Pseudomonas

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 fluorescent pseudomonads . Evidence for the presence of a pyrroloquinoline quinone (PQQ)-linked alcohol dehydrogenase in this organism is presented . Strain PM2 is, to our knowledge, the first example of a methylotrophic Pseudomonas to be characterized in detail . This novel type of metabolism in Pseudomonas broadens even further the metabolic versatility for which this genus is renowned.

Planta, 2004 Feb, 218(4), 606 - 14 Epub 2003 Oct 30.
Cloning of soybean genes induced during hypersensitive cell death caused by syringolide elicitor; Hagihara T et al.; Syringolide elicitors produced by bacteria expressing Pseudomonas syringae pv . glycinea avirulence gene D (avrD) induce hypersensitive cell death (HCD) only in soybean (Glycine max {L.} Merr.) plants carrying the Rpg4 disease resistance gene . Employing a differential display method, we isolated 13 gene fragments induced in cultured cells of a soybean cultivar Harosoy (Rpg4) treated with syringolides . Several genes for isolated fragments were induced by syringolides in an rpg4 cultivar Acme as well as in Harosoy; however, the genes for seven fragments designated as SIH (for syringolide-induced/ HCD associated) were induced exclusively or strongly in Harosoy . cDNA clones for SIH genes were obtained from a cDNA library of Harosoy treated with syringolide . Several sequences are homologous to proteins associated with plant defense responses . The SIH genes did not respond to a non-specific beta-glucan elicitor, which induces phytoalexin accumulation but not HCD, suggesting that the induction of the SIH genes is specific for the syringolide-Harosoy interaction . HCD and the induction of SIH genes by syringolides were independent of H(2)O(2) . On the other hand, Ca(2+) was required for HCD and the induction of some SIH genes . These results suggest that the induction of SIH genes by syringolides could be activated through the syringolide-specific signaling pathway and the SIH gene products may play an important role(s) in the processes of HCD induced by syringolides.

Eur J Immunol, 2003 Nov, 33(11), 3080 - 90
Intrapulmonary targeting of RANTES/CCL5-responsive cells prevents chronic fungal asthma; Schuh JM et al.; Regulated upon activation in normal T cells, expressed, and secreted (RANTES)/CCL5 is abundantly expressed during atopic asthma, suggesting that it is an important mediator of this disease . The contribution of intrapulmonary RANTES/CCL5-sensitive cells during Aspergillus fumigatus-induced airway disease in mice was assessed in this study . The intranasal delivery of a chimeric protein comprised of RANTES/CCL5 and a truncated version of Pseudomonas exotoxin A (RANTES-PE38) significantly attenuated serum IgE, peribronchial eosinophilia, and airway hyperreactivity when it was administered from day 0 to 15 after intratracheal conidia challenge in A . fumigatus-sensitized mice but had little effect when delivered from day 15 to 30 after conidia challenge . Intranasal RANTES-PE38 treatment enhanced macrophage recruitment and accelerated fungal clearance in the lungs of RANTES-PE38-treated mice . These data reveal a major role for RANTES/CCL5 and its receptors in the development of fungal asthma yet reveal only a modest role in the chronic remodeling of the allergic airway in this disease.

Z Naturforsch {C}, 2003 Sep-Oct, 58(9-10), 740 - 5
A novel cyclopeptide from a bacterium associated with the marine sponge Ircinia muscarum; Mitova M et al.; A new cyclotetrapeptide 1, together with three known cyclopeptides were isolated from the exo-cellular extract of Pseudomonas sp . a bacterium associated with the sponge Ircinia muscarum . The structure of 1 was suggested on the basis of spectroscopic analytical data and chemical degradation.

Plant Cell, 2003 Nov, 15(11), 2636 - 46 Epub 2003 Oct 23.
A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1; Zhang Y et al.; Plants have evolved sophisticated defense mechanisms against pathogen infections, during which resistance (R) genes play central roles in recognizing pathogens and initiating defense cascades . Most of the cloned R genes share two common domains: the central domain, which encodes a nucleotide binding adaptor shared by APAF-1, certain R proteins, and CED-4 (NB-ARC), plus a C-terminal region that encodes Leu-rich repeats (LRR) . In Arabidopsis, a dominant mutant, suppressor of npr1-1, constitutive 1 (snc1), was identified previously that constitutively expresses pathogenesis-related (PR) genes and resistance against both Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 . The snc1 mutation was mapped to the RPP4 cluster . In snc1, one of the TIR-NB-LRR-type R genes contains a point mutation that results in a single amino acid change from Glu to Lys in the region between NB-ARC and LRR . Deletions of this R gene in snc1 reverted the plants to wild-type morphology and completely abolished constitutive PR gene expression and disease resistance . The constitutive activation of the defense responses was not the result of the overexpression of the R gene, because its expression level was not altered in snc1 . Our data suggest that the point mutation in snc1 renders the R gene constitutively active without interaction with pathogens . To analyze signal transduction pathways downstream of snc1, epistasis analyses between snc1 and pad4-1 or eds5-3 were performed . Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid (SA) synthesis, suggesting that snc1 activates both SA-dependent and SA-independent resistance pathways.

Cryo Letters, 2003 Sep-Oct, 24(5), 323 - 30
The effect of antifreeze proteins and poly(vinyl alcohol) on the nucleation of ice: a preliminary study; Holt CB; Three substances have been tested for ice nucleation inhibition . These were an antifreeze protein AFP III from the fish Macrozoarces americanus, an antifreeze glycoprotein AFGP from the fish Dissostichus mawsoni, and an 80% hydrolysed poly(vinyl alcohol) with a molecular weight of 9 to 10 kD . A nucleation spectrometer was used to test nucleation inhibition at a range of concentrations against two types of ice nuclei: those present in tap water and a bacterial nucleator from Pseudomonas syringae . The PVA reduced the nucleation temperature of tap water and the bacterial dispersions at all the concentrations which were tested . The AFGP reduced the nucleation temperature of tap water but enhanced the nucleation activity of the bacterial nucleators . At low concentrations the AFP III reduced the nucleation temperature of both tap water and the bacterial nucleator . At high concentrations the AFP III enhanced the nucleation temperature of the bacterial nucleator and broadened the nucleation spectrum of the tap water to encompass the nucleation spread of the control . The possible mechanisms of nucleation suppression and enhancement are discussed.

Hum Pathol, 2003 Sep, 34(9), 929 - 38
Pseudomonas pneumonia in infants: an autopsy study; Bonifacio SL et al.; Pseudomonas pneumonia is an uncommon but serious infection in infants, occurring mainly in infants of low birth weight . In this retrospective clinicopathologic correlation study, we reviewed the clinical records and analyzed postmortem lung pathology in 8 infants with pneumonia due to P . aeruginosa . From the histopathology, 2 different pneumonic patterns emerged: a distinctive paucicellular coagulative confluent bronchopneumonia with perivascular bacillary infiltration (7 cases) and a more usual cellular pneumonia without evidence of perivascular organisms (1 case) . Clinically, infants with the first type could be considered immunocompromised and had a precipitous course characterized by signs of sepsis, whereas the infant with the second type (who likely had a more normal immune system) had a relatively protracted course with respiratory failure . We conclude that (1) the pattern of pneumonic inflammation correlates with the immune state of infants, similar to what has been reported in adults; (2) among immunocompromised infants, histopathologic signs of bacteremia are prevalent; and (3) the clinical signs do not correlate with the severity of the pathology at autopsy and may reflect sepsis rather than pneumonia . We speculate that the histopathology in this population reflects the virulence of the organism, as well as the immune status of the host.

