Invest Ophthalmol Vis Sci, 2004 Feb, 45(2), 522 - 30 Pseudomonas keratitis: protease IV gene conservation, distribution, and production relative to virulence and other Pseudomonas proteases; Caballero A et al.; PURPOSE: To determine the distribution of the protease IV gene, the production of this and other proteases by multiple strains of Pseudomonas, and the virulence of a mutant specifically deficient in protease IV . METHODS: The protease IV gene was cloned, its sequence analyzed, and its chromosomal location determined by pulse-field gel electrophoresis . Three PCR reactions were used to detect the protease IV gene in 30 Pseudomonas isolates and protease production was determined by Western blot analysis, colorimetric assay, and zymography . An allelic replacement mutant deficient in the protease IV gene was analyzed for enzyme production, corneal growth, and corneal virulence . RESULTS: The protease IV gene was identified in all P . aeruginosa, but none of the non-aeruginosa strains tested . The protease IV genes of strains PA103-29 and PAO1 were in a common chromosomal site and had 98.5% sequence identity with variations occurring mainly in the promoter region . The protease IV activity of the 23 wild-type P . aeruginosa strains tested varied from 2.3 to 221.5 x 10(-3) U/mg protein in the culture supernatant . Protease IV was produced by all P . aeruginosa wild-type strains . A protease IV-deficient mutant derived from strain PA103-29 had reduced virulence compared with its parent strain and unexpectedly produced alkaline protease . CONCLUSIONS: The protease IV gene and its product are common to P . aeruginosa, but not to other Pseudomonas species . Protease IV activity varies among P . aeruginosa strains, and a mutant specifically deficient in this activity produced alkaline protease and had reduced corneal virulence. Plant Cell, 2004 Feb, 16(2), 309 - 18 Epub 2004 Jan 23. Convergent evolution of disease resistance gene specificity in two flowering plant families; Ashfield T et al.; Plant disease resistance (R) genes that mediate recognition of the same pathogen determinant sometimes can be found in distantly related plant families . This observation implies that some R gene alleles may have been conserved throughout the diversification of land plants . To address this question, we have compared R genes from Glycine max (soybean), Rpg1-b, and Arabidopsis thaliana, RPM1, that mediate recognition of the same type III effector protein from Pseudomonas syringae, AvrB . RPM1 has been cloned previously, and here, we describe the isolation of Rpg1-b . Although RPM1 and Rpg1-b both belong to the coiled-coil nucleotide binding site (NBS) Leu-rich repeat (LRR) class of R genes, they share only limited sequence similarity outside the conserved domains characteristic of this class . Phylogenetic analyses of A . thaliana and legume NBS-LRR sequences demonstrate that Rpg1-b and RPM1 are not orthologous . We conclude that convergent evolution, rather than the conservation of an ancient specificity, is responsible for the generation of these AvrB-specific genes. Br J Anaesth, 2004 Mar, 92(3), 429 - 31 Epub 2004 Jan 22. Cerebrospinal fluid-cutaneous fistula and pseudomonas meningitis complicating thoracic epidural analgesia; Abaza KT et al.; We report a case of delayed cerebrospinal fluid-cutaneous fistula that developed in a patient following removal of a thoracic epidural catheter used for perioperative analgesia . It was further complicated by the development of bacterial meningitis . Predisposing factors and management of this rare iatrogenic complication are discussed and the literature reviewed for similar reports. J Biotechnol, 2004 Feb 19, 108(1), 51 - 9 Characterisation of steryl esterase activities in commercial lipase preparations; Kontkanen H et al.; Triglycerides, steryl esters, resin acids, free fatty acids and sterols are lipophilic extractives of wood (commonly referred to as pitch or wood resin) and have a negative impact on paper machine runnability and quality of paper . Thus, enzymes capable of modifying these compounds would be potential tools for reducing pitch problems during paper manufacture . In this work, 19 commercial lipase preparations were tested for their ability to degrade steryl esters, which may play a significant role in the formation and stabilisation of pitch particles . Six lipase preparations were shown to be able to degrade steryl esters . Lipase preparations of Pseudomonas sp., Chromobacterium viscosum and Candida rugosa were shown to have the highest steryl esterase activities . The enzymes were able to hydrolyse steryl esters totally in the presence of a surfactant (Thesit) . Up to 80% of the steryl esters were degraded in aqueous dispersion . Preliminary characterisation of the enzymatic activities revealed that the lipase preparation of Pseudomonas sp . could be the most potential enzyme in industrial applications . The steryl esterase activity of this preparation was stable over a broad pH range and the enzyme was able to act efficiently at pH 6-10 and at temperatures up to 70 degrees C. Pediatr Nephrol, 2004 Apr, 19(4), 438 - 41 Epub 2004 Jan 23. A boy with consecutive development of SLE and Wegener granulomatosis; Erdogan O et al.; An 11-year-old boy with consecutive development of systemic lupus erythematosus (SLE) and Wegener granulomatosis (WG) is presented . He was first admitted to the hospital with the findings of SLE, including crescentic glomerulonephritis, Coombs' test-positive hemolytic anemia, hypocomplementemia, antinuclear antibody (ANA) positivity, and elevated levels of anti-double-stranded (ds) DNA antibodies . He was treated successfully with steroids, cyclophosphamide, and peritoneal dialysis . One month after his discharge he developed an apparent viral infection . Three weeks afterwards he was readmitted with the findings of lower respiratory tract involvement, maxillary sinusitis, nasal septum perforation, p- and c-antineutrophil cytoplasmic antibody (ANCA) positivity, but normal complement, ANA, and anti-ds DNA levels, suggesting the diagnosis of WG . He did not respond to anti-infectious and immunosuppressive treatment, and he died of Pseudomonas sepsis. Appl Biochem Biotechnol, 2004 Jan, 112(1), 55 - 62 Lipase-catalyzed solvent-free transesterification of wood sterols; Martinez I et al.; Eighteen commercial lipase preparations, either immobilized or crude enzyme powders, were screened for the transesterification of wood sterols . The reactions were carried out in a solvent-free system, at the optimum temperature of the enzyme preparations as reported by the manufacturer and at the pressure of 2 mbar, with 5 or 10% in weight of the enzyme relative to the wood sterol content of the reacting mixture . Methyl esters of sunflower fatty acids were used as transesterifying agent . Of all the enzymes assayed, only Lipase TL from Pseudomonas stutzeri PL-836 (Meito Sangyo) exhibited any significant transesterifying capacity, 85 and 95% of conversion after 2 and 8 h of reaction, respectively, when 10% in weight of enzyme was used. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 638 - 45 Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate; Wlodawer A et al.; High-resolution crystallographic analysis of a complex of the serine-carboxyl proteinase sedolisin with pseudo-iodotyrostatin revealed two molecules of this inhibitor bound in the active site of the enzyme, marking subsites from S3 to S3(') . The mode of binding represents two products of the proteolytic reaction . Substrate specificity of sedolisin was investigated using peptide libraries and a new peptide substrate for sedolisin, MCA-Lys-Pro-Pro-Leu-Glu#Tyr-Arg-Leu-Gly-Lys(DNP)-Gly, was synthesized based on the results of the enzymatic and crystallographic studies and was shown to be efficiently cleaved by the enzyme . The kinetic parameters for the substrate, measured by the increase in fluorescence upon relief of quenching, were: k(cat)=73+/-5 s(-1), K(m)=0.12+/-0.011 microM, and k(cat)/K(m)=608+/-85 s(-1)microM(-1). Lipids, 2003 Nov, 38(11), 1197 - 206 Lipase-catalyzed methanolysis of triricinolein in organic solvent to produce 1,2(2,3)-diricinolein; Turner C et al.; The objective of this study was to find the optimal parameters for lipase-catalyzed methanolysis of triricinolein to produce 1,2(2,3)-diricinolein . Four different immobilized lipases were tested, Candida antarctica type B (CALB), Rhizomucor miehei (RML), Pseudomonas cepacia (PCL), and Penicillium roquefortii (PRL) . n-Hexane and diisopropyl ether (DIPE) were examined as reaction media at three different water activities (a(w)), 0.11, 0.53, and 0.97 . The consumption of triricinolein and the formation of 1,2(2,3)-diricinolein, methyl ricinoleate, and ricinoleic acid were followed for up to 48 h . PRL gave the highest yield of 1,2(2,3)-diricinolein . Moreover, this lipase showed the highest specificity for the studied reaction, i.e., high selectivity for the reaction with triricinolein but low for 1,2(2,3)-diricinolein . Recoveries of 93 and 88% DAG were obtained using PRL in DIPE at a(w) of 0.11 and 0.53, respectively . Further, NMR studies showed that a higher purity of the 1,2(2,3)-isomer vs . the 1,3-isomer was achieved at higher a(w) (88% at a(w) = 0.53), compared to lower a(w) (71% at a(w) = 0.11) . The DAG obtained was acylated by the DAG acyltransferase from Arabidopsis thaliana . Therefore, this enzymatic product is a useful enzyme substrate for lipid biosynthesis . Accordingly, the use of PRL in DIPE at a(w) 0.53 is considered optimal for the synthesis of 1,2(2,3)-diricinolein from triricinolein. Trends Cell Biol, 1992 Feb, 2(2), 35 - 7 Chloride channels, Golgi pH and cystic fibrosis; Barasch J et al.; Cystic fibrosis (CF) is associated with a defect in a cAMP-activated chloride channel in secretory epithelia, which leads to decreased fluid secretion . In addition, many mucus glycoproteins show decreased sialylation but increased sulphation . We have recently shown that the pH of intracellular organelles is elevated in CF cells, due to defective chloride conductance in the vesicle membranes . We postulate that this may affect the activity of sialyl-, fucosyl- and sulphotransferases, and thus explain the abnormal glycosylation . Defects in sialylation of glycolipids might also generate receptors for Pseudomonas, which infects the respiratory tract of CF patients. Plant Cell, 2004 Feb, 16(2), 465 - 77 Epub 2004 Jan 16. The Arabidopsis thaliana dihydroxyacetone phosphate reductase gene SUPPRESSSOR OF FATTY ACID DESATURASE DEFICIENCY1 is required for glycerolipid metabolism and for the activation of systemic acquired resistance; Nandi A et al.; Systemic acquired resistance (SAR) is a broad-spectrum resistance mechanism in plants that is activated in naive organs after exposure of another organ to a necrotizing pathogen . The organs manifesting SAR exhibit an increase in levels of salicylic acid (SA) and expression of the PATHOGENESIS-RELATED1 (PR1) gene . SA signaling is required for the manifestation of SAR . We demonstrate here that the Arabidopsis thaliana suppressor of fatty acid desaturase deficiency1 (sfd1) mutation compromises the SAR-conferred enhanced resistance to Pseudomonas syringae pv maculicola . In addition, the sfd1 mutation diminished the SAR-associated accumulation of elevated levels of SA and PR1 gene transcript in the distal leaves of plants previously exposed to an avirulent pathogen . However, the basal resistance to virulent and avirulent strains of P . syringae and the accumulation of elevated levels of SA and PR1 gene transcript in the pathogen-inoculated leaves of sfd1 were not compromised . Furthermore, the application of the SA functional analog benzothiadiazole enhanced disease resistance in the sfd1 mutant plants . SFD1 encodes a putative dihydroxyacetone phosphate (DHAP) reductase, which complemented the glycerol-3-phosphate auxotrophy of the DHAP reductase-deficient Escherichia coli gpsA mutant . Plastid glycerolipid composition was altered in the sfd1 mutant plant, suggesting that SFD1 is involved in lipid metabolism and that an SFD1 product lipid(s) is important for the activation of SAR. Appl Microbiol Biotechnol, 2004 Jun, 64(6), 840 - 7 Epub 2004 Jan 15. Inhibition of matrix metalloproteinase-2 activity by siderophores of Pseudomonas