neck tumor cell lines (YCUMS861 and KB), devoid of the transcripts of this chain, were transfected with IL-13Ralpha2 cDNA and then tested for cytotoxicity . Transient transfection of the IL-13Ralpha2 chain highly sensitized these cells to IL-13 toxin as compared with mock-transfected control cells . Thus, our results indicate that IL-13Ralpha2 is present in 50% SCCHN tumor cell lines; of these, 19% are high expresser for this chain and respond to IL-13 cytotoxin . Thus, IL-13 cytotoxin may be a useful agent for high IL-13R-expressing SCCHN.

Physiol Plant, 2001 Oct, 113(2), 267 - 274
Characterization of an ethylene-induced esterase gene isolated from Citrus sinensis by competitive hybridization; Zhong GY et al.; A simple new method, competitive hybridization, for identification of differentially regulated genes was used to isolate novel genes induced by ethylene in citrus (Citrus sinensis {L.} Osbeck cv . Shamouti) leaves . One of the isolated genes, an ethylene-induced esterase gene (EIE), was further characterized . The deduced protein sequence of this gene shows a similarity to those of several plant alpha/beta hydrolase gene family members, which are known to be involved in secondary metabolism . Northern blot analysis demonstrated that EIE mRNA was induced by ethylene within 4 h and accumulated to a very high level 24 h after the initiation of ethylene treatment . Induction of EIE by ethylene could be counteracted by 1-methylcyclopropene, a potent ethylene perception inhibitor, indicating that the expression of EIE is ethylene-dependent . The bacterially expressed protein of EIE was recognized by antiserum against Pir7b, a naphthol AS esterase induced in rice by the non-host pathogen, Pseudomonas syringae pv . syringae . The EIE protein was identified in ethylene-treated leaves using anti-Pir7b antibodies . An alpha-naphthyl acetate esterase accumulated concomitantly with the increase in EIE protein in ethylene-treated citrus leaves . An enzyme activity assay followed by western analysis confirmed that the esterase was EIE.

Plant J, 2002 Jan, 29(1), 23 - 32
Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi; Berrocal-Lobo M et al.; Infection of a plant by a pathogen induces a variety of defense responses that imply the action of several signaling molecules, including salicylic acid (SA), jasmonic acid (JA) and ethylene (E) . Here we describe the role of ETHYLENE-RESPONSE-FACTOR1 (ERF1) as a regulator of ethylene responses after pathogen attack in Arabidopsis . The ERF1 transcript is induced on infection by Botrytis cinerea, and overexpression of ERF1 in Arabidopsis is sufficient to confer resistance to necrotrophic fungi such as B . cinerea and Plectosphaerella cucumerina . A positive co-operation between E and SA pathways was observed in the plant response to P . cucumerina . Infection by Pseudomonas syringae tomato DC3000, however, does not affect ERF1 expression, and activation of ethylene responses by ERF1 overexpression in Arabidopsis plants reduces tolerance against this pathogen, suggesting negative crosstalk between E and SA signaling pathways, and demonstrating that positive and negative interactions between both pathways can be established depending on the type of pathogen.

Biochemistry (Mosc), 2002 May, 67(5), 558 - 65
Structure of the O-polysaccharide of Pseudomonas syringae pv . delphinii NCPPB 1879(T) having side chains of 3-acetamido-3,6-dideoxy-D-galactose residues; Zdorovenko EL et al.; The O-polysaccharide (OPS) was obtained from the lipopolysaccharide of Pseudomonas syringae pv . delphinii NCPPB 1879(T) and studied by sugar and methylation analyses, Smith degradation, and (1)H- and (13)C-NMR spectroscopy . The OPS was found to contain residues of L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), and the following structure of the major (n = 2) and minor (n = 3) heptasaccharide repeating units of the OPS was established: {carbohydrate structure: see text} . The OPS is distinguished by the presence of oligosaccharide side chains consisting of three D-Fuc3NAc residues that are connected to each other by the (alpha 1-->2)-linkage . The OPS is characterized by a structural heterogeneity due to a different position of substitution of one of the four L-rhamnose residues in the main chain of the repeating unit as well as to the presence of oligosaccharide units with an incomplete side chain.