J Mol Biol, 2003 Oct 24, 333(3), 573 - 85
Tertiary structure of thiopurine methyltransferase from Pseudomonas syringae, a bacterial orthologue of a polymorphic, drug-metabolizing enzyme; Scheuermann TH et al.; In humans, the enzyme thiopurine methyltransferase (TPMT) metabolizes 6-thiopurine (6-TP) medications, including 6-thioguanine, 6-mercaptopurine and azathioprine, commonly used for immune suppression and for the treatment of hematopoietic malignancies . S-Methylation by TPMT prevents the intracellular conversion of these drugs into active 6-thioguanine nucleotides (6-TGNs) . Genetic polymorphisms in the TPMT protein sequence have been associated with decreased tissue enzymatic activities and an increased risk of life-threatening myelo-suppression from standard doses of 6-TP medications . Biochemical studies have demonstrated that TPMT deficiency is primarily associated with increased degradation of the polymorphic proteins through an ubiquitylation and proteasomal-dependent pathway . We have now determined the tertiary structure of the bacterial orthologue of TPMT from Pseudomonas syringae using NMR spectroscopy . Bacterial TPMT similarly catalyzes the S-adenosylmethionine (SAM)-dependent transmethylation of 6-TPs and shares 45% similarity (33% identity) with the human enzyme . Initial studies revealed an unstructured N terminus, which was removed for structural studies and subsequently determined to be required for enzymatic activity . Despite lacking sequence similarity to any protein of known three-dimensional structure, the tertiary structure of bacterial TPMT reveals a classical SAM-dependent methyltransferase topology, consisting of a seven-stranded beta-sheet flanked by alpha-helices on both sides . However, some deviations from the consensus topology, along with multiple insertions of structural elements, are evident . A review of the many experimentally determined tertiary structures of SAM-dependent methyltransferases demonstrates that such structural deviations from the consensus topology are common and often functionally important.

Eye Contact Lens, 2003 Oct, 29(4), 196 - 200
Ocular complications associated with the use of cosmetic contact lenses from unlicensed vendors; Steinemann TL et al.; PURPOSE: To call attention to the unauthorized sale of cosmetic contact lenses, resulting in ocular complications.DESIGN Observational case report . METHODS: Retrospective, observational, clinical practice setting . RESULTS: Six patients (five female and one male) were seen urgently for acute eye pain and redness after wearing cosmetic plano contact lenses . None of the patients had previously worn a contact lens or spectacle correction . None of the lenses were dispensed by eye care professionals . One patient developed pseudomonal keratitis, ultimately requiring penetrating keratoplasty for visual rehabilitation . CONCLUSIONS: Colored contact lenses are being dispensed without a prescription or fitting from unlicensed vendors, such as cosmetics, convenience, and accessory stores . Lenses are sold individually and without care instructions . Consequently, uninformed lens wearers are experiencing acute, vision-threatening infections and inflammation.

Plant Cell, 2003 Nov, 15(11), 2551 - 65 Epub 2003 Oct 10.
The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis; Mengiste T et al.; The molecular and cellular mechanisms involved in plant resistance to the necrotrophic fungal pathogen Botrytis cinerea and their genetic control are poorly understood . Botrytis causes severe disease in a wide range of plant species, both in the field and in postharvest situations, resulting in significant economic losses . We have isolated the BOS1 (BOTRYTIS-SUSCEPTIBLE1) gene of Arabidopsis based on a T-DNA insertion allele that resulted in increased susceptibility to Botrytis infection . The BOS1 gene is required to restrict the spread of another necrotrophic pathogen, Alternaria brassicicola, suggesting a common host response strategy against these pathogens . In the case of the biotrophic pathogens Pseudomonas syringae pv tomato and the oomycete parasite Peronospora parasitica, bos1 exhibits enhanced disease symptoms, but pathogen growth is similar in bos1 and wild-type plants . Strikingly, bos1 plants have impaired tolerance to water deficit, increased salinity, and oxidative stress . Botrytis infection induces the expression of the BOS1 gene . This increased expression is severely impaired in the coi1 mutant, suggesting an interaction of BOS1 with the jasmonate signaling pathway . BOS1 encodes an R2R3MYB transcription factor protein, and our results suggest that it mediates responses to signals, possibly mediated by reactive oxygen intermediates from both biotic and abiotic stress agents.

Appl Occup Environ Hyg, 2003 Nov, 18(11), 966 - 75
DNA-based methodologies for rapid detection, quantification, and species- or strain-level identification of respiratory pathogens (Mycobacteria and Pseudomonads) in metalworking fluids; Yadav JS et al.; Mycobacteria and pseudomonads occurring in modern metalworking fluids (MWF) have been implicated in occupational health hazards as causal agents for hypersensitivity pneumonitis (HP) and other respiratory illnesses in machine workers exposed to these fluids and their aerosols . Unlike the conventional cultural and biochemical methods, which are often slow and ambiguous and detect only culturable cells, DNA-based methods offer a time-saving alternative for reliable detection and identification of both culturable and nonculturable bacteria in MWF and for selective quantification of individual genera of pathogens of interest in these fluids . This is the first report on DNA-based direct detection of mycobacteria and pseudomonads in MWF without culturing . Genus-specific PCR approach was successfully applied for screening of field MWF samples originating from different industrial users for detection of mycobacteria or pseudomonads including both culturable and nonculturable cells . PCR in combination with amplicon DNA sequencing led to the identification of Mycobacterium chelonae, Pseudomonas nitroreducens, and an undefined Pseudomonas species from these fluids . Genome fingerprinting by pulsed-field gel electrophoresis (PFGE) on Mycobacterium isolates further showed that the isolates represented three strains of M . chelonae although the possibility of one of the strains being clonal with M . immunogenum cannot be excluded . In parallel efforts, a quantitative competitive PCR method developed based on the Pseudomonas-specific PCR was applied to quantify total P . fluorescens cells in contaminated metalworking fluid and MWF aerosol without culturing . The DNA-based protocols developed in this study will allow rapid screening of field MWF samples for the presence of both culturable and nonculturable cells and thus facilitate effective fluid management and timely exposure assessment.

FEMS Microbiol Lett, 2003 Sep 26, 226(2), 347 - 53
Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen; Kang SM et al.; Cell surface expression of protein has been widely used to display enzymes and antigens . Here we show that Pseudomonas syringae ice nucleation protein with a deletion of internal repeating domain (INC) can be used in Escherichia coli to display peptide in a conformationally active form on the outside of the folded protein by fusing to the C-terminus of INC . Diagnostic potential of this technology was demonstrated by effective mapping of antigenic epitopes derived from hepatitis C virus (HCV) core protein . Amino acids 1-38 and 26-53 of HCV core protein were found to react more sensitively in a native conformation with the HCV patient sera than commercial diagnostic antigen, c22p (amino acids 10-53) by display-ELISA . These results demonstrate that the bacterial cell surface display using INC is useful for peptide presentation and thus epitope mapping of antigen.

FEMS Microbiol Lett, 2003 Sep 26, 226(2), 251 - 5
Tn5060 from the Siberian permafrost is most closely related to the ancestor of Tn21 prior to integron acquisition; Kholodii G et al.; A Tn21-related mercury resistance transposon, Tn5060, has been isolated from Pseudomonas sp . strain A19-1 from a 8,000-10,000-year-old Siberian permafrost sample, and sequenced . Like Tn21, the element transposes to different plasmids at a frequency of 10(-2)-10(-3) per target plasmid transfer . Comparison of the complete Tn5060 DNA sequence (8,667 bp) with that of Tn21 (19,672 bp) shows that Tn5060 does not contain integron In2 and deviates from Tn21 in four nucleotide positions . These and other comparative data demonstrate that Tn5060 is the most closely related of the characterized mercury resistances to the as yet hypothetical immediate ancestor of Tn21, TnX.

J Biochem Mol Biol, 2003 Sep 30, 36(5), 433 - 41
Characterization of a salicylic acid- and pathogen-induced lipase-like gene in Chinese cabbage; Lee KA et al.; A cDNA clone for a salicylic acid-induced gene in Chinese cabbage (Brassica rapa subsp . pekinensis) was isolated and characterized . The cabbage gene, designated Br-sil1 (for Brassica rapa salicylate-induced lipase-like 1 gene), encodes a putative lipase that has the family II lipase motif GDSxxDxG around the active site serine . A database search showed that plant genomes have a large number of genes that contain the family II lipase motif . The lipase-like proteins include a myrosinase-associated protein, an anther-specific proline-rich protein APG, a pollen coat protein EXL, and an early nodule-specific protein . The Br-sil1 gene is strongly induced by salicylic acid and a nonhost pathogen, Pseudomonas syringae pv . tomato, that elicits a hypersensitive response in Chinese cabbage . Treatment of the cabbage leaves with BTH, methyl jasmonate, or ethephon showed that the Br-sil1 gene expression is induced by BTH, but not by methyl jasmonate or ethylene . This indicates that the cabbage gene is activated via a salicylic acid-dependent signaling pathway . An examination of the tissue-specific expression revealed that the induction of the Br-sil1 gene expression by BTH occurs in leaves and stems, but not in roots and flowers . Without the BTH treatment, however, the Br-sil1 gene is not expressed in any of the tissues that were examined.