species; Shinozaki Y et al.; To obtain a novel matrix metalloproteinase (MMP) inhibitor produced by bacteria, we have focused on the chelating activity of siderophores . Several siderophore-producing bacteria were isolated from soil using chrome azurol S agar plates and then the effect of siderophores on MMP-2 activity was assayed by gelatin zymography . The results showed that partially purified siderophores from ten isolated strains inhibited MMP-2 activity . Among these strains, two were non-fluorescent and eight were fluorescent Pseudomonas species . From these eight strains, pyoverdine-type siderophores were detected . The Zn(2+)-chelating activity of these siderophores correlated with the inhibition of MMP-2 activity . Therefore, it is considered that siderophores such as pyoverdines inhibit MMP-2 activity by chelating Zn(2+) on the active site of MMP-2. Biotechnol Lett, 2003 Dec, 25(23), 1977 - 81 Immobilization of Pseudomonas delafieldii with magnetic polyvinyl alcohol beads and its application in biodesulfurization; Shan GB et al.; Pseudomonas delafieldii was immobilized in magnetic polyvinyl alcohol (PVA) beads using a hydrophilic magnetic fluid, which was prepared by a co-precipitation method . The beads had distinct super-paramagnetic properties and were compared with immobilized cells in non-magnetic PVA beads . Their desulfurizing activity was increased slightly from 8.7 to 9 mmol sulfur kg(-1) (dry cell) h(-1) . The main advantages was that the magnetic immobilized cells maintain a high desulfurization activity and remain in good shape after 7 times of repeated use, while the non-magnetic immobilized cells could only be used for 5 times . Furthermore, the magnetic immobilized cells could be easily collected or separated magnetically from the biodesulfurization reactor. Mol Plant Microbe Interact, 2004 Jan, 17(1), 90 - 7 Functional analysis of genes involved in the synthesis of syringolin A by Pseudomonas syringae pv . syringae B301 D-R; Amrein H et al.; Strains of the phytopathogenic bacterium Pseudomonas syringae pv . syringae secrete a family of structurally closely related peptide derivatives dubbed syringolins, of which syringolin A is the major variant . The function of syringolins in the interaction of P . syringae pv . syringae with their host plants presently is unknown . It is hypothesized that they may constitute virulence factors . However, syringolins are determinants recognized and reacted to by nonhost plant species, and syringolin A has been shown to induce hypersensitive death of cells colonized by powdery mildew in wheat and, thus, to reprogram a compatible interaction into an incompatible one . Syringolin A is an unusual derivative of a tripeptide that contains a 12-membered ring consisting of the amino acids 5-methyl-4-amino-2-hexenoic acid and 3,4-dehydrolysine, two nonproteinogenic amino acids . Here we report the cloning, sequencing, and analysis of genes involved in the biosynthesis of syringolin A . The genes encode proteins consisting of modules typical for nonribosomal peptide synthetases and type I polyketide synthetases, as well as proteins likely involved in the transcriptional regulation of syringolin A biosynthesis and in syringolin A export . The structure and arrangement of the modules lead to the formulation of a model explaining the synthesis of the tripeptide, including the formation of the two nonproteinogenic amino acids in the ring structure of syringolin A. Genetika, 2003 Nov, 39(11), 1454 - 60 {Construction of a genetic map of Pseudomonas mendocina bacteria}; Vasilenko SL et al.; Based on the results of matings with interrupted conjugation and analysis of marker joint inheritance frequencies, distances between 26 genetic determinants were estimated and a genetic map of Pseudomonas mendocina bacteria was constructed. Appl Environ Microbiol, 2004 Jan, 70(1), 483 - 9 Molecular and metabolic characterization of cold-tolerant alpine soil Pseudomonas sensu stricto; Meyer AF et al.; Alpine soils undergo dramatic temporal changes in their microclimatic properties, suggesting that the bacteria there encounter uncommon shifting selection gradients . Pseudomonads constitute important members of the alpine soil community . In order to characterize the alpine Pseudomonas community and to assess the impact of shifting selection on this community, we examined the ability of cold-tolerant Pseudomonas isolates to grow on a variety of carbon sources, and we determined their phylogenetic relationships based on 16S ribosomal DNA sequencing . We found a high prevalence of Pseudomonas in our soil samples, and isolates from these soils exhibited extensive metabolic diversity . In addition, our data revealed that many of our isolates form a unique cold-adapted clade, representatives of which are also found in the Swedish tundra and Antarctica . Our data also show a lack of concordance between the metabolic properties and 16S phylogeny, indicating that the metabolic diversity of these organisms cannot be predicted by phylogeny. Appl Environ Microbiol, 2004 Jan, 70(1), 346 - 55 Frequency, size, and localization of bacterial aggregates on bean leaf surfaces; Monier JM et al.; Using epifluorescence microscopy and image analysis, we have quantitatively described the frequency, size, and spatial distribution of bacterial aggregates on leaf surfaces of greenhouse-grown bean plants inoculated with the plant-pathogenic bacterium Pseudomonas syringae pv . syringae strain B728a . Bacterial cells were not randomly distributed on the leaf surface but occurred in a wide range of cluster sizes, ranging from single cells to over 10(4) cells per aggregate . The average cluster size increased through time, and aggregates were more numerous and larger when plants were maintained under conditions of high relative humidity levels than under dry conditions . The large majority of aggregates observed were small (less than 100 cells), and aggregate sizes exhibited a strong right-hand-skewed frequency distribution . While large aggregates are not frequent on a given leaf, they often accounted for the majority of cells present . We observed that up to 50% of cells present on a leaf were located in aggregates containing 10(3) cells or more . Aggregates were associated with several different anatomical features of the leaf surface but not with stomates . Aggregates were preferentially associated with glandular trichomes and veins . The biological and ecological significance of aggregate formation by epiphytic bacteria is discussed. Microb Ecol, 2003 Aug, 46(2), 216 - 27 Comparison of subsurface and surface soil bacterial communities in California grassland as assessed by terminal restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes; LaMontagne MG et al.; The integrated biomass beneath the surface horizon in unsaturated soils is large and potentially important in nutrient and carbon cycling . Compared to surface soils, the ecology of these subsurface soils is weakly understood, particularly in terms of the composition of bacterial communities . We compared soil bacterial communities along two vertical transects by terminal restriction fragment length polymorphisms (TRFLPs) of PCR-amplified 16S rRNA genes to determine how surface and deep bacterial communities differ . DNA yield from soils collected from two Mediterranean grassland transects decreased exponentially from the surface to 4 m deep . Richness, as assessed by the number of peaks obtained after restriction with HhaI, MspI, RsaI, or HaeIII, and diversity, as assessed by the Shannon diversity indices, were lowest in the deepest sample . Lower diversity at depth is consistent with species-energy theory, which would predict relatively low diversity in the low organic matter horizons . Principal components analysis suggested that, in terms of HhaI and HaeIII generated TRFLPs, bacterial communities differed between depths . The most abundant amplicons cloned from the deepest sample contained sequences with restriction sites consistent with the largest peaks observed in TRFLPs generated from deep samples . These more abundant operational taxonomic units (OTUs) appeared related to Pseudomonas and Variovorax . Several OTUs were more related to each other than any previously described ribotypes . These OTUs showed similarity to bacteria from the divisions Actinobacteria and Firmicutes. Biotechnol Bioeng, 2004 Jan 20, 85(2), 214 - 21 Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein; Li L et al.; We investigated the ability of the N-terminal domain of InaK, an ice nucleation protein from Pseudomonas syringae KCTC1832, to act as an anchoring motif for the display of foreign proteins on the Escherichia coli cell surface . Total expression level and surface display efficiency of green fluorescent protein (GFP) was compared following their fusion with either the N-terminal domain of InaK (InaK-N), or with the known truncated InaK containing both N- and C-terminal domains (InaK-NC) . We report that the InaK-N/GFP fusion protein showed a similar cell surface display efficiency ( approximately 50%) as InaK-NC/GFP, demonstrating that the InaK N-terminal region alone can direct translocation of foreign proteins to the cell surface and can be employed as a potential cell surface display motif . Moreover, InaK-N/GFP showed the highest levels of total expression and surface display based on unit cell density . InaK-N was also successful in directing cell surface display of organophosphorus hydrolase (OPH), confirming its ability to act as a display motif . J Bacteriol, 2004 Jan, 186(2), 543 - 55 Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter; Schechter LM et al.; Pseudomonas syringae pv . tomato strain DC3000 is a pathogen of tomato and Arabidopsis: The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity . In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues . Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P . syringae TTSS effectors into plant cells . This system includes a cloned P . syringae hrp gene cluster and the model plant Nicotiana benthamiana . Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation . AvrB, tested because it is poorly secreted in cultures by the P . syringae Hrp system, was translocated into plant cells as effectively as AvrPto . The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA . We also confirmed that HopPtoK, HopPtoC, and AvrPphE(Pto) are translocated into plant cells . These results increased the number of Hrp system-secreted proteins in DC3000 to 40 . Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics . Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P . syringae. J Biol Chem, 2004 Mar 19, 279(12), 11546 - 52 Epub 2003 Dec 29. The X-ray structure of trans-3-chloroacrylic acid dehalogenase reveals a novel hydration mechanism in the tautomerase superfamily; de Jong RM et al.; Isomer-specific 3-chloroacrylic acid dehalogenases function in the bacterial degradation of 1,3-dichloropropene, a compound used in agriculture to kill plant-parasitic nematodes . The crystal structure of the heterohexameric trans-3-chloroacrylic acid dehalogenase (CaaD) from Pseudomonas pavonaceae 170 inactivated by 3-bromopropiolate shows that Glu-52 in the alpha-subunit is positioned to function as the water-activating base for the addition of a hydroxyl group to C-3 of 3-chloroacrylate and 3-bromopropiolate, whereas the nearby Pro-1 in the beta-subunit is positioned to provide a proton to C-2 . Two arginine residues, alphaArg-8 and alphaArg-11, interact with the C-1 carboxylate groups, thereby polarizing the alpha,beta-unsaturated acids . The reaction with 3-chloroacrylate results in the production of an unstable halohydrin, 3-chloro-3-hydroxypropanoate, which decomposes into the products malonate semialdehyde and HCl . In the inactivation mechanism, however, malonyl bromide is produced, which irreversibly alkylates the betaPro-1 . CaaD is related to 4-oxalocrotonate tautomerase, with which it shares an N-terminal proline . However, in 4-oxalocrotonate tautomerase, Pro-1 functions as a base participating in proton transfer within a hydrophobic active site, whereas in CaaD, the acidic proline is stabilized in a hydrophilic active site . The altered active site environment of CaaD thus facilitates a previously unknown reaction in the tautomerase superfamily, the hydration of the alpha,beta-unsaturated bonds of trans-3-chloroacrylate and 3-bromopropiolate . The mechanism for these hydration reactions represents a novel catalytic strategy that results in carbon-halogen bond cleavage. Carbohydr Res, 2004 Jan 22, 339(2), 393 - 400 Synthesis of the pentasaccharide repeating unit of the major O-antigen component from Pseudomonas syringae pv . ribicola NVPPB 1010; Bedini E et al.; The synthesis of the repeating unit of the major O-antigen component from Pseudomonas syringae pv . ribicola NVPPB 1010 is reported . The strategy used was based on the successive coupling of a trisaccharide rhamnosyl trichloroacetimidate with a rhamnosyl acceptor with a free hydroxyl group on C-2 . The pentasaccharide was then obtained by coupling with a N-Troc-tri-O-acetyl-glucosamine trichloroacetimidate . The synthesis allowed the oligomerisation of the repeating unit. Gene, 2004 Jan 21, 325, 137 - 43 The global regulator GacS of a biocontrol bacterium Pseudomonas chlororaphis O6 regulates transcription from the rpoS gene encoding a stationary-phase sigma factor and affects survival in oxidative stress; Kang BR et al.; The global regulator, GacS (global activator for antibiotics and cyanide sensor kinase), of the rhizosphere bacterium Pseudomonas chlororaphis O6 (Pc O6) was required for increased resistance to hydrogen peroxide as cultures mature . Specific bands of peroxidase and catalase activity were absent in the stationary-phase cells of the Pc O6 gacS mutant, whereas a manganese superoxide dismutase (MnSOD) isozyme was expressed earlier and to a greater extent than in the wild-type . In the wild-type cell, transcript accumulation of rpoS was higher in late logarithmic (log)-phase cells than cells from mid log-phase or stationary-phase . Transcript abundance from rpoS was reduced in the gacS mutant throughout the growth phase compared to the wild-type expression . The sequence of a small RNA, rsmZ, found downstream of rpoS in other pseudomonads was lacking in Pc O6 . This RNA is implicated in the control of genes activated by the GacS system . Thus, the mechanism by which GacS mediates the activation of genes under its control requires further investigation in Pc O6. Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 555 - 8 Affinity labeled glutaryl-7-amino cephalosporanic acid acylase C130 can hydrolyze the inhibitor during crystallization; Zhang W et al.; 7Beta-bromoacetyl amino cephalosporanic acid (BA-7-ACA), an analog of glutaryl-7-amino cephalosporanic acid (GL-7-ACA), can inhibit and specifically alkylate GL-7-ACA acylase (C130) from Pseudomonas sp.130, forming a carbon-carbon bond between BA-7-ACA and the C-2 on indole ring of Trp-beta4 residue of C130 . Here we reported that BA-7-ACA labeled C130 (BA-C130) could self-catalyze the hydrolysis of BA-7-ACA during crystallization process . The hydrolysis was confirmed to be a reaction analogous to the one of GL-7-ACA by comparative matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry analysis . BA-C130 was inactive at room temperature, but in the process of crystallization at 18 degrees C it catalyzed the hydrolysis of BA-7-ACA, and thus made the latter become a substrate . Meanwhile, in crystals, 7-ACA was released but the acetic acid still bound with Trp-beta4, and as a result, the enzyme remained to be inactive . These results demonstrated that Trp-beta4 in the alphabetabetaalpha motif was critical and sensitive for the activity of C130 and also suggested that there was a conformational change induced by deacylation during the process of crystallization. Chembiochem, 2004 Jan 3, 5(1), 93 - 8 Unprecedented diversity of catalytic domains in the first four modules of the putative pederin polyketide synthase; Piel J et al.; Polyketides of the pederin group are highly potent antitumor compounds found in terrestrial beetles and marine sponges . Pederin is used by beetles of the genera Paederus and Paederidus as a chemical defense . We have recently identified a group of putative pederin biosynthesis genes and localized them to the genome of an as yet unculturable Pseudomonas sp . symbiont, the likely true pederin producer . However, this polyketide synthase cluster lacks several genes expected for pederin production . Here we report an additional polyketide synthase encoded on a separate region of the symbiont genome . It contains at least three novel catalytic domains that are predicted to be involved in pederin chain initiation and the formation of an unusual exomethylene bond . The region is bordered by mobility pseudogenes; this suggests that gene transposition led to the disjointed cluster organization . With this work, all putative pederin genes have been identified . Their heterologous expression in a culturable bacterium will provide important insights into how sustainable sources of invertebrate-derived drug candidates can be created. Clin Cancer Res, 2003 Dec 15, 9(17), 6516 - 22 Antitumor therapy with bacterial DNA and toxin: complete regression of established tumor induced by liposomal CpG oligodeoxynucleotides plus interleukin-13 cytotoxin; Ishii KJ et al.; Despite urgent need, no single strategy has been widely effective at controlling the growth of rapidly progressive solid tumors . We demonstrate here a potent antitumor therapy using modified bacterial DNA and toxin . Treatment of human head and neck cancer established as xenografts in athymic mice with immunostimulatory CpG oligodeoxynucleotides encapsulated in sterically stabilized cationic liposome {(CpG ODN)(SSCL)} and recombinant interleukin-13 Pseudomonas exotoxin (IL13-PE) significantly reduced the tumor growth followed by complete regression in most animals . The antitumor activity of (CpG ODN)(SSCL) was dependent on natural killer cells that infiltrated within tumors . Interestingly, IL13-PE enhanced (CpG ODN)(SSCL)-induced natural killer cell activity and cytokine production in vivo and in vitro . These data strongly suggest that a combination of innate immune activation by (CpG ODN)(SSCL) and tumor-directed targeting by IL13-PE is a novel approach for human cancer immunotherapy. Proc Natl Acad Sci U S A, 2004 Jan 6, 101(1), 70 - 5 Epub 2003 Dec 23. Structure of HrcQB-C, a conserved component of the bacterial type III secretion systems; Fadouloglou VE et al.; Type III secretion systems enable plant and animal bacterial pathogens to deliver virulence proteins into the cytosol of eukaryotic host cells, causing a broad spectrum of diseases including bacteremia, septicemia, typhoid fever, and bubonic plague in mammals, and localized lesions, systemic wilting, and blights in plants . In addition, type III secretion systems are also required for biogenesis of the bacterial flagellum . The HrcQ(B) protein, a component of the secretion apparatus of Pseudomonas syringae with homologues in all type III systems, has a variable N-terminal and a conserved C-terminal domain (HrcQ(B)-C) . Here, we report the crystal structure of HrcQ(B)-C and show that this domain retains the ability of the full-length protein to interact with other type III components . A 3D analysis of sequence conservation patterns reveals two clusters of residues potentially involved in protein-protein interactions . Based on the analogies between HrcQ(B) and its flagellum homologues, we propose that HrcQ(B)-C participates in the formation of a C-ring-like assembly. Dermatol Surg, 2004 Jan, 30(1), 58 - 62; discussion 62 The incidences of chondritis and perichondritis associated with the surgical manipulation of auricular cartilage; Kaplan AL et al.; BACKGROUND: The cartilage and soft tissues of the ear are frequently employed as donor sites for tissue used in the repair of defects of the nose and external ear after Mohs surgery . Enthusiasm for using these auricular donor sites is occasionally tempered by surgeons' concerns for the development of Pseudomonal suppurative chondritis, a complication that has been described to follow cartilage manipulation . OBJECTIVE: To quantify the incidence of postoperative perichondritis and chondritis after Mohs reconstructions involving auricular cartilage manipulations . METHODS: We retrospectively reviewed 341 Mohs reconstructions that involved cartilage and soft-tissue donor sites located on the ear . Procedures included full-thickness skin grafts (295) harvested from the conchal bowl and flap repairs (46) incorporating cartilage batten grafts from conchal or anthelix donor sites . When the perichondrium was compromised, patients were routinely prescribed perioperative prophylactic antibiotics with Pseudomonal coverage . Postoperative examinations were performed at 1 week and 4 to 12 weeks . Patients not seen in clinic were interviewed by telephone regarding complications . RESULTS: Complete follow-up information was obtained in 337 of 341 (98.8%) cases . Inflammatory perichondritis was observed in 19 (5.6%) patients . There were no cases of suppurative chondritis . CONCLUSION: The incidence of inflammatory perichondritis is low after Mohs reconstructions involving auricular cartilage manipulation . When prophylactic antibiotics and appropriate operative technique are used, the historic concern for suppurative chondritis associated with these procedures is unwarranted. Appl Microbiol Biotechnol, 2004 Apr, 64(2), 154 - 74 Epub 2003 Dec 20. Divergence of mobile genetic elements involved in the distribution of xenobiotic-catabolic capacity; Nojiri H et al.; Bacteria adapt rapidly to environmental stimuli, such as exposure to xenobiotics . Mobile genetic elements (MGEs) play a major role in such bacterial adaptation, via the dispersal of catabolic capacity; and, in fact, genes that encode the degradation enzymes for xenobiotics are often located on MGEs . The list of reported catabolic MGEs keeps growing as researchers continue to isolate and characterize xenobiotic degraders and the corresponding catabolic genes . Major catabolic MGEs include (conjugative) plasmids, transposons, and conjugative transposons . Catabolic transposons can be divided into class I elements (composite transposons) and class II elements (Tn 3 family transposons) . This review includes a comprehensive list of naturally occurring discrete catabolic MGEs, together with a brief description for each . While MGEs are often rather large, genome-wide or large-scale sequence analyses have provided useful information on the whole genetic structure of MGEs, with clues to their function (transfer, maintenance, catabolism, etc.) and behavior in a complex natural environment . This review also gives an insight into MGE functions, based on the complete sequencing of several catabolic plasmids and two Pseudomonas genomes. Planta, 2004 Feb, 218(4), 668 - 72 Epub 2003 Dec 18. Induction of 3'-O-beta-D-ribofuranosyl adenosine during compatible, but not during incompatible, interactions of Arabidopsis thaliana or Lycopersicon esculentum with Pseudomonas syringae pathovar tomato; Bednarek P et al.; All hitherto identified aromatic compounds accumulating in leaves of Arabidopsis thaliana (L.) Heynh . upon infection with virulent or avirulent strains of Pseudomonas syringae pathovar tomato ( Pst) were indolic metabolites . We now report the strong accumulation of a novel type of natural product, 3'-O-beta-D-ribofuranosyl adenosine (3'RA), exclusively during compatible interactions . In contrast to the various indolic metabolites, 3'RA was undetectable in incompatible interactions of A . thaliana leaves with an avirulent Pst strain, as well as in uninfected control leaves . A similar, strong induction of 3'RA was observed in compatible but, again, not in incompatible interactions of Pst with its natural host, Lycopersicon esculentum . The strength of the effect and its confinement to compatible interactions suggests that it may be applicable as a diagnostic tool. Protein Expr Purif, 2003 Nov, 32(1), 35 - 43 Purification and characterization of 2'aminobiphenyl-2,3-diol 1,2-dioxygenase from Pseudomonas sp . LD2; Gibbs PR et al.; Carbazole is a nitrogen-containing heteroaromatic compound that occurs as a widespread and mutagenic environmental pollutant . The 2'aminobiphenyl-2,3-diol 1,2-dioxygenase involved in carbazole degradation was purified to near electrophoretic homogeneity from Pseudomonas sp . LD2 by a combination of ion-exchange chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography . This purification was challenging due to the great instability of the enzyme under many standard conditions . The enzyme was also purified to electrophoretic homogeneity from recombinant Escherichia coli expressing the 2'aminobiphenyl-2,3-diol 1,2-dioxygenase-encoding gene cloned from Pseudomonas sp . LD2 . The molecular mass of the native enzyme was determined by gel filtration to be 70 kDa . The subunit molecular masses were determined to be 25 and 8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase is an {alpha2beta2} heterotetramer . The optimal temperature and pH for the enzymatic production of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) from 2,3-dihydroxybiphenyl were determined to be 40 degrees C and 8.0, respectively . The maximum observed specific activity on 2,3-dihydroxybiphenyl was 48.1 mmol HOPDA min(-1) mg(-1) . This indicated a maximum observed turnover rate of 360,000 molecules HOPDA enz(-1) s(-1) . The K'm inhibition constant Ks and Vmax on 2,3 dihydroxybiphenyl were determined to be 5 microM, 37 microM, and 44 mmol min(-1) mg(-1), respectively . These results show that 2'aminobiphenyl-2,3-diol 1,2-dioxygenase is a meta-cleavage enzyme related to the 4,5-protocatechuate dioxygenase family, with comparable purification challenges posed by intrinsic enzyme instability. Mikrobiologiia, 2003 Sep-Oct, 72(5), 672 - 80 {Identification of the key genes of naphthalene catabolism in soil DNA}; Mavrodi DV et al.; The key genes nahAc and xylE of the naphthalene catabolism of fluorescent Pseudomonas spp . in the total soil DNA samples were detected by the polymerase chain reaction (PCR) technique . The collection of fluorescent Pseudomonas spp . was screened for the occurrence of these genes . The results obtained show the possibility of using this approach in the goal-directed search for plasmid-containing naphthalene-degrading fluorescent pseudomonads in soil . The distribution of the naphthalene catabolism genes in soils contaminated with creosote and petroleum products was also studied. Mikrobiologiia, 2003 Sep-Oct, 72(5), 645 - 50 {The production of antifungal metabolites by Pseudomonas chlororaphis grown on different nutrient sources}; Shtark OIu et al.; It was found that the antifungal activity of Pseudomonas chlororaphis SPB1217 is due to phenazine-1-carboxylic acid, phenazine-1-carboxamide, and two unidentified exometabolites . The carbon source used for the growth of this bacterial strain and iron ions present in the medium considerably influenced the proportion between the antifungal metabolites . The maximum production of phenazines was observed in the media enriched in amino acids and iron ions . The absence of correlation between the production of phenazines and antifungal activity indicates that phenazines are not the only antifungal metabolites of the strain . Organic acids as nutrient sources provide for more intense production of exometabolites and for a higher level of antifungal activity than do sugars. J Bacteriol, 2004 Jan, 186(1), 146 - 53 In Pseudomonas syringae pv . phaseolicola, expression of the argK gene, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase, is regulated indirectly by temperature and directly by a precursor resembling carbamoylphosphate; Lopez-Lopez K et al.; Pseudomonas syringae pv . phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin . ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C . To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase {SOCT}), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis) . The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT . This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT . Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P . syringae pv . phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C . These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin. J Bacteriol, 2004 Jan, 186(1), 35 - 42 Characterization of CmaA, an adenylation-thiolation didomain enzyme involved in the biosynthesis of coronatine; Couch R et al.; Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond . The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected . These efforts allowed overproduction of P . syringae pv . glycinea PG4180 CmaA in P . syringae pv . syringae FF5 as a FLAG-tagged protein and overproduction of P . syringae pv . tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form . Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain) . ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine . Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate . This covalent species was detected by incubating CmaA with ATP and L-{G-(3)H}allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA . The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid. Cancer Res, 2003 Dec 1, 63(23), 8414 - 9 Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells; Tur MK et al.; CD64, the high affinity receptor for IgG (FcgammaRI) is expressed on acute myeloid leukemia blast cells and has recently been described as a specific target for immunotherapy . To generate a recombinant immunotoxin, the anti-CD64 single chain fragment (scFv) m22 was cloned into the bacterial expression vector pBM1.1 and fused to a deletion mutant of Pseudomonas exotoxin A (ETA') . Genetically modified Escherichia coli BL21 Star (DE3) were grown under osmotic stress conditions in the presence of compatible solutes . After isopropyl beta-D-thiogalactoside induction, the 70-kDa His(10)-tagged m22(scFv)-ETA' was directed into the periplasmic space and purified by a combination of metal-ion affinity and molecular size-chromatography . The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays, using CD64-positive AML cells . Binding specificity of m22(scFv)-ETA' was verified by competition with the parental anti-CD64 monoclonal antibody m22 . The recombinant immunotoxin showed significant toxicity toward the CD64-positive cell lines HL-60 and U937 reaching 50% inhibition of cell proliferation at a concentration (IC(50)) of 11.6 ng/ml against HL-60 cells and 12.9 ng/ml against U937 cells . Approximately 41% of primary leukemia cells from a patient with CD64-positive AML were driven into early apoptosis by m22(scFv)-ETA' as measured by flow cytometric analysis . This is the first article documenting the specific cytotoxicity of a novel recombinant immunotoxin with major implications for immunotherapy of CD64-positive diseases. Biotechnol Lett, 2003 Nov, 25(21), 1863 - 7 Stereochemistry of a diastereoisomeric amphiphile and the species of the lipase influence enzyme activity in the transesterification catalyzed by a lipase-co-lyophilizate with the amphiphile in organic media; Mine Y et al.; Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, D- and L-2-(3-{bis-{3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl}-carbamoyl}-propionylamino)-pentanedioic acid didodecyl ester (D- and L-BIG2C12CA); D- and L-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (D- and L-2C12GE) . Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated . Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19-48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the L-BIG2C12CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68-78 fold. Plant J, 2003 Dec, 36(6), 905 - 17 Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato; Ekengren SK et al.; The tomato Pto kinase confers resistance to the causative agent of bacterial speck disease, Pseudomonas syringae pv . tomato, by recognizing the pathogen effector proteins AvrPto or AvrPtoB . Pto-mediated resistance requires multiple signal transduction pathways and has been shown to activate many defense responses including an oxidative burst, rapid changes in the expression of over 400 genes, and localized cell death . We have tested the role in Pto-mediated resistance in tomato of a set of 21 genes from other species known to be involved in defense-related signaling . Expression of each gene was suppressed by virus-induced gene silencing (VIGS) and the effect on disease symptoms and bacterial growth during the tomato-Pseudomonas incompatible interaction was determined . We found that Pto-mediated resistance was compromised by silencing of genes encoding two mitogen-activated protein (MAP) kinase kinases, MEK1 and MEK2, two MAP kinases, NTF6 and wound-induced protein kinase (WIPK), a key regulator of systemic acquired resistance (SAR), NPR1, and two transcription factors, TGA1a and TGA2.2 . A lesser impact on Pto-mediated resistance was observed in plants silenced for RAR1 and COI1 . The identification of nine genes that play a role in resistance to bacterial speck disease both advances our knowledge of Pto signal transduction and demonstrates the conservation of many defense signaling components among diverse plant species. Hum Gene Ther, 2003 Dec 10, 14(18), 1787 - 98 Retroviral immunotoxin gene therapy of leukemia in mice using leukemia-specific T cells transduced with an interleukin-3/Bax fusion protein gene; Vallera DA et al.; In past studies, we showed that T cells transduced with retroviral diphtheria immunotoxin (IT) target genes could serve as vehicles for delivering IT to tumors in vivo . We took advantage of the observation that antigen-specific T cells are able to penetrate tumors to design an approach delivering combined cellular and humoral therapy directly to the tumor site . To improve tumor specificity, we selected interleukin (IL)-3 as a ligand because its receptor is selectively overexpressed on myeloid leukemia progenitors . Because Bcl-2 family proteins show structural similarity to diphtheria toxin (DT), we constructed a unique retroviral IT using Bax, a proapoptotic member of the Bcl-2 family, in place of DT . Bax was chosen because several studies showed that its transduction induces lethal apoptosis in different cancers . The retroviral construct for gene therapy included IL-3 positioned downstream of its 80 amino acid leader, and permitted cotranslational protein synthesis of hybrid IL-3/human Bax fusion protein . Other vectors were constructed with IL-3 fused to DT or Pseudomonas exotoxin . Retroviral vectors were used to transiently transduce C8, a CD4(+) T cell clone that specifically recognized FBL-3, a lethal myeloid leukemia . Supernatants collected from transduced cells showed proapoptotic activity and selectively inhibited FBL-3 cells in vitro . Intraperitoneal injection of transduced but not nontransduced C8 into mice with subcutaneous tumors or systemic cancer significantly inhibited tumor growth . These results indicate that retroviral IT made with IL-3 and various toxic proteins may be useful in patients with acute myelogenous leukemia (AML) . Furthermore, the Bax construct may be particularly useful as a nonimmunogenic substitute for bacterial toxins in retIT. Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15977 - 82 Epub 2003 Dec 09. Differential survival of solitary and aggregated bacterial cells promotes aggregate formation on leaf surfaces; Monier JM et al.; The survival of individual Pseudomonas syringae cells was determined on bean leaf surfaces maintained under humid conditions or periodically exposed to desiccation stress . Cells of P . syringae strain B728a harboring a GFP marker gene were visualized by epifluorescence microscopy, either directly in situ or after recovery from leaves, and dead cells were identified as those that were stained with propidium iodide in such populations . Under moist, conducive conditions on plants, the proportion of total live cells was always high, irrespective of their aggregated state . In contrast, the proportion of the total cells that remained alive on leaves that were periodically exposed to desiccation stress decreased through time and was only approximately 15% after 5 days . However, the fraction of cells in large aggregates that were alive on such plants in both condition was much higher than more solitary cells . Immediately after inoculation, cells were randomly distributed over the leaf surface and no aggregates were observed . However, a very aggregated pattern of colonization was apparent within 7 days, and >90% of the living cells were located in aggregates of 100 cells or more . Our results strongly suggest that, although conducive conditions favor aggregate formation, such cells are much more capable of tolerating environmental stresses, and the preferential survival of cells in aggregates promotes a highly clustered spatial distribution of bacteria on leaf surfaces. Appl Environ Microbiol, 2003 Dec, 69(12), 7401 - 8 Transporter-mediated uptake of 2-chloro- and 2-hydroxybenzoate by Pseudomonas huttiensis strain D1; Yuroff AS et al.; We investigated the mechanisms of uptake of 2-chlorobenzoate (2-CBa) and 2-hydroxybenzoate (2-HBa) by Pseudomonas huttiensis strain D1 . Uptake was monitored by assaying intracellular accumulation of 2-{UL-ring-14C}CBa and 2-{UL-ring-14C}HBa . Uptake of 2-CBa showed substrate saturation kinetics with an apparent Km of 12.7 +/- 2.6 micromoles and a maximum velocity (Vmax) of 9.76 +/- 0.78 nmol min-1 mg of protein-1 . Enhanced rates of uptake were induced by growth on 2-CBa and 2-HBa, but not by growth on benzoate or 2,5-di-CBa . Intracellular accumulations of 2-CBa and 2-HBa were 109- and 42-fold greater, respectively, than the extracellular concentrations of these substrates and were indicative of uptake mediated by a transporter rather than driven by substrate catabolism ("metabolic drag") . Results of competitor screening tests indicated that the substrate range of the transporter did not include other o-halobenzoates that serve as growth substrates for strain D1 and for which the metabolism was initiated by the same dioxygenase as 2-CBa and 2-HBa . This suggested that multiple mechanisms for substrate uptake were coupled to the same catabolic enzyme . The preponderance of evidence from tests with metabolic inhibitors and artificial electrochemical gradients suggested that 2-CBa uptake was driven by ATP hydrolysis . If so, the 2-CBa transporter would be the first of the ATP binding cassette type implicated in uptake of haloaromatic acids. Appl Environ Microbiol, 2003 Dec, 69(12), 7248 - 56 Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments; Schonfeld J et al.; Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops . A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established . Based on the first fliC gene sequence of R . solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R . solanacearum strains tested . However, R . pickettii and four environmental Ralstonia isolates also yielded amplicons . The Ral_fliC PCR products obtained with 12 strains (R . solanacearum, R . pickettii, and environmental isolates) were sequenced . By sequence alignment, Rsol_fliC primers specific for R . solanacearum were designed . With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R . solanacearum tested . Six strains of R . pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain . A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P . syzygii from R . solanacearum . The Rsol_fliC PCR system was applied to detect R . solanacearum in soil . PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1) . The system was applied to survey soils from different geographic origins for the presence of R . solanacearum. Appl Environ Microbiol, 2003 Dec, 69(12), 7108 - 15 Efficient degradation of 2,4,6-Trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4); Matus V et al.; 2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant . Several aerobic bacteria are known to degrade this compound . One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and beta-ketoadipate . Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway . Here we provide evidence that all these tcp genes are clustered in the R . eutropha JMP134(pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD . We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains . One type includes strains belonging to the Ralstonia genus and possessing a set of tcp-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source . The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tcp-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP . Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences. Gene, 2003 Dec 24, 323, 125 - 31 Transcriptional regulation and mutational analysis of a dctA gene encoding an organic acid transporter protein from Pseudomonas chlororaphis O6; Nam HS et al.; A dctA gene encoding a protein with identity to a C(4)-dicarboxylic acid/H(+) symporter was cloned from a beneficial root colonizer, Pseudomonas chlororaphis O6 (PcO6) . Expression of the dctA gene was induced in minimal medium by several organic acids and was repressed by glucose . Highest expression was observed in early-logarithmic (log) cells grown on fumarate, acetate or succinate with decline as cells approached late-log growth phase . The dctA transcript accumulated weakly when cells were grown on malate, but strong expression was observed with benzoate . Expression of the dctA transcript was repressed in early-log cells upon addition of glucose to fumarate, but was detected as the cell culture aged . A dctA-deficient mutant of PcO6, constructed by marker exchange mutagenesis, did not grow on minimal medium containing succinate, benzoate, acetate or fumarate and growth on malate was delayed . The dctA mutant and wild-type grew equally on citrate, glucose, fructose, sucrose or inositol . We conclude that the transporter protein encoded by dctA is essential for utilization of certain organic acids and its expression is controlled by the availability of sugars. FEMS Microbiol Lett, 2003 Dec 5, 229(1), 31 - 6 Purification and characterization of an aldehyde oxidase from Pseudomonas sp . KY 4690; Uchida H et al.; An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp . KY 4690, a soil isolate, to an electrophoretically homogeneous state . The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa . The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family . The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa . Molecular oxygen served as the sole electron acceptor . These results suggest that aldehyde oxidase from Pseudomonas sp . KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of {2Fe-2S} clusters per mol of enzyme protein . The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability. Ying Yong Sheng Tai Xue Bao, 2003 Aug, 14(8), 1343 - 6 {Factors restricting growth of heterotrophic bacteria in the water body of West Lake, Hangzhou}; Wu G et al.; Restricting factors of bacteria growth were studied by pure culture and natural culture test . The results showed that organic carbon source available for bacteria was more important than (NH4)2SO4 and KH2PO4, while higher pH, and rich biomass of phytoplanktons and zooplanktons in the water restrained the growth of heterotrophic bacteria . Under natural culture experiment, Azotobacter increased after 0.5% glucose was added, and a lot of mildew grew after adding 0.5% glucose with 0.1% (NH4)2SO4 and 0.1% KH2PO4, while Pseudomonas enriched 30-57% after adding 0.01% beef extract . It was also showed that bacteria growth potentiality in natural water could reach to 10(5) cfu.ml-1. Apoptosis, 1997, 2(2), 192 - 8 Effects of BCL-2 overexpression on the sensitivity of MCF-7 breast cancer cells to ricin, diphtheria and Pseudomonas toxin and immunotoxins; Brinkmann U et al.; Immunotoxins are presently being evaluated as novel agents for cancer therapy . The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents . Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents . It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis . We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor alpha (TNF) . We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3-10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudomonas exotoxin, diphtheria toxin and ricin . We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2 . Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins. J Neurooncol, 2003 Oct, 65(1), 37 - 48 Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside; Husain SR et al.; Central nervous system malignant neoplasias, in particular, glioblastoma multiforme (GBM) have defied all current therapeutic modalities . New therapies involving tumor targeting approach are being explored . This approach relies on the identification of unique or over-expressed cell surface receptors or antigens on tumor cells . In that regard, we have identified receptor for an immune regulatory cytokine, interleukin-13 (IL-13), which is over-expressed on human malignant glioma cell lines and primary tumor cell cultures . To target IL-13 receptors (IL-13R) for cancer therapy, we have developed a recombinant fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR or IL-13 cytotoxin) . The IL-13 cytotoxin was found to be highly selective and potent in killing human GBM cells in vitro while normal cells including immune cells, endothelial cells and normal brain cells were generally spared the cytotoxic effect of IL-13 cytotoxin . This is because these cells either expressed none or expressed low levels of IL-13R . Consistent with in vitro cytotoxic activity, IL-13 cytotoxin mediated remarkable anti-tumor activity to human glioma in animal xenograft models . The direct injection of IL-13 cytotoxin into subcutaneous human GBM tumors grown in nude mice produced complete and durable regression of established tumors . Intravenous and intraperitoneal administration of IL-13 cytotoxin also reduced tumor burden significantly with fewer complete responders . All animals tolerated therapy well with minimal toxicity to vital organs . Pre-clinical safety and toxicity studies were performed in mice, rats and monkeys . Systemic administration of IL-13 cytotoxin appeared to be well tolerated at high doses (up to 50 microg/kg) . Intrabrain parenchyma administration of IL-13 cytotoxin at doses up to 100 microg/ml was very well tolerated without any evidence of gross or microscopic necrosis, whereas at 500 microg/ml dose, localized necrosis was observed in normal rat brain . Based on these encouraging pre-clinical studies, three Phase I/II clinical trials in adults with malignant glioma have been initiated . The first clinical trial involves convection-enhanced delivery (CED) of IL-13 cytotoxin into recurrent malignant glioma . This route of IL-13 cytotoxin administration appears to be fairly well tolerated with no neurotoxicity . The second clinical trial involves infusion of IL-13 cytotoxin by CED following tumor resection . The initial stage of the second study assessed histologic effect of drug administered prior to resection . In third one, IL-13 cytotoxin is infused by CED followed by tumor resection . All three clinical trials are currently ongoing. J Neurooncol, 2003 Oct, 65(1), 27 - 35 Progress report of a Phase I study of the intracerebral microinfusion of a recombinant chimeric protein composed of transforming growth factor (TGF)-alpha and a mutated form of the Pseudomonas exotoxin termed PE-38 (TP-38) for the treatment of malignant brain tumors; Sampson JH et al.; TP-38 is a recombinant chimeric targeted toxin composed of the EGFR binding ligand TGF-alpha and a genetically engineered form of the Pseudomonas exotoxin, PE-38 . After in vitro and in vivo animal studies that showed specific activity and defined the maximum tolerated dose (MTD), we investigated this agent in a Phase I trial . The primary objective of this study was to define the MTD and dose limiting toxicity of TP-38 delivered by convection-enhanced delivery in patients with recurrent malignant brain tumors . Twenty patients were enrolled in the study and doses were escalated from 25 ng/mL to 100 with a 40 mL infusion volume delivered by two catheters . One patient developed Grade IV fatigue at the 100 ng/mL dose, but the MTD has not been established . The overall median survival after TP-38 for all patients was 23 weeks whereas for those without radiographic evidence of residual disease at the time of therapy, the median survival was 31.9 weeks . Overall, 3 of 15 patients, with residual disease at the time of therapy, have demonstrated radiographic responses and one patient with a complete response and has survived greater than 83 weeks. J Neurooncol, 2003 Oct, 65(1), 15 - 25 Interleukin-4-Pseudomonas exotoxin chimeric fusion protein for malignant glioma therapy; Kawakami M et al.; Human malignant glioma cell lines, primary cell cultures, and tumor specimens derived from surgical samples have been shown to overexpress high-affinity receptors (R) for interleukin-4 (IL-4) in vitro and in situ . The