Mol Plant Microbe Interact, 2002 Jun, 15(6), 608 - 16
NHL25 and NHL3, two NDR1/HIN1-1ike genes in Arabidopsis thaliana with potential role(s) in plant defense; Varet A et al.; The Arabidopsis genome contains 28 genes with sequence homology to the Arabidopsis NDR1 gene and the tobacco HIN1 gene . Expression analysis of eight of these genes identified two (NHL25 and NHL3 for NDR1/HIN1-like) that show pathogen-dependent mRNA accumulation . Transcripts did not accumulate during infection with virulent Pseudomonas syringae pv . tomato DC3000 but did accumulate specifically when the bacteria carried any of the four avirulence genes avrRpm1, avrRpt2, avrB, or avrRps4 . Furthermore, expression of avrRpt2 in plants containing the corresponding resistance gene, RPS2, was sufficient to induce transcript accumulation . However, during infection with an avirulent oomycete, Peronospora parasitica isolate Cala-2, only NHL25 expression was reproducibly induced . Salicylic acid (SA) treatment can induce expression of NHL25 and NHL3 . Studies performed on nahG plants showed that, during interaction with avirulent bacteria, only the expression of NHL25 but not that of NHL3 was affected . This suggests involvement of separate SA-dependent and SA-independent pathways, respectively, in the transcriptional activation of these genes . Bacteria-induced gene expression was not abolished in ethylene- (etrl-3 and ein2-1) and jasmonate- (coil-1) insensitive mutants or in mutants impaired in disease resistance (ndr1-1 and pad4-1) . Interestingly, NHL3 transcripts accumulated after infiltration with the avirulent hrcC mutant of Pseudomonas syringae pv . tomato DC3000 and nonhost bacteria but not with the virulent Pseudomonas syringae pv . tomato DC3000, suggesting that virulent bacteria may suppress NHL3 expression during pathogenesis . Hence, the expression patterns and sequence homology to NDR1 and HIN1 suggest one or more potential roles for these genes in plant resistance.

Mol Plant Microbe Interact, 2002 Jun, 15(6), 557 - 66
Characterization of a novel, defense-related Arabidopsis mutant, cir1, isolated by luciferase imaging; Murray SL et al.; In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants . The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes . Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv . tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2 . Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways . While resistance against both P . syringae pv . tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background . Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways . While jar1 and ein2 reduced resistance against P . syringae pv . tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2 . Thus, JA and ET sensitivity are required for cir1-mediated resistance against P . syringae pv . tomato DC3000 but not Peronospora parasitica Noco2 . Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.

Folia Microbiol (Praha), 2002, 47(2), 137 - 43
Plant disease suppression and growth promotion by a fluorescent Pseudomonas strain; Boruah HP et al.; An antibiotic- and siderophore-producing Pseudomonas strain isolated from virgin soils (with forest trees) displayed in vitro antibiosis against many plant pathogenic fungi . The presence of iron had no effect on this in vitro antibiosis . Seed bacterization improved germination, shoot height, root length, fresh and dry mass, enhanced yield and chlorophyll content of leaves in the five test crop plants under field conditions . Seed bacterization also reduced the number of infected brinjal plants grown in soil infested with Rhizoctonia solani . The strain produced a yellowish green siderophore in the standard succinate medium and both siderophore and a yellow viscous antibiotic compound in King's B medium . The results confirmed that the plant growth promotion was due to siderophore production whereas the disease suppression was due to the antibiotic substance.