J Org Chem, 2003 Oct 17, 68(21), 8136 - 41
Using a lipase as a high-throughput screening method for measuring the enantiomeric excess of allylic acetates; Onaran MB et al.; This report describes a high-throughput method for measuring the enantiomeric excess of allylic acetates . Such methods are useful tools for screening libraries of potential catalysts for enantioselective reactions . This technique, which is called EMDee for an enzymatic method for determining enantiomeric excess, uses the lipase from Pseudomonas cepacia to hydrolyze the (R) enantiomer of an allylic acetate, while the (S) enantiomer does not react . The rate of the reaction is monitored by measuring the acetic acid that is produced during the hydrolysis reaction with a pH indicator . Using the Michaelis-Menten equation, the rate of the reaction can be correlated with the concentration of the (R) enantiomer . This method can process 88 samples in less that 30 min.

Appl Environ Microbiol, 2003 Oct, 69(10), 5793 - 801
Conditional survival as a selection strategy to identify plant-inducible genes of Pseudomonas syringae; Marco ML et al.; A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of Pseudomonas syringae that are induced during growth on bean leaves . This strategy is based on the complementation of metXW, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of P . syringae on bean leaves exposed to conditions of low humidity . We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type P . syringae metXW genes . Only with an active promoter fused to metXW on pTrap did this plasmid restore methionine prototrophy to the P . syringae metXW mutant B7MX89 and survival of this strain on bean leaves . To test this method, a partial library of P . syringae genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves . This resulted in the enrichment of five clones, each with a unique RsaI restriction pattern of their DNA insert . Sequence analysis of these clones revealed those P . syringae genes for which putative plant-inducible activity could be assigned . Promoter activity experiments with a gfp reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium . Based on green fluorescent protein fluorescence levels, it appears that many P . syringae genes have relatively low expression levels and that the metXW HIRS strategy is a sensitive method to detect weakly expressed P . syringae genes that are active on plants . Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for P . syringae expressing metXW than exposed sites . Thus, the metXW HIRS strategy should lead to the identification of P . syringae genes that are expressed primarily in these areas on the leaf.

Acta Neurochir Suppl, 2003, 88, 105 - 11
Convection enhanced delivery of IL13-PE38QQR for treatment of recurrent malignant glioma: presentation of interim findings from ongoing phase 1 studies; Kunwar S; IL13PE38QQR is a recombinant toxin composed of the enzymatically-active portion of Pseudomonas Exotoxin A conjugated with human IL13 . Binding of IL13-PE38 to the IL13 receptor (IL13R) permits internalization of the recombinant toxin resulting in selective and potent cytoxicity at nanomolar concentrations . Normal brain tissue expresses little or no IL13R, but malignant gliomas overexpress IL13R conferring the selective cytotoxicity to the agent . Convection-enhanced delivery (CED), a novel direct drug delivery method to tumor and peritumoral region uses positive pressure infusion to generate a pressure gradient that optimizes distribution of macromolecules within the brain . Three phase I studies have been initiated to investigate IL13-PE38QQR as an anti-tumor agent for the treatment of patients with recurrent malignant gliomas . As of January 2003 a total of 46 patients have been treated . The presentation at the March 2003 EANS Local Therapy of Glioma meeting reflects adverse event findings through January 2003 and survival data through March 2003 . Intratumoral infusion with or without resection is fairly well-tolerated with corticosteroids prophylaxis particularly for patients with raised intracranial pressure . Post-resection infusion into the peritumoral brain parenchyma also appears to be very well tolerated . Histopathological tumor effect was seen at drug concentrations of 0.5-2.0 microg/mL . Although phase I studies do not focus on efficacy evaluation, prolonged survival times have been observed in this select population of patients . The preclinical data and details and preliminary results of the three clinical trials are reviewed.

Acta Neurochir Suppl, 2003, 88, 93 - 103
Local convection enhanced delivery of IL4-Pseudomonas exotoxin (NBI-3001) for treatment of patients with recurrent malignant glioma; Weber FW et al.; PURPOSE: This was an open-label, dose-escalation trial of intratumoral administration of IL-4 Pseudomonas Exotoxin (NBI-3001) in patients with recurrent malignant glioma . PATIENTS AND METHODS: A total of 31 patients with histologically verified supratentorial grade 3 and 4 astrocytoma were studied . Of these, twenty-five patients were diagnosed with glioblastoma multiforme (GBM) while six were diagnosed with anaplastic astrocytoma (AA) . Patients were over 18 years of age and had Karnofsky performance scores > or = 60 . Patients were assigned to one of four dose groups in a dose-escalation fashion: 6 microg/ml x 40 ml, 9 microg/ml x 40 ml, 15 microg/ml x 40 ml, or 9 microg/ml x 100 ml of NBI-3001 administered intratumorally via stereotactically placed catheters . Patients were followed with serial MRI scans and clinical assessments every four weeks for the first 16 weeks and then every eight weeks until week 26 . RESULTS: No drug-related systemic toxicity, as evident by lack of hematological or serum chemical changes, was apparent in any patients; treatment-related adverse effects were limited to the central nervous system . No deaths were attributable to treatment . Drug-related Grade 3 or 4 toxicity was seen in 39% of patients in all dose groups and 22% of patients at the maximum tolerated dose of 6 microg/ml x 40 ml . The overall median survival was 8.2 months with a median survival of 5.8 months for the GBM patients . Six-month survival was 52% and 48%, respectively . Gadolinium-enhanced magnetic resonance imaging of the brain showed areas of decreased signal intensity within the tumor consistent with tumor necrosis following treatment in many patients . CONCLUSIONS: NBI-3001 appears to have an acceptable safety and toxicity profile when administered intratumorally in patients with recurrent malignant glioma.

Hong Kong Med J, 2003 Oct, 9(5), 363 - 8
Thrombotic thrombocytopenic purpura as a rare complication in childhood systemic lupus erythematosus: case report and literature review; Chak WK et al.; Thrombotic thrombocytopenic purpura is a rare but serious condition in childhood . It can be idiopathic or a complication of other diseases or drug therapy . We report on a 12-year-old Chinese girl who presented with fulminant systemic lupus erythematosus with progressive renal failure, pancytopenia, and cerebral dysfunction due to thrombotic thrombocytopenic purpura . The patient also had Pneumocystis carinii pneumonia, Pseudomonas septicaemia, and Herpes zoster infections as a result of immunosuppressive treatment . She responded to combined therapy with pulse methylprednisolone, cyclophosphamide, plasmapheresis, and intensive care support, and completely recovered renal and neurological function . A review of the English-language medical literature since 1968 identified 20 other paediatric cases of systemic lupus erythematosus and thrombotic thrombocytopenic purpura . Clinical features, treatment, and outcome of these cases are presented and discussed . Early recognition is important, and although plasmapheresis is not of proven benefit in severe cases of systemic lupus erythematosus, it is life-saving in lupus-related thrombotic thrombocytopenic purpura and must be instituted early to avoid a poor outcome.

Biochemistry, 2003 Oct 14, 42(40), 11604 - 14
Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design; Woodyer R et al.; Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity . Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity . All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+) . The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant . Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein . HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate . Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+) . The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity . The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.