significance of IL-4R expression on malignant glioma cells is still unclear . However, IL-4 has been reported to mediate functional effects in several solid tumor cell lines . These activities include inhibition of cell proliferation, regulation of adhesion molecules, and induction of signal transduction through the JAK/STAT pathway . To target IL-4Rs on tumor cells, we have produced a chimeric recombinant fusion protein consisting of a binding ligand, circularly permuted IL-4 and a mutated form of Pseudomonas exotoxin . This molecule is termed IL4(38-37)-PE38KDEL, cpIL4-PE, or IL-4 cytotoxin . Recombinant cpIL4-PE is highly and specifically cytotoxic to glioma cell lines in vitro, while it is not cytotoxic or less cytotoxic to hematopoietic and normal brain cells . In a nude mouse model, cpIL4-PE showed significant antitumor activity and partial or complete regression of small or large established human glioblastoma tumors . Encouraging preclinical efficacy, safety, and tolerability studies lead to testing of this agent in patients with recurrent glioblastoma . Based on these pilot studies, an extended Phase I/II clinical trial is currently ongoing to determine safety, tolerability, and efficacy of cpIL4-PE when injected stereotactically directly into the recurrent glioma by convection enhanced delivery . Preliminary clinical results suggest that cpIL4-PE can cause pronounced necrosis of recurrent glioma tumors without systemic toxicity . The central nervous system toxicities observed were attributed to the volume of infusion and/or nonspecific toxicity . Ongoing clinical trials will reveal antitumor activities of IL-4 cytotoxin in recurrent malignant glioma. Burns, 2003 Dec, 29(8), 854 - 6 Subeschar clysis in deep burns; Sinha R et al.; Six hundred thirteen patients with deep burn of up to 50% total body surface area (TBSA) were treated with 0.25% povidone iodine subeschar clysis (PVP-SEC) in addition to surface application of povidone iodine + Neosporin in the form of "crust" . The results were compared with those of 595 age, sex and percentage of burn, matched patients treated only by "crust application" . The quantitative bacterial count showed significantly less incidence of infection on the 7th and 8th days post treatment (P<0.001) . The organisms identified were predominately Staphylcocous aureus and Pseudomonas aeroginosa . Significantly more number of patients, with burns up to 50% TBSA, could be grafted within 20 days in the SEC group . The graft acceptance rate in this group was 90%. Mol Genet Genomics, 2004 Jan, 270(6), 462 - 76 Epub 2003 Nov 21. Complete nucleotide sequence and analysis of pPSR1 (72,601 bp), a pPT23A-family plasmid from Pseudomonas syringae pv . syringae A2; Sundin GW et al.; Plasmid pPSR1 is a conjugative plasmid originally isolated from Pseudomonas syringae pv . syringae A2, and is a member of the recently described pPT23A plasmid family . We have determined the complete sequence of pPSR1 and found the plasmid to be 72,601 bp in length, encoding 55 ORFs . Putative functions were assigned to 49 ORFs; of these, 24 (49.0%) are involved in plasmid replication, maintenance or conjugation, 17 (34.7%) have roles in virulence or ecological fitness, and eight (16.3%) encode transposase functions as part of mobile elements . pPSR1 carries the effector gene orf34, the mutagenic DNA repair operon rulAB which confers tolerance to ultraviolet radiation, and two genes for methyl-accepting chemotaxis proteins, one of which was located within the novel transposon Tn 5395 . The streptomycin resistance transposon Tn 5393a, which carries a strA-strB determinant, was found inserted immediately downstream of the pPSR1 repA gene . Functional analysis of the replication region of pPSR1 indicated that the repA gene and flanking upstream and downstream sequences are required for autonomous replication in P . syringae . Hybridization analyses of the distribution of 11 of the pPSR1 ORFs indicated that many of the ecologically important ORFs were confined to the pathovar P . syringae pv . syringae -either to strains from the local population from which pPSR1 was originally isolated, or strains from a worldwide collection . Conjugative transfer genes and a gene encoding a transcriptional regulator were more widely distributed among several P . syringae pathovars . The sequence analysis of pPSR1 suggests that pPT23A-family plasmids evolve by accumulating genes that are important for host-pathogen interactions or growth on plant hosts, which are incorporated onto a conserved backbone encoding conjugation and stability determinants. Plant Cell, 2003 Dec, 15(12), 3033 - 50 Epub 2003 Nov 20. The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements; Chakravarthy S et al.; The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes . We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box-containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii . To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants . SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues . Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs) . Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes . Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box . Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element . Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4 . Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs. Curr Microbiol, 2003 Oct, 47(4), 290 - 4 Purification and characterization of a phytase from Pseudomonas syringae MOK1; Cho JS et al.; A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography . The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . The optimal activity occurred at pH 5.5 and 40 degrees C . The Michaelis constant (Km) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively . The enzyme was strongly inhibited by Cu2+, Cd2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA) . It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates . The enzyme efficiently released orthophosphate from wheat bran and soybean meal. Nucleic Acids Res, 2003 Dec 1, 31(23), 6996 - 7002 A key role for the mRNA leader structure in translational control of ribosomal protein S1 synthesis in gamma-proteobacteria; Tchufistova LS et al.; The translation initiation region (TIR) of the Escherichia coli rpsA mRNA coding for ribosomal protein S1 is characterized by a remarkable efficiency in driving protein synthesis despite the absence of the canonical Shine-Dalgarno element, and by a strong and specific autogenous repression in the presence of free S1 in trans . The efficient and autoregulated E.coli rpsA TIR comprises not less than 90 nt upstream of the translation start and can be unambiguously folded into three irregular hairpins (HI, HII and HIII) separated by A/U-rich single-stranded regions (ss1 and ss2) . Phylogenetic comparison revealed that this specific fold is highly conserved in the gamma-subdivision of proteobacteria (but not in other subdivisions), except for the Pseudomonas group . To test phylogenetic predictions experimentally, we have generated rpsA'-'lacZ translational fusions by inserting the rpsA TIRs from various gamma-proteobacteria in-frame with the E.coli chromosomal lacZ gene . Measurements of their translation efficiency and negative regulation by excess protein S1 in trans have shown that only those rpsA TIRs which share the structural features with that of E.coli can govern efficient and regulated translation . We conclude that the E.coli-like mechanism for controlling the efficiency of protein S1 synthesis evolved after divergence of Pseudomona. J Basic Microbiol, 2003, 43(6), 534 - 8 Influence of carbon source on pyrimidine synthesis in Pseudomonas mendocina; Santiago MF et al.; The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was studied . When glucose was the carbon source, orotic acid supplementation of P . mendocina cells produced the greatest depression of aspartate transcarbamoylase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities while P . mendocina cells grown in the presence of uracil caused the maximal decrease in dihydroorotase and OMP decarboxylase activities . After the pyrimidine starvation of an orotate phosphoribosyltransferase mutant strain of P . mendocina grown on glucose, the pyrimidine biosynthetic pathway enzyme activities were generally diminished . With respect to pyrimidine starvation studies, the carbon source glucose appeared to lessen regulation at the level of enzyme synthesis compared to what has been observed when succinate served as the carbon source . The regulation of the pyrimidine biosynthetic pathway by carbon source in P . mendocina appeared to differ from how carbon source influenced the control of pyrimidine biosynthesis in the closely-related species Pseudomonas stutzeri. Bioconjug Chem, 2003 Nov-Dec, 14(6), 1107 - 14 Diphtheria toxin-epidermal growth factor fusion protein and Pseudomonas exotoxin-interleukin 13 fusion protein exert synergistic toxicity against human glioblastoma multiforme cells; Liu TF et al.; The cytotoxicity of combinations of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) and a Pseudomonas exotoxin-human interleukin 13 fusion protein (IL13PE38QQR) was tested against 14 human glioma cell lines . After cells were cultured for 48 h with various concentrations of the fusion proteins, the percentage reductions in thymidine incorporation were determined . Seven of fourteen cell lines were highly sensitive to DAB(389)EGF alone, and six cell lines were highly sensitive to IL13PE38QQR alone with IC(90)'s < 100 pM . When combined, synergistic cell killing was observed for seven of the cell lines based upon concave isobolograms and combination indices (CI's) of 0.2 to 0.7 . Supraadditive cytotoxicity was confirmed by measurements of induction of apoptosis . Receptor expression was assessed by flow cytometry and confocal microscopy . Marked heterogeneity of expression of EGFR and IL13Ralpha2 was seen on all the glioma cell lines . This heterogeneity may contribute to incomplete cell killing with the individual fusion proteins and synergistic cell kill with the combination . These results suggest that both fusion proteins may yield antitumor effects in patients with recurrent gliomas and that combination fusion protein intracranial therapy of malignant gliomas may yield an improved therapeutic index. Biofouling, 2003 Apr, 19 Suppl, 197 - 205 The development of a marine natural product-based antifouling paint; Burgess JG et al.; Problems with tin and copper antifouling compounds have highlighted the need to develop new environmentally friendly antifouling coatings . Bacteria isolated from living surfaces in the marine environment are a promising source of natural antifouling compounds . Four isolates were used to produce extracts that were formulated into ten water-based paints . All but one of the paints showed activity against a test panel of fouling bacteria . Five of the paints were further tested for their ability to inhibit the settlement of barnacle larvae, Balanus amphitrite, and algal spores of Ulva lactuca, and for their ability to inhibit the growth of U . lactuca . Two paints caused a significant decrease in the number of settled barnacles . One paint containing extract of Pseudomonas sp . strain NUDMB50-11, showed excellent activity in all assays . The antifouling chemicals responsible for the activity of the extract were isolated, using bioassay guided fractionation, and their chemical structures determined. J Bacteriol, 2003 Dec, 185(23), 6790 - 800 New bacterial pathway for 4- and 5-chlorosalicylate degradation via 4-chlorocatechol and maleylacetate in Pseudomonas sp . strain MT1; Nikodem P et al.; Pseudomonas sp . strain MT1 is capable of degrading 4- and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway . 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases . However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone . Protoanemonin is obviously a dead-end product of the pathway . A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates . Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in . As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation . The maleylacetate formed in this way is reduced by maleylacetate reductase . Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH. Plant J, 2003 Nov, 36(3), 342 - 52 Genetic evidence that expression of NahG modifies defence pathways independent of salicylic acid biosynthesis in the Arabidopsis-Pseudomonas syringae pv . tomato interaction; Heck S et al.; The salicylic acid (SA)-induction deficient (sid) mutants of Arabidopsis, eds5 and sid2 accumulate normal amounts of camalexin after inoculation with Pseudomonas syringae pv . tomato (Pst), while transgenic NahG plants expressing an SA hydroxylase that degrades SA have reduced levels of camalexin and exhibit a higher susceptibility to different pathogens compared to the sid mutants . SID2 encodes an isochorismate synthase necessary for the synthesis of SA . NahG was shown to act epistatically to the sid mutant phenotype regarding accumulation of camalexin after inoculation with Pst in eds5NahG and sid2NahG plants . The effect of the pad4 mutation on the sid mutant phenotype was furthermore tested in eds5pad4 and sid2pad4 double mutants, and it was demonstrated that PAD4 acts epistatically to EDS5 and SID2 regarding the production of camalexin after inoculation with Pst . NahG plants and pad4 mutants were also found to produce less ethylene (ET) after infection with Pst in comparison to the wild type (WT) and sid mutants . Both PAD4 and NahG acted epistatically to SID regarding the Pst-dependent production of ET that was found to be necessary for the accumulation of camalexin . Early production of jasmonic acid (JA) 12 h after inoculation with Pst/avrRpt2 was absent in all plants expressing NahG compared to the other mutants tested here . These genetic studies unravel pleiotropic changes in defence signalling of NahG plants that are unlikely to result from their low SA content . This adds unexpected difficulties in the interpretation of earlier findings based solely on NahG plants. Plant J, 2003 Nov, 36(4), 485 - 99 Virulence systems of Pseudomonas syringae pv . tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway; Zhao Y et al.; Pseudomonas syringae pv . tomato strain DC3000 (Pst DC3000) causes bacterial speck disease on tomato . The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of the plant hormone jasmonic acid (JA) . We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type (WT) plants . Treatment of WT plants with exogenous methyl-JA (MeJA) complemented the virulence defect of a bacterial mutant deficient in COR production, but not a mutant defective in the type III secretion system . Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA and wound response genes that have been implicated in defense against herbivores . Concomitant with the induction of JA and wound response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected WT plants . Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound response genes . These results indicate that COR promotes bacterial virulence by activating the host's JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway in susceptible tomato plants. Clin Microbiol Infect, 2003 Aug, 9(8), 846 - 51 Infrequent detection of acquired metallo-beta-lactamases among carbapenem-resistant Pseudomonas isolates in a Greek hospital; Tsakris A et al.; OBJECTIVE: To study the possible distribution of metallo-beta-lactamases among nosocomial Pseudomonas isolates in a Greek hospital with a recent high prevalence of carbapenem-resistant Pseudomonas isolates . METHODS: All carbapenem-resistant (imipenem- and/or meropenem-resistant) (MICs > 8 mg/L) Pseudomonas non-replicate isolates recovered from clinical infections in the Microbiology Laboratory of Saint Demetrios Hospital, Thessaloniki, Greece, from April 1998 to November 2000 were studied for the presence of metallo-beta-lactamases . They were tested by a disk diffusion test, PCR analysis, and nucleotide sequencing . DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA . RESULTS: In total, 24 carbapenem-resistant isolates (23 P . aeruginosa and one P . putida) were recovered . The serotypes observed among the P . aeruginosa isolates were, in order of decreasing frequency, O:11 (52%), O:3 and O:12 (17% each), and O:6 (13%) . PFGE grouped 17 of the P . aeruginosa isolates into four clusters, each containing from two to seven isolates, while the remaining isolates exhibited unique genotypes . blaVIM-2 was detected in the P . putida isolate and a P . aeruginosa serotype O:3 isolate . The latter strain was genotypically distinct from other contemporaneous or older carbapenem-resistant P . aeruginosa Greek isolates . CONCLUSION: These findings suggest that, although the prevalence of metallo-beta-lactamases is low, the integron-associated blaVIM genes can spread to P . aeruginosa serotypes that have not been previously associated with carbapenem resistance in our region, as well as to other pseudomonal species. Planta, 2004 Feb, 218(4), 552 - 61 Epub 2003 Nov 12. PCC1: a merging point for pathogen defence and circadian signalling in Arabidopsis; Sauerbrunn N et al.; Using a cDNA-array we identified expressed sequence tag 163B24T7 as rapidly up-regulated in Arabidopsis thaliana (L.) Heynh . after pathogen exposure . Detailed expression analysis revealed that the corresponding gene is up-regulated not only after exposure to avirulent Pseudomonas syringae pv . tomato but also to virulent strains . This up-regulation is dependent on functional salicylic acid defence-signalling pathways . Moreover, we found the gene was circadian-regulated, showing peaks of expression at the end of the day . Using plants overexpressing the clock component CCA1, we showed that the PCC1 gene is regulated by the inner clock of Arabidopsis . Accordingly, we named the gene PCC1, for pathogen and circadian controlled . PCC1 is a member of a novel family of six small polypeptides in Arabidopsis . A functional role for PCC1 in plant defence was demonstrated since plants overexpressing PCC1 are resistant against normally virulent oomycetes . Thus, PCC1 demonstrates a potential interrelationship between pathogen and circadian signalling pathways. BMC Struct Biol . 2003 Nov 11;3(1):8. A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases; Wlodawer A et al.; BACKGROUND: Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases) . In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis . Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted . RESULTS: We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species . Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow . Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra), as well as in frogs (Xenopus tropicalis) . A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp . 101 sedolisin . CONCLUSION: CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles . The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes . This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated . This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2. Nucleosides Nucleotides Nucleic Acids, 2003 Oct, 22(10), 1939 - 52 Synthesis, protonation behavior, conformational analysis, and regioselective enzymatic acylation of the novel diamino analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU); Lavandera I et al.; (E)-3',5'-Diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU . The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy . This study allows the determination of the basicity constants and the stepwise protonation sites . Thus, the main species at physiological pH is the monoprotonated form . The conformational analysis of this nucleoside analogue was also carried out through 1H NMR spectroscopy . In addition, a convenient synthesis of N-3' and N-5' acylated derivatives was developed by regioselective enzymatic acylation . Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5'-amino group, thus furnishing nucleosides 8 . On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3'-amino group, thus affording derivatives 9. Structure (Camb), 2003 Nov, 11(11), 1413 - 22 The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral; Sewell BT et al.; The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique . The structure is a spiral comprising 14 subunits with 2-fold symmetry . Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits . Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope . The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly. Appl Environ Microbiol, 2003 Nov, 69(11), 6864 - 74 Development and application of a dapB-based in vivo expression technology system to study colonization of rice by the endophytic nitrogen-fixing bacterium Pseudomonas stutzeri A15; Rediers H et al.; Pseudomonas stutzeri A15 is a nitrogen-fixing bacterium isolated from paddy rice . Strain A15 is able to colonize and infect rice roots . This strain may provide rice plants with fixed nitrogen and hence promote plant growth . In this article, we describe the use of dapB-based in vivo expression technology to identify P . stutzeri A15 genes that are specifically induced during colonization and infection (cii) . We focused on the identification of P . stutzeri A15 genes that are switched on during rice root colonization and are switched off during free-living growth on synthetic medium . Several transcriptional fusions induced in the rice rhizosphere were isolated . Some of the corresponding genes are involved in the stress response, chemotaxis, metabolism, and global regulation, while others encode putative proteins with unknown functions or without significant homology to known proteins. Appl Environ Microbiol, 2003 Nov, 69(11), 6560 - 8 Population structure of Alexandrium (Dinophyceae) cyst formation-promoting bacteria in Hiroshima Bay, Japan; Adachi M et al.; A total of 31 bacterial isolates that have potential Alexandrium cyst formation-promoting activity (Alex-CFPB) were isolated from Hiroshima Bay (Japan), which is characterized by seasonal blooms of the toxic dinoflagellate Alexandrium tamarense . The population structure of Alex-CFPB was analyzed by means of restriction fragment length polymorphism analysis of the 16S rRNA genes (16S rDNA) . Fourteen ribotypes, A to N, were observed among the 31 isolates of Alex-CFPB by using four restriction enzymes, MboI, HhaI, RsaI and BstUI . Among them, seven isolates, which were obtained from the seawater samples taken during the peak and termination periods of the A . tamarense bloom in 1998, belonged to ribotype A . This result suggests that bacterial strains of ribotype A may be dominant in the Alex-CFPB assemblages during these periods . The partial 16S rDNA-based phylogenetic tree of 10 ribotypes studied showed that nine of them fell into the Rhodobacter group of the alpha subclass of the Proteobacteria: Eight of nine ribotypes of the Rhodobacter group fell into the lineage of the Roseobacter subgroup, and one fell into the Rhodobacter subgroup . The non-Rhodobacter group type fell into the Marinobacterium-Neptunomonas-Pseudomonas group of the gamma-Proteobacteria: Isolates of Alex-CFPB ribotypes A and C do not have clear growth-promoting activities but have strong cyst formation-promoting activities (CFPAs) under our laboratory conditions . These results show that the Alex-CFPB assemblage may consist of various bacteria that belong mainly to the Roseobacter group and have strong CFPAs . These results suggest that not only the Alexandrium cyst formation-inhibiting bacteria (Alex-CFIB) reported previously but also Alex-CFPB, especially bacteria of ribotype A, may play significant roles in the process of encystment and bloom dynamics of Alexandrium in the natural environment. Appl Environ Microbiol, 2003 Nov, 69(11), 6464 - 74 Low-temperature isolation of disease-suppressive bacteria and characterization of a distinctive group of pseudomonads; Johansson PM et al.; The influence of environmental factors during isolation on the composition of potential biocontrol isolates is largely unknown . Bacterial isolates that efficiently suppressed wheat seedling blight caused by Fusarium culmorum were found by isolating psychrotrophic, root-associated bacteria and by screening them in a bioassay that mimicked field conditions . The impact of individual isolation factors on the disease-suppressive index (DSI) of almost 600 isolates was analyzed . The bacteria originated from 135 samples from 62 sites in Sweden and Switzerland . The isolation factors that increased the probability of finding isolates with high DSIs were sampling