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 com