Syst Appl Microbiol, 2003 Sep, 26(3), 390 - 403
Polyphasic characterization of Pseudomonas stutzeri CLN100 which simultaneously degrades chloro- and methylaromatics: a new genomovar within the species; Garcia-Valdes E et al.; Strain CLN100 was isolated after enrichment on mineral medium with chloronaphthalene as the only carbon and energy source . It was able to use simultaneously and productively chloro- and methyl-derivatives of naphthalene and salicylate through a chromosomally encoded meta pathway . Phenotypic, chemotaxonomic and genotypic characterization classified strain CLN100 as a member of the species Pseudomonas stutzeri . DNA-DNA hybridizations, 16S rDNA, gyrB, rpoD sequences, and molecular fingerprinting indicate that strain CLN100 is a representative of a new genomovar (genomovar 10) within the species.

Mol Cell, 2003 Sep, 12(3), 603 - 13
Retroviral insertional mutagenesis identifies a small protein required for synthesis of diphthamide, the target of bacterial ADP-ribosylating toxins; Liu S et al.; Retroviral insertional mutagenesis was used to produce a mutant Chinese hamster ovary cell line that is completely resistant to several different bacterial ADP-ribosylating toxins . The gene responsible for toxin resistance, termed diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) sensitivity required gene 1 (DESR1), encodes two small protein isoforms of 82 and 57 residues . DESR1 is evolutionally conserved and ubiquitously expressed . Only the longer isoform is functional because the mutant cell line can be complemented by transfection with the long but not the short isoform . We demonstrate that DESR1 is required for the first step in the posttranslational modification of elongation factor-2 at His(715) that yields diphthamide, the target site for ADP ribosylation by DT and ETA . KTI11, the analog of DESR1 in yeast, which was originally identified as a gene regulating the sensitivity of yeast to zymocin, is also required for diphthamide biosynthesis, implicating DESR1/KTI11 in multiple biological processes.

Annu Rev Phytopathol, 2003, 41, 215 - 43
Molecular basis of Pto-mediated resistance to bacterial speck disease in tomato; Pedley KF et al.; The Pto gene in tomato confers gene-for-gene resistance to Pseudomonas syringae pv . tomato, the causative agent of bacterial speck disease . Pto was first introgressed from a wild species of tomato into cultivated tomato varieties over 60 years ago and is now widely used to control speck disease . Cloning of the Pto gene revealed that it encodes a cytoplasmically localized serine-threonine protein kinase . The molecular basis of gene-for-gene recognition in this pathosystem is the direct physical interaction of the Pto kinase with either of two Pseudomonas effector proteins, AvrPto and AvrPtoB . Upon recognition of AvrPto or AvrPtoB, the Pto kinase acts in concert with Prf, a leucine-rich repeat-containing protein, to activate multiple signal transduction pathways . There has been much progress in understanding the evolutionary origin of the Pto gene, structural details about how the Pto kinase interacts with AvrPto and AvrPtoB, signaling steps downstream of Pto, and defense responses activated by the Pto pathway . Future work on this model system will focus on how the interaction of the Pto kinase with bacterial effector proteins activates signal transduction, defining the specific role of signaling components, and ultimately, determining which host defense responses are most responsible for inhibiting growth of the pathogen and suppressing symptoms of bacterial speck disease.

Plant Physiol, 2003 Nov, 133(3), 1072 - 82 Epub 2003 Oct 02.
Cleavage of the Pseudomonas syringae type III effector AvrRpt2 requires a host factor(s) common among eukaryotes and is important for AvrRpt2 localization in the host cell; Jin P et al.; Many phytopathogenic bacteria use a type III secretion system to deliver type III effector proteins into the host plant cell . The Pseudomonas syringae type III effector AvrRpt2 is cleaved at a specific site when translocated into the host cell . In this study, we first demonstrate that the factor(s) required for AvrRpt2 cleavage is present in extracts from animal and yeast cells, as well as plant cells . The cleavage factor in animal and plant cell extracts was heat labile but relatively insensitive to protease inhibitors . Second, mutational analysis of AvrRpt2 was applied to identify features important for its cleavage . In addition to two of the amino acid residues in the immediate vicinity of the cleavage site, a large part of the region C-terminal to the cleavage site was required when AvrRpt2 was cleaved in animal cell extract . Most of these features were also important when AvrRpt2 was cleaved in plant cells . Third, we investigated the effect of cleavage in interactions of AvrRpt2 with plant cells . Cleavage of AvrRpt2 appeared to be important for proper interactions with Arabidopsis cells that lack the resistance gene product corresponding to AvrRpt2, RPS2 . In addition, removal of the region N-terminal to the cleavage site was important for the correct localization of the C-terminal effector region of the protein in the host cell . We speculate that the virulence function of AvrRpt2 requires removal of the N-terminal region to redirect the effector protein to a specific subcellular location in the host cell after translocation of the protein.

Blood, 2004 Apr 1, 103(7), 2718 - 26 Epub 2003 Oct 02.
Induction of caspase-dependent programmed cell death in B-cell chronic lymphocytic leukemia by anti-CD22 immunotoxins; Decker T et al.; B cells of chronic lymphocytic leukemia (CLL) are long-lived in vivo, possibly because of defects in apoptosis . We investigated BL22, an immunotoxin composed of the Fv portion of an anti-CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment . B cells from 22 patients with CLL were immunomagnetically enriched (96% purity) and were cultured with BL22 or an immunotoxin that does not recognize hematopoietic cells . The antileukemic activity of BL22 was correlated with CD22 expression, as determined by flow cytometry . BL22 induced caspase-9 and caspase-3 activation, poly(adenosine diphosphate {ADP}-ribose)polymerase (PARP) cleavage, DNA fragmentation, and membrane flipping . Cell death was associated with the loss of mitochondrial membrane potential and the down-regulation of Mcl-1 and X-chromosomal inhibitor of apoptosis protein (XIAP) . Furthermore, BL22 induced a proapoptotic 18-kDa Bax protein and conformational changes of Bax . Z-VAD.fmk abrogated apoptosis, confirming that cell death was executed by caspases . Conversely, interleukin-4, a survival factor, inhibited spontaneous death in culture but failed to prevent immunotoxin-induced apoptosis . BL22 cytotoxicity was markedly enhanced when combined with anticancer drugs including vincristine . We also investigated HA22, a newly engineered immunotoxin, in which BL22 residues are mutated to improve target binding . HA22 was more active than BL22 . In conclusion, these immunotoxins induce caspase-mediated apoptosis involving mitochondrial damage . Combination with chemotherapy is expected to improve the efficacy of immunotoxin treatment.

Microbiology, 2003 Oct, 149(Pt 10), 2891 - 900
Clonal population structure of Pseudomonas avellanae strains of different origin based on multilocus enzyme electrophoresis; Scortichini M et al.; To assess the genetic diversity and genetic relationships of Pseudomonas avellanae, the causative agent of hazelnut decline, a total of 102 strains, obtained from central Italy (provinces of Viterbo and Rome) and northern Greece, were studied using multilocus enzyme electrophoresis (MLEE) . Their allelic variation in 10 loci was determined . All loci were polymorphic and 53 electrophoretic types (ETs) were identified from the total sample . The mean genetic diversity (H) was 0.65 and this value ranged from 0.37 for the least polymorphic to 0.82 for the most polymorphic locus . The dendrogram originated from MLEE data indicated two main groups of ETs, A and B . The groups do not appear to be correlated to the geographic origin of the strains, although all the ETs from northern Greece clustered into subgroup B1 . Pseudomonas syringae pv . actinidiae and P . syringae pv . theae, included in the analysis as outgroups, clustered apart . The index of association (I(A)) for P . avellanae was 0.90 . The I(A) values were always significantly different from zero for the population subsets studied and no epidemic structure was found . These results would indicate that the population structure of P . avellanae is clonal either in northern Greece or in central Italy . The recent outbreaks of the bacterium in new areas of hazelnut cultivation would explain the current clonal structure that is persisting over decades.