from arable land, Swiss origin of samples, and origination of isolates from plants belonging to the family Brassicaceae . The colony morphology of the isolates was characterized and compared to DSIs, which led to identification of a uniform morphological group containing 57 highly disease-suppressive isolates . Isolates in this group were identified as Pseudomonas sp.; they were fluorescent on King's medium B and had characteristic crystalline structures in their colonies . These isolates were morphologically similar to seven strains that had previously been selected for suppression of barley net blotch caused by Drechslera teres . Members of this morphological group grow at 1.5 degrees C and produce an antifungal polyketide (2,3-deepoxy-2,3-didehydrorhizoxin {DDR}) . They have similar two-dimensional polyacrylamide gel electrophoresis protein profiles, phenotypic characteristics, and in vitro inhibition spectra of pathogens . In summary, in this paper we describe some isolation factors that are important for obtaining disease-suppressive bacteria in our system, and we describe a novel group of biocontrol pseudomonads. Mol Plant Microbe Interact, 2003 Nov, 16(11), 1003 - 12 Biocontrol traits of Pseudomonas spp . are regulated by phase variation; van den Broek D et al.; Of 214 Pseudomonas strains isolated from maize rhizosphere, 46 turned out to be antagonistic, of which 43 displayed clear colony phase variation . The latter strains formed both opaque and translucent colonies, designated as phase I and phase II, respectively . It appeared that important biocontrol traits, such as motility and the production of antifungal metabolites, proteases, lipases, chitinases, and biosurfactants, are correlated with phase I morphology and are absent in bacteria with phase II morphology . From a Tn5luxAB transposon library of Pseudomonas sp . strain PCL1171 phase I cells, two mutants exhibiting stable expression of phase II had insertions in gacS . A third mutant, which showed an increased colony phase variation frequency was mutated in mutS . Inoculation of wheat seeds with PCL1171 bacteria of phase I morphology resulted in efficient suppression of take-all disease, whereas disease suppression was absent with phase II bacteria . Neither the gacS nor the mutS mutant was able to suppress take-all, but biocontrol activity was restored after genetic complementation of these mutants . Furthermore, in a number of cases, complementation by gacS of wild-type phase II sectors to phase I phenotype could be shown . A PCL1171 phase I mutant defective in antagonistic activity appeared to have a mutation in a gene encoding a lipopeptide synthetase homologue and had lost its biocontrol activity, suggesting that biocontrol by strain PCL1171 is dependent on the production of a lipopeptide . Our results show that colony phase variation plays a regulatory role in biocontrol by Pseudomonas bacteria by influencing the expression of major biocontrol traits and that the gacS and mutS genes play a role in the colony phase variation process . Therefore phase variation not only plays a role in escaping animal defense but it also appears to play a much broader and vital role in the ecology of bacteria producing exoenzymes, antibiotics, and other secondary metabolites. Mol Plant Microbe Interact, 2003 Nov, 16(11), 962 - 72 Nitric oxide does not trigger early programmed cell death events but may contribute to cell-to-cell signaling governing progression of the Arabidopsis hypersensitive response; Zhang C et al.; Nitric oxide (NO) has been suggested to play a role in the hypersensitive response (HR) . Single- and double-label fluorescence microscopy experiments were conducted using Arabidopsis leaves infected with Pseudomonas syringae pv . tomato DC3000 carrying either avrB or avrRpt2 . Kinetics of NO production were followed by measurement of green 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) triazole fluorescence in leaves coinfiltrated with DAF-FM diacetate . Kinetics of hypersensitive cell death were followed by measurement of cytoplasmic red fluorescence following internalization of coinfiltrated propidium iodide through compromised plasma membranes . Neither NO accumulation nor cell death was seen until approximately 3 h postinoculation of Columbia leaves with DC3000.avrB or approximately 5.5 h post-inoculation with DC3000.avrRpt2 . Subsequent NO accumulation kinetics closely paralleled HR progression in both Columbia and ndr1-1 mutant plants . These data established that NO accumulation does not happen sufficiently early for NO to be a signaling component controlling HR triggering . NO accumulation did contribute to the HR, as proven by an approximately 1-h delay in cell death kinetics caused by an NO scavenger or an NO synthase inhibitor . NO was first seen as punctate foci at the cell surface . Subsequent NO accumulation patterns were consistent with NO being an intercellular signal that functions in cell-to-cell spread of the HR. Microbiology, 2003 Nov, 149(Pt 11), 3279 - 87 Characterization of a soil-derived bacterial consortium degrading 4-chloroaniline; Radianingtyas H et al.; A bacterial consortium comprising four different species was isolated from an Indonesian agricultural soil using a mixture of aniline and 4-chloroaniline (4CA) as principal carbon sources . The four species were identified as Chryseobacterium indologenes SB1, Comamonas testosteroni SB2, Pseudomonas corrugata SB4 and Stenotrophomonas maltophilia SB5 . Growth studies on aniline and 4CA as single and mixed substrates demonstrated that the bacteria preferred to grow on and utilize aniline rather than 4CA, although both compounds were eventually depleted from the culture supernatant . However, despite 100 % disappearance of the parent substrates, the degradation of 4CA was always characterized by incomplete dechlorination and 4-chlorocatechol accumulation . This result suggests that further degradation of 4-chlorocatechol may be the rate-limiting step in the metabolism of 4CA by the bacterial consortium . HPLC-UV analysis showed that 4-chlorocatechol was further degraded via an ortho-cleavage pathway by the bacterial consortium . This hypothesis was supported by the results from enzyme assays of the crude cell extract of the consortium revealing catechol 1,2-dioxygenase activity which converted catechol and 4-chlorocatechol to cis,cis-muconic acid and 3-chloro-cis,cis-muconic acid respectively . However, the enzyme had a much higher conversion rate for catechol {156 U (g protein)(-1)} than for 4-chlorocatechol {17.2 U (g protein)(-1)}, indicating preference for non-chlorinated substrates . Members of the bacterial consortium were also characterized individually . All isolates were able to assimilate aniline . P . corrugata SB4 was able to grow on 4CA solely, while S . maltophilia SB5 was able to grow on 4-chlorocatechol . These results suggest that the degradation of 4CA in the presence of aniline by the bacterial consortium was a result of interspecies interactions. Microbiology, 2003 Nov, 149(Pt 11), 3265 - 77 3- and 4-alkylphenol degradation pathway in Pseudomonas sp . strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase; Jeong JJ et al.; The enzymes and genes responsible for the catabolism of higher alkylphenols have not been characterized in aerobic bacteria . Pseudomonas sp . strain KL28 can utilize a wide range of alkylphenols, which include the 4-n-alkylphenols (C(1)-C(5)) . The genes, designated as lap (for long-chain alkylphenols), encoding enzymes for the catabolic pathway were cloned from chromosomal DNA and sequenced . The lap genes are located in a 13.2 kb region with 14 ORFs in the order lapRBKLMNOPCEHIFG and with the same transcriptional orientation . The lapR gene is transcribed independently and encodes a member of the XylR/DmpR positive transcriptional regulators . lapB, the first gene in the lap operon, encodes catechol 2,3-dioxygenase (C23O) . The lapKLMNOP and lapCEHIFG genes encode a multicomponent phenol hydroxylase (mPH) and enzymes that degrade derivatives of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates, respectively . The P(lapB) promoter contains motifs at positions -24(GG) and -12(GC) which are typically found in sigma(54)-dependent promoters . A promoter assay using a P(lapB) : : gfp transcriptional fusion plasmid showed that lapB promoter activity is inducible and that it responds to a wide range of (alkyl)phenols . The structural genes encoding enzymes required for this catabolism are similar (42-69 %) to those encoded on a catabolic pVI150 plasmid from an archetypal phenol degrader, Pseudomonas sp . CF600 . However, the lap locus does not include genes encoding HMS hydrolase and ferredoxin . The latter is known to be functionally associated with C23O for use of 4-alkylcatechols as substrates . The arrangement of the lap catabolic genes is not commonly found in other meta-cleavage operons . Substrate specificity studies show that mPH preferentially oxidizes 3- and 4-alkylphenols to 4-alkylcatechols . C23O preferentially oxidizes 4-alkylcatechols via proximal (2,3) cleavage . This indicates that these two key enzymes have unique substrate preferences and lead to the establishment of the initial steps of the lap pathway in strain KL28. J Bacteriol, 2003 Nov, 185(22), 6658 - 65 Flagellin glycosylation island in Pseudomonas syringae pv . glycinea and its role in host specificity; Takeuchi K et al.; The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv . tabaci and P . syringae pv . glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different . The reason for the difference seems to depend on the posttranslational modification of the flagellins . To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P . syringae pv . glycinea (glycosylation island); then defective mutants with mutations in these genes were generated . There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3 . orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively . Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes . In contrast, these mutants failed to cause symptoms on original host soybean leaves . These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species. EMBO J, 2003 Nov 3, 22(21), 5690 - 9 High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance; Lu R et al.; Virus-induced gene silencing was used to assess the function of random Nicotiana benthamiana cDNAs in disease resistance . Out of 4992 cDNAs tested from a normalized library, there were 79 that suppressed a hypersensitive response (HR) associated with Pto-mediated resistance against Pseudomonas syringae . However, only six of these clones blocked the Pto-mediated suppression of P.syringae growth . The three clones giving the strongest loss of Pto resistance had inserts corresponding to HSP90 and also caused loss of Rx-mediated resistance against potato virus X and N-mediated tobacco mosaic virus resistance . The role of HSP90 as a cofactor of disease resistance is associated with stabilization of Rx protein levels and could be accounted for in part by SGT1 and other cofactors of disease resistance acting as co-chaperones . This approach illustrates the potential benefits and limitations of RNA silencing in forward screens of gene function in plants. EMBO J, 2003 Nov 3, 22(21), 5679 - 89 Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein; Hubert DA et al.; The Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system . We demonstrate that specific mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function . These mutations do not affect the function of related disease resistance proteins . RPM1 associates with HSP90 in plant cells . The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones . Each associates with HSP90 in plant cells . Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors . Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes. FEMS Microbiol Lett, 2003 Oct 24, 227(2), 279 - 85 Strain PM2, a novel methylotrophic fluorescent Pseudomonas sp; Pacheco CC et al.; A novel bacterial strain, PM2, capable of growing on methanol, was isolated in alkaline conditions from a soil inoculum . This bacterium was characterized at the physiological, biochemical and molecular level . Based on biochemical and molecular data strain PM2 was classified as a novel member of the group of fluores