J Bacteriol, 2002 Jul, 184(13), 3682 - 8
Alteration of regiospecificity in biphenyl dioxygenase by active-site engineering; Suenaga H et al.; Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation step during the metabolism of biphenyl . The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds, including polychlorinated biphenyls (PCBs) . Based on crystallographic analyses of naphthalene dioxygenase (B . Kauppi, K . Lee, E . Carredano, R . E . Parales, D . T . Gibson, H . Eklund, and S . Ramaswamy, Structure 6:571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of Pseudomonas pseudoalcaligenes KF707 . Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in Escherichia coli . The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox . In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2',5'-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2'-trichlorobiphenyl and 2,5,4'-trichlorobiphenyl . Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2',3'-dihydroxy biphenyl with concomitant dechlorination.

Microbiology, 2002 Jun, 148(Pt 6), 1699 - 708
The membrane-bound respiratory chain of Pseudomonas pseudoalcaligenes KF707 cells grown in the presence or absence of potassium tellurite; Di Tomaso G et al.; The respiratory chain of Pseudomonas pseudoalcaligenes KF707 in membranes isolated from cells grown in the presence or absence of the toxic oxyanion tellurite (TeO3(2-)) was examined . Aerobic growth in the absence of tellurite shows an NADH-dependent respiration which is 80% catalysed by the cytochrome (cyt) bc1-containing pathway leading to two terminal membrane-bound cyt c oxidases inhibited by different concentrations of KCN (IC50 0.2 and 1 microM) . A third oxidase, catalysing the remaining 20% of the cyanide-resistant respiration and fully inhibited by 2-3 mM KCN, is also present; this latter pathway accounts for 60-70% of the total NADH-dependent respiration in membranes from cells grown in LB medium supplemented with potassium tellurite (35 microg x ml(-1)) . Two high-potential b-type haems (E(m,7) +395 and 318 mV) are redox centres of a membrane-bound cyt c oxidase (possibly of the cbb3 type) which shows a 50% decrease of its activity in parallel with a similar decrease of the c-type haem content (mostly soluble cyt c) in membranes from tellurite-grown cells; the latter type of cells specifically contain a cyt b type at +203 mV (pH 9.0) which is likely to be involved in cyanide-resistant respiration . Comparison of the growth curve of KF707 cells in parallel with tellurite uptake showed that intracellular accumulation of tellurium (Te(0)) crystallites starts from the mid-exponential growth phase, whereas tellurite-induced changes of the respiratory chain are already evident during the early stages of growth . These data were interpreted as showing that reduction of tellurite to tellurium and tellurite-dependent modifications of the respiratory chain are unrelated processes in P . pseudoalcaligenes KF707.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 739 - 47
Phylogenetic study of the genus Oceanospirillum based on 16S rRNA and gyrB genes: emended description of the genus Oceanospirillum, description of Pseudospirillum gen . nov., Oceanobacter gen . nov . and Terasakiella gen . nov . and transfer of Oceanospirillum jannaschii and Pseudomonas stanieri to Marinobacterium as Marinobacterium jannaschii comb . nov . and Marinobacterium stanieri comb . no; Satomi M et al.; The phylogenetic relationships of Oceanospirillum strains were analysed by using the nucleotide sequences of 16S rRNA and gyrB genes . Results from sequence analysis demonstrated that the Oceanospirillum core group consisted of four species, Oceanospirillum linum, Oceanospirillum maris, Oceanospirillum beijerinckii and Oceanospirillum multiglobuliferum, with enough distance to separate them as different species . However, four other Oceanospirillum species occupied taxonomic positions separate from the Oceanospirillum core group: Oceanospirillum jannaschii, Oceanospirillum japonicum and Oceanospirillum kriegii in the gamma-Proteobacteria and Oceanospirillum pusillum in the alpha-Proteobacteria . Oceanospirillum jannaschii clustered with Marinobacterium georgiense, Pseudomonas iners and Pseudomonas stanieri on the basis of phylogenetic analysis of 16S rRNA and gyrB genes . The other three species did not cluster with known genera . Also, the sequence similarity values of the gyrB genes between the three subspecies of Oceanospirillum maris and those between the two subspecies of Oceanospirillum beijerinckii were above 99% . The close relationships between the subspecies of Oceanospirillum maris and of Oceanospirillum beijerinckii were further supported by similar physiological properties and high DNA-DNA hybridization values, suggesting that these subspecies should not be regarded as valid . From these results, Oceanospirillum sensu stricto should be defined to consist of Oceanospirillum linum, Oceanospirillum maris, Oceanospirillum beijerinckii and Oceanospirillum multiglobuliferum . We propose to create the following new genera: Pseudospirillum gen . nov . for Oceanospirillum japonicum as Pseudospirillum japonicum comb . nov.; Oceanobacter gen . nov . for Oceanospirillum kriegii as Oceanobacter kriegii comb . nov.; and Terasakiella gen . nov . for Oceanospirillum pusillum as Terasakiella pusilla comb . nov . The transfer is proposed of Oceanospirillum jannaschii and Pseudomonas stanieri to Marinobacterium as Marinobacterium jannaschii comb . nov . and Marinobacterium stanieri comb . nov . Furthermore, Pseudomonas iners should be reclassified as a strain of Marinobacterium georgiense . Finally, the subspecies of Oceanospirillum maris (O . maris subsp . maris, O . maris subsp . hiroshimense and O . maris subsp . williamsae) and Oceanospirillum beijerinckii (O . beijerinckii subsp . beijerinckii and O . beijerinckii subsp . pelagicum) should be combined as Oceanospirillum maris and Oceanospirillum beijerinckii, respectively.