J Biol Chem, 2003 Dec 19, 278(51), 51796 - 805 Epub 2003 Sep 30.
Activation of the fatty acid alpha-dioxygenase pathway during bacterial infection of tobacco leaves . Formation of oxylipins protecting against cell death; Hamberg M et al.; A pathogen-induced oxygenase showing homology to prostaglandin endoperoxide synthases-1 and -2 was recently characterized by in vitro experiments as a fatty acid alpha-dioxygenase catalyzing formation of unstable 2(R)-hydroperoxy fatty acids . To study the activity of this enzyme under in vivo conditions and to elucidate the fate of enzymatically produced 2-hydroperoxides, leaves of tobacco were analyzed for the presence of alpha-dioxygenase-generated compounds as well as for lipoxygenase (LOX) products and free fatty acids . Low basal levels of 2-hydroxylinolenic acid (0.4 nmol/g leaves fresh weight) and 8,11,14-heptadecatrienoic acid (0.1 nmol/g) could be demonstrated . These levels increased strongly upon infection with the bacterium Pseudomonas syringae pv syringae (548 and 47 nmol/g, respectively) . Transgenic tobacco plants overexpressing alpha-dioxygenase were developed, and incompatible infection of such plants led to a dramatic elevation of 2-hydroxylinolenic acid (1778 nmol/g) and 8,11,14-heptadecatrienoic acid (86 nmol/g), whereas the levels of LOX products were strongly decreased . Further analysis of oxylipins in infected leaves revealed the presence of a number of 2-hydroxy fatty acids differing with respect to chain length and degree of unsaturation as well as two new doubly oxygenated oxylipins identified as 2(R),9(S)-dihydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 2(R),9(S)-dihydroxy-10(E),12(Z)-octadecadienoic acid . alpha-Dioxygenase-generated 2-hydroxylinolenic acid, and to a lesser extent lipoxygenase-generated 9-hydroxyoctadecatrienoic acid, exerted a tissue-protective effect in bacterially infected tobacco leaves.

Microbiol Res, 2003, 158(3), 265 - 70
Interaction between gfp-tagged Pseudomonas tolaasii P12 and Pleurotus eryngii; Russo A et al.; Pseudomonas sp., (formerly reported as strain P12) which produces brown blotch disease symptoms on Pleurotus eryngii, has been identified as P . tolaasii based on its biochemical, physiological properties and 16S rDNA sequence analysis . This pathogen is able to infect basidiocarps when surface-inoculated on mushroom casing soil . However, infected basidiocarps develop the brown blotch disease symptoms when the pathogen concentration in the fruiting body tissues is higher than 10(4) cfu/g d.w . Using gfp-tagged cells and confocal laser scanning microscopy, it was possible to show that the pathogen has the ability to tightly attach to the hyphae of Pleurotus eryngii.

Biosci Biotechnol Biochem, 2003 Sep, 67(9), 1970 - 5
The dual functions of biphenyl-degrading ability of Pseudomonas sp . KKS102: energy acquisition and substrate detoxification; Delawary M et al.; The bph operon of Pseudomonas sp . KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation . Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner . We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity . Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation . The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl . These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.

Biotechnol Lett, 2003 Aug, 25(15), 1255 - 61
Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676; Shintani M et al.; The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed > 99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1 . Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.

J Ind Microbiol Biotechnol, 2003 Sep, 30(9), 561 - 7 Epub 2003 Aug 30.
Characterisation of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid; Smejkal CW et al.; The aim of this study was to enrich and characterise bacterial consortia from soils around a herbicide production plant through their capability to degrade the herbicides 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) and 4-(4-chloro-2-methylphenoxy) butyric acid (MCPB) . Partial 16S rRNA gene sequencing revealed members of the genera Stenotrophomonas, Brevundimonas, Pseudomonas, and Ochrobactrum in the 2,4-DB- and MCPB-degrading communities . The degradation of 2,4-DB and MCPB was facilitated by the combined activities of the community members . Some of the members were able to utilise other herbicides from the family of chlorophenoxyalkanoic acids . During degradation of 2,4-DB and MCPB, phenol intermediates were detected, indicating ether cleavage of the side chain as the initial step responsible for the breakdown . This was also verified using an indicator medium . Repeated attempts to amplify putatively conserved tfd genes by PCR indicated the absence of tfd genes among the consortia members . First step cleavage of the chlorophenoxybutyric acid herbicides is by ether cleavage in bacteria and is encoded by divergent or different tfd gene types . The isolation of mixed cultures capable of degrading 2,4-DB and MCPB will aid future investigations to determine both the metabolic route for dissimilation and the fate of these herbicides in natural environments.

Plant Cell, 2003 Oct, 15(10), 2408 - 20 Epub 2003 Sep 24.
ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response; Lu H et al.; The previously reported Arabidopsis dominant gain-of-function mutant accelerated cell death6-1 (acd6-1) shows spontaneous cell death and increased disease resistance . acd6-1 also confers increased responsiveness to the major defense signal salicylic acid (SA) . To further explore the role of ACD6 in the defense response, we cloned and characterized the gene . ACD6 encodes a novel protein with putative ankyrin and transmembrane regions . It is a member of one of the largest uncharacterized gene families in higher plants . Steady state basal expression of ACD6 mRNA required light, SA, and an intact SA signaling pathway . Additionally, ACD6 mRNA levels were increased in the systemic, uninfected tissue of Pseudomonas syringae-infected plants as well as in plants treated with the SA agonist benzothiazole (BTH) . A newly isolated ACD6 loss-of-function mutant was less responsive to BTH and upon P . syringae infection had reduced SA levels and increased susceptibility . Conversely, plants overexpressing ACD6 showed modestly increased SA levels, increased resistance to P . syringae, and BTH-inducible and/or a low level of spontaneous cell death . Thus, ACD6 is a necessary and dose-dependent activator of the defense response against virulent bacteria and can activate SA-dependent cell death.

Plant Cell, 2003 Oct, 15(10), 2383 - 98 Epub 2003 Sep 24.
Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase; Nandi A et al.; A loss-of-function mutation in the Arabidopsis SSI2/FAB2 gene, which encodes a plastidic stearoyl-acyl-carrier protein desaturase, has pleiotropic effects . The ssi2 mutant plant is dwarf, spontaneously develops lesions containing dead cells, accumulates increased salicylic acid (SA) levels, and constitutively expresses SA-mediated, NPR1-dependent and -independent defense responses . In parallel, jasmonic acid-regulated signaling is compromised in the ssi2 mutant . In an effort to discern the involvement of lipids in the ssi2-conferred developmental and defense phenotypes, we identified suppressors of fatty acid (stearoyl) desaturase deficiency (sfd) mutants . The sfd1, sfd2, and sfd4 mutant alleles suppress the ssi2-conferred dwarfing and lesion development, the NPR1-independent expression of the PATHOGENESIS-RELATED1 (PR1) gene, and resistance to Pseudomonas syringae pv maculicola . The sfd1 and sfd4 mutant alleles also depress ssi2-conferred PR1 expression in NPR1-containing sfd1 ssi2 and sfd4 ssi2 plants . By contrast, the sfd2 ssi2 plant retains the ssi2-conferred high-level expression of PR1 . In parallel with the loss of ssi2-conferred constitutive SA signaling, the ability of jasmonic acid to activate PDF1.2 expression is reinstated in the sfd1 ssi2 npr1 plant . sfd4 is a mutation in the FAD6 gene that encodes a plastidic omega6-desaturase that is involved in the synthesis of polyunsaturated fatty acid-containing lipids . Because the levels of plastid complex lipid species containing hexadecatrienoic acid are depressed in all of the sfd ssi2 npr1 plants, we propose that these lipids are involved in the manifestation of the ssi2-conferred phenotypes.

Curr Microbiol, 2003 Aug, 47(2), 125 - 8
Mercury decreases culturability of Pseudomonas frederiksbergensis JAJ 28 in soil microcosms; Johnsen K et al.; Mercury is a biologically potent heavy metal, which has been found to change the diversity of culturable bacteria . Therefore, we investigated whether Hg kills bacteria in soil or reduces culturability . Soil microcosms were inoculated with Pseudomonas frederiksbergensis JAJ 28 and were sampled regularly during 28 days . The total number of acridine orange-stained cells was relatively constant, and Hg reduced the number on only one sampling day . However, the fraction of culturable cells on 1/10 tryptic soy agar was lowered on days 6, 13, and 21 . The number of microcolony forming units, which represents viable cells, was also affected by Hg, but this effect was delayed compared with the effects on CFUs . The amount of headspace CO2 per cell was overall increased by Hg, another indication of the toxic effects of Hg on the bacterial cells . Our results thus emphasize the need to take culturability into account when studying the effects of heavy metals on bacterial diversity.