FEMS Microbiol Lett, 2002 May 21, 211(1), 43 - 9
Sphingomonas sp . strain KA1, carrying a carbazole dioxygenase gene homologue, degrades chlorinated dibenzo-p-dioxins in soil; Habe H et al.; Hybridization analysis showed that a newly isolated carbazole (CAR)-degrading bacterium Sphingomonas sp . strain KA1 did not possess the gene encoding the terminal oxygenase component (carAa) of CAR 1,9a-dioxygenase at high homology (more than 90% identity) to that of another CAR-degrader, Pseudomonas resinovorans strain CA10 . However, PCR experiments using the primers for amplifying the internal fragment of the carAa gene (810 bp for strain CA10) showed that a PCR product of unexpected size (1100 bp) was amplified . Sequence analysis revealed that this DNA region contained the portion of two possible ORFs, which showed moderate homology to CarAa and CarBa from strain CA10 (61% and 40% identities at the amino acid level, respectively) . Inoculation of strain KA1 into dioxin-contaminated model soil resulted in 96% and 70% degradation of 2-mono- and 2,3-dichlorinated dibenzo-p-dioxin, respectively, after 7-day incubation.

Arch Biochem Biophys, 2002 Jun 1, 402(1), 24 - 30
Analysis of the roles of amino acid residues in the flavoprotein tryptophan 2-monooxygenase modified by 2-oxo-3-pentynoate: characterization of His338, Cys339, and Cys511 mutant enzymes; Sobrado P et al.; The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide . His338, Cys339, and Cys511 of the Pseudomonas savastanoi enzyme were previously identified as possible active-site residues by modification with 2-oxo-3-pentynoate ({G . Gadda, L.J . Dangott, W.H . Johnson Jr., C.P . Whitman, P.F . Fitzpatrick, Biochemistry 38 (1999) 5822-5828}) . The H338N, C339A, and C511S enzymes have been characterized to determine the roles of these residues in catalysis . The steady-state kinetic parameters with both tryptophan and methionine decrease only slightly in the case of the H338N and C339A enzymes; the decrease in activity is greater for the C511S enzyme . Only in the case of the C511S enzyme do deuterium kinetic isotope effects on kinetic parameters indicate a significant change in catalytic rates . The structural bases for the effects of the mutations can be interpreted by identification of L-amino acid oxidase and tryptophan monooxygenase as homologous proteins.

FEMS Microbiol Lett, 2002 May 7, 210(2), 239 - 44
Purification and characterization of an erythromycin esterase from an erythromycin-resistant Pseudomonas sp; Kim YH et al.; An erythromycin esterase (molecular mass 51200 Da) was purified from Pseudomonas sp . GD100, which was isolated from a salmon hatchery sediment sample from Washington State . The pI of the protein was 4.5-4.8 . The enzyme was inhibited by 1 mM mercuric acid, and had the substrate specificity for structurally related 14-membered macrolides, which decreased in the order of oleandomycin, erythromycin A and erythromycin A enol ether . The activity for erythromycin A varied with temperature, but the effect of pH was minimal at pH 6.0-9.0 . The half-life of the enzyme was estimated to be 8.9 h at 35 degrees C and 0.23 h at 55 degrees C, and the activation energy of the catalytic reaction of erythromycin A was estimated at 16.2 kJ mol(-1).

Appl Environ Microbiol, 2002 Jun, 68(6), 2637 - 43
Protection of tomato seedlings against infection by Pseudomonas syringae pv . tomato by using the plant growth-promoting bacterium Azospirillum brasilense; Bashan Y et al.; Pseudomonas syringae pv . tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium Azospirillum brasilense were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively . The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored . When inoculated onto separate plants, the A . brasilense population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of P . syringae pv . tomato (10(7) versus 10(5) CFU/g {dry weight} of root) . Under mist chamber conditions, the leaf population of P . syringae pv . tomato was 1 order of magnitude greater than that of A . brasilense (10(7) versus 10(6) CFU/g {dry weight} of leaf) . Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the A . brasilense population, the prevention of bacterial speck disease development, and improved plant growth . Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the P . syringae pv . tomato population and significantly decreased disease severity . Challenge with P . syringae pv . tomato after A . brasilense was established in the leaves further reduced both the population of P . syringae pv . tomato and disease severity and significantly enhanced plant development . Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10(5) to 10(6) CFU/g {dry weight} of root) . However, P . syringae pv . tomato did not survive in the rhizosphere in the presence of A . brasilense . Foliar inoculation of A . brasilense after P . syringae pv . tomato was established on the leaves did not alleviate bacterial speck disease, and A . brasilense did not survive well in the phyllosphere under these conditions, even in a mist chamber . Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of A . brasilense (>10(8) CFU/g {dry weight} of leaf), decreased the population of P . syringae pv . tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants . Based on our results, we propose that A . brasilense be used in prevention programs to combat the foliar bacterial speck disease caused by P . syringae pv . tomato.

Microb Ecol, 2002 Apr, 43(3), 298 - 306 Epub 2002 Mar 13.
Contribution of cytophaga-like bacteria to the potential of turnover of carbon, nitrogen, and phosphorus by bacteria in the rhizosphere of barley (Hordeum vulgare L.); Johansen JE et al.; The functional potential of bacteria isolated from the rhizosphere of barley (Hordeum vulgare L.) in May, July, and August and cultivated on nutrient-rich substrate (1/10 TSBA) and nutrient-poor substrate (cold soil extract agar) was determined . There was no significant difference in numbers of CFU when counted on nutrient rich or poor substrate . Bacterial numbers increased approximately 3-fold in the rhizosphere soil from May to August but was unchanged in bulk soil over the same period . A total of 4474 randomly isolated bacteria were screened for enzymatic activities involved in carbon turnover (amylase, cellulase, mannanase, xylanase, and chitinase), nitrogen turnover (protease, nitrate and nitrite reductase), and phosphate turnover (phosphatase) . In the rhizosphere soil, bacteria carrying C and P turnover enzymes were not stimulated by the growing plant whereas protease and nitrate and nitrite reductase were stimulated by the growing plant . No changes were observed in the bulk soil . Two taxonomic groups were followed: Cytophaga-like bacteria (CLB) and fluorescent pseudomonads, the latter being abundant in the rhizosphere and important contributors to the cycling of organic matter in soil . Unexpectedly in the spring samples, CLB were around 25% of all bacteria isolated, whereas fluorescent pseudomonads made up less than 10% . The relative proportion of these bacterial groups then decreased during the plant growth season but at all times showing a clear rhizosphere effect . Furthermore, up to 70% of the isolates carrying enzymes involved in the turnover of carbon, in the May sample, were identified as CLB, indicating the importance of this group in early colonization of the rhizosphere . The fluorescent pseudomonad group contributed less than 3%.