J Biol Chem, 2003 Dec 5, 278(49), 48674 - 83 Epub 2003 Sep 23.
Mechanistic characterization of a bacterial malonate semialdehyde decarboxylase: identification of a new activity on the tautomerase superfamily; Poelarends GJ et al.; Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde . The enzyme is found in a degradative pathway for the xenobiotic nematocide trans-1,3-dichloropropene . MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro1-(X)8 -Gly-Arg11-X-Asp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily . Sequence analysis identified Pro1 and Arg75 as potential active site residues that might be involved in the MSAD activity . The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily . MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily . Malonate semialdehyde is analogous to a beta-keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions . A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme . The site of modification is Pro1 . These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism . Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro1 and the conserved arginine in all tautomerase superfamily members characterized thus far, two mechanistic scenarios are proposed for the MSAD-catalyzed reaction in which Pro1 and Arg75 play prominent roles.

Bioprocess Biosyst Eng, 2003 Mar, 25(5), 279 - 84 Epub 2003 Feb 04.
Hydrolases in supercritical CO2 and their use in a high-pressure membrane reactor; Knez Z et al.; The thermal stability and activity of enzymes in supercritical carbon dioxide (SC CO(2)) and near-critical propane were studied at a pressure of 300 bar in the temperature range 20-90 degrees C . Proteinase from Carica papaya was incubated in microaqueous SC CO(2) at atmospheric pressure in a nonaqueous system . Lipase stability in an aqueous medium at atmospheric pressure and in SC CO(2) as well as near-critical propane at 100 bar and 40 degrees C was studied . In order to investigate the impact of solvent on lipases, these were chosen from different sources: Pseudomonas fluorescences, Rhizpous javanicus, Rhizopus niveus and porcine pancreas . On the basis of our previous study on lipase activities in dense gases, a high-pressure continuous flat-shape membrane reactor was designed . The hydrolysis of sunflower oil in SC CO(2) was performed as a model reaction in this reactor . The reaction was catalyzed by the lipase preparation Lipolase 100T and was performed at 50 degrees C and 200 bar.

Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11777 - 82 Epub 2003 Sep 22.
HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in Arabidopsis; Takahashi A et al.; RAR1 and its interacting partner SGT1 play a central role in plant disease resistance triggered by a number of resistance (R) proteins . We identified cytosolic heat shock protein 90 (HSP90), a molecular chaperone, as another RAR1 interacting protein by yeast two-hybrid screening . RAR1 interacts with the N-terminal half of HSP90 that contains the ATPase domain . HSP90 also specifically interacts with SGT1 that contains a tetratricopeptide repeat motif and a domain with similarity to the cochaperone p23 . In Arabidopsis, the HSP90 inhibitor geldanamycin reduces the hypersensitive response and abolishes resistance triggered by the R protein RPS2 against Pseudomonas syringae pv . tomato DC3000 (avrRpt2) . One of four Arabidopsis cytosolic HSP90 isoforms, AtHSP90.1 is required for full RPS2 resistance and is rapidly induced upon pathogen challenge . We propose that RAR1 and SGT1 function closely with HSP90 in chaperoning roles that are essential for disease resistance.

Biochemistry, 2003 Sep 30, 42(38), 11263 - 71
Complex interactions of carbon monoxide with reduced cytochrome cbb3 oxidase from Pseudomonas stutzeri; Pitcher RS et al.; Cytochrome cbb(3) oxidase, from Pseudomonas stutzeri, contains a total of five hemes, two of which, a b-type heme in the active site and a hexacoordinate c-type heme, can bind CO in the reduced state . By comparing the cbb(3) oxidase complex and the isolated CcoP subunit, which contains the ligand binding bishistidine-coordinated c-type heme, we have deconvoluted the contribution made by each center to CO binding . A combination of rapid mixing and flash photolysis experiments, coupled with computer simulations, reveals the kinetics of the reaction of c-type heme with CO to be complex as a result of the need to displace an endogenous axial ligand, a property shared with nonsymbiotic plant hemoglobins and some heme-based gas sensing domains . The recombination of CO with heme b(3), unlike all other heme-copper oxidases, including mitochondrial cytochrome c oxidase, is independent of ligand concentration . This observation suggests a very differently organized dinuclear center in which CO exchange between Cu(B) and heme b(3) is significantly enhanced, perhaps reflecting an important determinant of substrate affinity.

Res Microbiol, 2003 Sep, 154(7), 521 - 6
Molecular cloning of the carboxylesterase gene and biochemical characterization of the encoded protein from Pseudomonas citronellolis ATCC 13674; Chao YP et al.; A genomic library of Pseudomonas citronellolis ATCC 13674 was constructed and screened for esterase activity in Escherichia coli using tributyrin-containing medium . One positive transformant was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.1-kb DNA fragment potentially carrying an esterase gene . The deduced nucleotide sequence of the DNA was found to contain an open reading frame encoding carboxylesterase and designated estA . Amino acid sequence analysis of estA showed the serine conservative motif, GDSAG, located between residues 208 and 212 . Together with Ser, residues 310 and 334 corresponding to aspartic acid and histidine, respectively, comprised the catalytic triad . With the aid of immobilized metal ion affinity chromatography, the carboxylesterase fused with poly His at its C-terminus was purified and shown to be strongly inhibited by the tryptophan modifier and mercuric ion, indicating the important role of conservative Trp (189) and cysteine (152 and/or 183) residues in maintaining the structural integrity of the protein . Further analyses showed that the carboxylesterase functioned optimally at 37-40 degrees C with pH ranging between 8 and 9 and displayed a broad substrate spectrum . The protein exhibited greater preference toward short-chain (C2-C4) than medium- and long-chain fatty acids . Higher substrate specificity on para-nitrophenol butyrate was observed in comparison with para-nitrophenol acetate as indicated by the higher kcat/Km value of the former.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1461 - 9
Fluorescent pseudomonads associated with the phyllosphere of grasses; Pseudomonas trivialis sp . nov., Pseudomonas poae sp . nov . and Pseudomonas congelans sp . nov; Behrendt U et al.; Strains of fluorescent pseudomonads, isolated from the phyllosphere of grasses, were analysed by a polyphasic approach in order to clarify their interspecific position . Classification on the basis of ribotyping revealed six genotypes; four of these, which could be differentiated clearly from each other and from Pseudomonas species with validly published names on the basis of phenotypic features, were chosen for detailed phylogenetic analysis . DNA-DNA hybridization studies among representative strains of the four genotypes and closely related Pseudomonas species, determined by comparison of 16S rDNA sequences, showed that three of the studied ribotypes represented novel species . Two of them were related to mainly saprophytic fluorescent pseudomonads and could be easily distinguished by a negative arginine dihydrolase reaction . One ribotype, also characterized by a negative arginine dihydrolase reaction, was closely related to potentially plant-pathogenic fluorescent pseudomonads and differed in certain phenotypic features from its phylogenetic neighbours . As a consequence of the phenotypic and phylogenetic analyses, Pseudomonas trivialis sp . nov . (type strain: P 513/19(T)=DSM 14937(T)=LMG 21464(T)), Pseudomonas poae sp . nov . (type strain: P 527/13(T)=DSM 14936(T)=LMG 21465(T)) and Pseudomonas congelans sp . nov . (type strain: P 538/23(T)=DSM 14939(T)=LMG 21466(T)) are proposed.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1357 - 61
Transfer of Pseudomonas elongata Humm 1946 to the genus Microbulbifer as Microbulbifer elongatus comb . nov; Yoon JH et al.; Phylogenetic analysis based on 16S rDNA sequences revealed that Pseudomonas elongata Humm 1946 is more closely related to the genus Microbulbifer than to authentic pseudomonads . The type strain of P . elongata (DSM 6810(T)) exhibited 16S rDNA similarity levels of 97.5 and 98.2 % to the type strains of Microbulbifer hydrolyticus and Microbulbifer salipaludis, respectively, but of less than approximately 92 % to Pseudomonas species with known 16S rDNA sequences . Respiratory lipoquinone and cellular fatty acid analyses showed that the type strain of P . elongata has characteristics similar to those of the genus Microbulbifer, not those of the genus PSEUDOMONAS: P . elongata DSM 6810(T) contained ubiquinone-8 as the predominant respiratory lipoquinone and iso-C(15 : 0) as the major fatty acid . DNA-DNA relatedness data indicate that P . elongata is a species distinct from M . hydrolyticus and M . salipaludis . Therefore, on the basis of these data, P . elongata Humm 1946 should be transferred to the genus Microbulbifer as Microbulbifer elongatus comb . nov.