Biosci Biotechnol Biochem, 2002 Apr, 66(4), 897 - 901
Organization and transcriptional characterization of catechol degradation genes involved in carbazole degradation by Pseudomonas resinovorans strain CA10; Nojiri H et al.; Pseudomonas resinovorans strain CA10 assimilates catechol, which is an intermediate of carbazole degradation, by ortho cleavage pathway enzymes encoded by the catR, catBCA operon . Cat proteins of strain CA10 were very similar to those of P . putida, although the relatedness in non-coding regions was not high . It was found that catBCA genes were induced in carbazole-grown cells as a single transcriptional unit.

Microb Ecol, 2001 Feb, 41(2), 132 - 139
Biological Control of Pseudomonas syringae pv . glycinea by Epiphytic Bacteria under Field Conditions; Volksch B et al.; The efficacy of a bacterial strain as a biocontrol agent in the field may be related to the ecological similarity between the biocontrol agent and the target pathogen . Therefore, a number of different Pseudomonas syringae strains were evaluated for their antagonistic activities in vitro (agar-diffusion assay) and in planta (greenhouse assay) against the target pathogen, Pseudomonas syringae pv . glycinea . Six strains of five different pathovars were found to be antagonistic in vitro as well as in planta . The epiphytic fitness of the antagonistic Pseudomonas syringae strain 22d/93 and its two antibiotic-resistant mutants were examined on soybean plants in the fields . After adaptation the parental strain and its mutants had the ability to establish and maintain large epiphytic populations (about 106 cfu/g FW) over the whole growing season after a single spray inoculation . The epiphytic behaviors of the mutants and the parent were not significantly different . The introduced bacteria did not influence the total bacterial population size . When the antagonist was coinoculated with the pathogen, the development of the pathogen was significantly reduced during the whole growing season . When the antagonistic strain was inoculated 4 weeks in advance of the pathogen, this antagonistic effect could be markedly enhanced . The final population size of the pathogen reached just 104 cfu/g FW and was significantly reduced to 0.12% compared to the pathogen alone . This study demonstrates that biological control of foliar pathogens through colonization of the host plants with near isogenic or ecologically similar antagonistical strains seems to be a realistic goal.

Microb Ecol, 2001 Feb, 41(4), 314 - 324
Growth of Pseudomonas aureofaciens PGS12 and the Dynamics of HHL and Phenazine Production in Liquid Culture, on Nutrient Agar, and on Plant Roots; Seveno NA et al.; The growth of Pseudomonas aureofaciens PGS12 was followed in nutrient broth (NB), on nutrient agar (NA), and on plant roots by monitoring cell numbers, the production of the autoinducer hexanoyl-homoserine lactone (HHL), and the antibiotic phenazine-1-carboxylic acid (PCA) . In NB, as the growth rate declined in transition phase, HHL synthesis increased rapidly, shortly followed by PCA production . During stationary phase, HHL concentration declined rapidly while PCA concentration continued to increase slowly . The luxAB reporter genes were inserted in the phzB gene of the phenazine operon and phenazine transcriptional activity was monitored using measurement of luminescence . Levels and pattern of light output were similar to HHL accumulation and indicated that gene expression was maximal in transition phase and silenced in stationary phase . PCA production continued in stationary phase, suggesting that the protein products of the phenazine operon were maintained in the cell after down regulation . HHL accumulation was 60 times higher on NA than in NB per equivalent volume because of a 60-fold increase in cell density on NA . Higher levels of PCA per cell (6.8 times) and per equivalent volume (360-fold) accumulated in a colony compared to that found in broth . HHL remained at a high concentration in a colony for a longer period compared to a short burst in NB, and this may explain the increased PCA production . In contrast, on wheat seedlings and bean plant roots, bacterial growth was observed, but neither HHL nor PCA was detected; however, transcriptional activity of the phzB::luxAB reporter occurred on the bean plant roots.

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