J Biol Chem, 2003 Nov 21, 278(47), 46601 - 6 Epub 2003 Sep 15.
Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum; Bard F et al.; The tyrosine kinase Src is present on the Golgi membranes . Its role, however, in the overall function and organization of the Golgi apparatus is unclear . We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed . The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells . Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum . Other Golgi-specific marker proteins were not affected under these conditions . Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src . Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src . Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition . We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.

Mol Plant Microbe Interact, 2003 Sep, 16(9), 817 - 26
Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes; Ashfield T et al.; Alleles or tightly linked genes at the soybean (Glycine max L . Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv . glycinea that express the avirulence genes avrB or avrRpm1 . We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F . Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases . Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region . Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine . Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency . At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.

Am J Physiol Heart Circ Physiol, 2004 Jan, 286(1), H340 - 5 Epub 2003 Sep 11.
Effect of nitric oxide on capillary hemodynamics and cell injury in the pancreas during Pseudomonas pneumonia-induced sepsis; Tribl B et al.; Sepsis-induced nitric oxide (NO) overproduction has been implicated in a redistribution of flow from the pancreas making it vulnerable to ischemic injury in septic shock . To test this hypothesis in a remote injury model of normotensive sepsis, we induced Pseudomonas pneumonia in the rat and used intravital video microscopy (IVVM) of the pancreas to measure functional capillary density, capillary hemodynamics {red blood cell (RBC) velocity, lineal density, and supply rate}, and lethal cellular damage (propidium iodine staining) at 6 and 24 h after the induction of pneumonia . With pneumonia, plasma nitrite/nitrate {NO2(-)/NO3(-)(NOx(-))} levels were doubled by 21 h (P < 0.05) . To assess the effect of NO overproduction on microvascular perfusion, N6-(1-iminoethyl)-L-lysine (L-NIL) was administered to maintain NOx(-) levels at baseline . Pneumonia did cause a decrease in RBC velocity of 23% by 6 h, but by 24 h RBC velocity and supply rate had increased relative to sham by 22 and 38%, respectively (P < 0.05) . L-NIL treatment demonstrated that this increase was due to NO overproduction . With pneumonia, there was no change in functional capillary density and only modest increases in cellular damage . We conclude that, in this normotensive pneumonia model of sepsis, NO overproduction was protective of microvascular perfusion in the pancreas.

Int J Oncol, 2003 Oct, 23(4), 1179 - 86
The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA' suppresses growth of a highly metastatic pancreatic carcinoma cell line; Bruell D et al.; Pancreatic carcinoma still has the highest mortality rate in comparison to any other malignancy . Major reasons are late detection of disease, highly aggressive tumor growth and the early formation of metastases . Thus, novel effective therapies are urgently needed to improve the outcome of the patients . Overexpression of the epidermal growth factor receptor (EGFR) and its ligands has been implicated in the oncogenesis of pancreatic carcinoma and associated with an unfavorable prognosis . Consequently, the EGFR represents a specific target antigen suitable for immunotherapy . We generated a recombinant immunotoxin by fusing the anti-EGFR single chain fragment 425(scFv) to a truncated mutant of Pseudomonas Exotoxin A (ETA') . Using the expression vector pBM1.1, functional 425(scFv)-ETA' was periplasmically expressed under osmotic stress conditions in the presence of compatible solutes . The 72 kDa His10-tagged fusion protein was purified by a combination of metal-ion affinity and molecular size chromatography . Binding activity and specificity of the immunotoxin to the EGFR-positive pancreatic carcinoma cell line L3.6pl was confirmed by flow cytometry and ELISA . Finally, 425(scFv)-ETA' showed significant toxicity toward this cell line reaching 50% inhibition of cell proliferation at a concentration (IC50) of 7.5 ng/ml . This is the first report documenting the specific cytotoxicity of a recombinant immunotoxin towards metastatic pancreatic carcinoma cells, suggesting that EGFR-specific antibody toxins may become valuable therapeutic reagents for the treatment of pancreatic carcinoma.

Cryobiology, 2003 Aug, 47(1), 21 - 9
Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis; Palmfeldt J et al.; The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated . P . chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium . Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%) . Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration . The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium . The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml . For cell concentrations outside this window more than 10 times lower survival values were observed . P . chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation . Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance . By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.

Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10842 - 7 Epub 2003 Sep 05.
Local Context Finder (LCF) reveals multidimensional relationships among mRNA expression profiles of Arabidopsis responding to pathogen infection; Katagiri F et al.; A major task in computational analysis of mRNA expression profiles is definition of relationships among profiles on the basis of similarities among them . This is generally achieved by pattern recognition in the distribution of data points representing each profile in a high-dimensional space . Some drawbacks of commonly used pattern recognition algorithms stem from their use of a globally linear space and/or limited degrees of freedom . A pattern recognition method called Local Context Finder (LCF) is described here . LCF uses nonlinear dimensionality reduction for pattern recognition . Then it builds a network of profiles based on the nonlinear dimensionality reduction results . LCF was used to analyze mRNA expression profiles of the plant host Arabidopsis interacting with the bacterial pathogen Pseudomonas syringae . In one case, LCF revealed two dimensions essential to explain the effects of the NahG transgene and the ndr1 mutation on resistant and susceptible responses . In another case, plant mutants deficient in responses to pathogen infection were classified on the basis of LCF analysis of their profiles . The classification by LCF was consistent with the results of biological characterization of the mutants . Thus, LCF is a powerful method for extracting information from expression profile data.

J Theor Biol, 2003 Oct 21, 224(4), 437 - 49
Deciphering environmental signal integration in sigma54-dependent promoters with a simple mathematical model; Van Dien SJ et al.; A mathematical model was developed to describe the physiological co-regulation of two Pseudomonas sigma54-dependent promoter/regulator systems, Pu/XylR and Po/DmpR of Pseudomonas strains mt2 and CF600, respectively . Five ordinary differential equations and six algebraic equations were developed to describe the following processes of transcription initiation: binding of the activator protein to the upstream activating sequence, union of the sigma factor with the core polymerase, formation of the open complex, and escape of the transcription machinery from the promoter region . In addition, growth-phase control of the integration host factor (IHF), sigma-70 regulation during stationary phase, and the contribution of (p)ppGpp to both sigma factor selectivity and promoter escape were hypothesized . By including any three of these four effects, the model predicted that expression from both promoters is repressed during exponential growth and sharply increases as the cells enter stationary phase . The difference in behavior of the two systems during overexpression of either sigma54 or (p)ppGpp could be explained by different values of two model parameters . To accurately represent the behavior of both promoters in (p)ppGpp null strains, an additional parameter must be varied . Although numerical data available for this system is scarce, the model has proved useful for helping to interpret the experimental observations and to evaluate four hypotheses that have been proposed to explain the phenomenon of exponential silencing.

J Ind Microbiol Biotechnol . 2003 Aug 30; {Epub ahead of print}
Characterisation of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid; Smejkal CW et al.; The aim of this study was to enrich and characterise bacterial consortia from soils around a herbicide production plant through their capability to degrade the herbicides 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) and 4-(4-chloro-2-methylphenoxy) butyric acid (MCPB) . Partial 16S rRNA gene sequencing revealed members of the genera Stenotrophomonas, Brevundimonas, Pseudomonas, and Ochrobactrum in the 2,4-DB- and MCPB-degrading communities . The degradation of 2,4-DB and MCPB was facilitated by the combined activities of the community members . Some of the members were able to utilise other herbicides from the family of chlorophenoxyalkanoic acids . During degradation of 2,4-DB and MCPB, phenol intermediates were detected, indicating ether cleavage of the side chain as the initial step responsible for the breakdown . This was also verified using an indicator medium . Repeated attempts to amplify putatively conserved tfd genes by PCR indicated the absence of tfd genes among the consortia members . First step cleavage of the chlorophenoxybutyric acid herbicides is by ether cleavage in bacteria and is encoded by divergent or different tfd gene types . The isolation of mixed cultures capable of degrading 2,4-DB and MCPB will aid future investigations to determine both the metabolic route for dissimilation and the fate of these herbicides in natural environments.

Lung, 2003, 181(2), 79 - 88
Proteolysis of surfactant protein D by cystic fibrosis relevant proteases; von Bredow C et al.; Surfactant protein D (SP-D) is an important innate host defense molecule that has been shown to interact with cystic fibrosis (CF)-associated pathogens . Previous studies demonstrated that rat SP-D is highly resistant to degradation by a wide range of proteolytic enzymes . The aim of this study was to examine whether human SP-D can be degraded by CF relevant proteases ex vivo and in vitro . Bronchoalveolar lavage fluids (BALFs) of 11 patients with CF in a stable clinical condition were examined for SP-D by immunoblotting . In vitro, purified human SP-D was treated with human leukocyte elastase, proteinase 3, cathepsin G or Pseudomonas elastase followed by immunoblotting with specific antibodies to SP-D . In BALF of 8 of the 11 patients investigated, proteolytic fragments or absence of SP-D were detected . In vitro proteolysis of SP-D was observed in a time-dependent manner for each protease applied . The presence of Ca(++) at a physiologic concentration delayed, but did not prevent the degradation . We conclude that SP-D is an important target of numerous proteases present in the CF lung . Host defense is probably impaired due to proteolysis of SP-D and may contribute to the suppurative lung disease in CF.

J Neurooncol, 2003 Aug-Sep, 64(1-2), 125 - 37
Safety, tolerability, and tumor response of IL4-Pseudomonas exotoxin (NBI-3001) in patients with recurrent malignant glioma; Weber F et al.; PURPOSE: This was an open-label, dose-escalation trial of intratumoral administration of IL-4 Pseudomonas exotoxin (NBI-3001) in patients with recurrent malignant glioma . PATIENTS AND METHODS: A total of 31 patients with histologically verified supratentorial grades 3 and 4 astrocytoma were studied . Of these, 25 patients were diagnosed with glioblastoma multiforme (GBM) while six were diagnosed with anaplastic astrocytoma . Patients were over 18 years of age and had Karnofsky performance scores > or = 60 . Patients were assigned to one of four dose groups in a dose-escalation fashion: 6 microg/ml x 40 ml, 9 microg/ml x 40 ml, 15 microg/ml x 40 ml, or 9 microg/ml x 100 ml of NBI-3001 administered via convection-enhanced delivery intratumorally using stereotactically placed catheters . Patients were followed with serial MRI scans and clinical assessments every four weeks for the first 16 weeks and then every eight weeks until week 26 . RESULTS: No drug-related systemic toxicity, as evident by lack of hematological or serum chemical changes, was apparent in any patients; treatment-related adverse effects were limited to the central nervous system . No deaths were attributable to treatment . Drug-related grade 3 or 4 toxicity was seen in 39% of patients in all dose groups and 22% of patients at the maximum tolerated dose of 6 microg/ml x 40 ml . The overall median survival was 8.2 months with a median survival of 5.8 months for the GBM patients . Six-month survival was 52% and 48%, respectively . Gadolinium-enhanced magnetic resonance imaging of the brain showed areas of decreased signal intensity within the tumor consistent with tumor necrosis following treatment in many patients . CONCLUSIONS: NBI-3001 appears to have an acceptable safety and toxicity profile when administered intratumorally in patients with recurrent malignant glioma.

J Neurooncol, 2003 Aug-Sep, 64(1-2), 117 - 23
Molecular targeting with recombinant cytotoxins of interleukin-13 receptor alpha2-expressing glioma; Mintz A et al.; A restricted receptor for interleukin 13 (IL-13R alpha2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs . In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13R alpha2, we established an IL-13R alpha2-expressing syngeneic immunocompetent murine model of HGA . The model was obtained by transfecting G-26 murine glioma cells with IL-13R alpha2 . G-26-IL-13R alpha2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells . G-26-IL-13R alpha2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13R alpha2 in vivo . These characteristics of the G-26-IL-13R alpha2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy . A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative of Pseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments . G-26-IL-13R alpha2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion . To test the cytotoxin in vivo, G-26-IL-13R alpha2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50 mm3, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E . In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured . This study documents the establishment of an IL-13R alpha2-positive model of HGA in immunocompetent rodents . Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13R alpha2-expressing tumors in a more clinically relevant in vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.

FEMS Microbiol Lett, 2003 Aug 29, 225(2), 189 - 93
Serine racemase homologue of Saccharomyces cerevisiae has L-threo-3-hydroxyaspartate dehydratase activity; Wada M et al.; The NH(2)-terminal amino acid sequence of L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp . T62 showed significant similarity to that of the SRY1 gene product of Saccharomyces cerevisiae (serine racemase in yeast) . SRY1 was cloned and expressed in Escherichia coli, and the gene product was purified and partially characterized . The SRY1 gene product exhibited dehydratase activity specific for L-threo-3-hydroxyaspartate (K(m)=3.9 mM, V(max)=110 micromol min(-1) (mg protein)(-1)) but not for D-threo- or DL-erythro-3-hydroxyaspartate . The purified enzyme showed no detectable serine racemase activity . The activity of the enzyme was inhibited by hydroxylamine and EDTA, and was activated by Mg(2+), Ca(2+), and Mn(2+), suggesting that pyridoxal-5'-phosphate and divalent cations participate in the enzyme reaction . Gene disruption and overexpression indicated that SRY1 is responsible for the 3-hydroxyaspartate resistance of S . cerevisiae . To our knowledge, this is the first report of 3-hydroxyaspartate dehydratase activity in eukaryotic cells.

Mol Microbiol, 2003 Sep, 49(6), 1537 - 46
Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease; Axtell MJ et al.; Upon delivery to the plant cell during infection, the Pseudomonas syringae effector protein AvrRpt2 undergoes proteolytic processing, enhances pathogen virulence and causes the elimination of the Arabidopsis RIN4 protein . A structure-prediction method was employed in order to investigate possible biochemical functions of AvrRpt2 . Results of a secondary structure prediction algorithm suggest that the functional C-terminal portion of AvrRpt2 is a cysteine protease . Mutation of predicted catalytic residues within this portion of AvrRpt2 abolished in planta processing, elimination of Arabidopsis RIN4, and the ability to trigger an RPS2-specific resistance response . These data indicate that AvrRpt2 is most likely a sequence divergent cysteine protease whose activity is required for elimination of RIN4 during infection.

Croat Med J, 2003 Aug, 44(4), 455 - 62
Identification and characterization of interleukin-13 receptor in human medulloblastoma and targeting these receptors with interleukin-13-pseudomonas exotoxin fusion protein; Joshi BH et al.; AIM: To identify and characterize the subunit structure of interleukin-13 receptor (IL-13R) in human medulloblastoma cell lines and target them with a chimeric fusion protein consisting of interleukin-13 and Pseudomonas exotoxin (termed as IL-13 cytotoxin) . METHODS: Five human medulloblastoma cell lines were examined for the expression of IL-13R subunits at the mRNA and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR) and indirect immunofluorescence studies, respectively . In addition, IL-13 cytotoxin-induced cytotoxicity was examined in these medulloblastoma cell lines by measuring protein synthesis inhibition . RESULTS: All five medulloblastoma cell lines expressed mRNA and proteins for IL-4Ra and IL-13Ra1 chains, whereas three of the cell lines also showed the presence of IL-13Ra2 . mRNA or protein for IL-2gc chain was not detected in any of the cell lines . Consistent with the expression of IL-13Ra2 chain, IL-13 cytotoxin was highly and specifically cytotoxic to three of five medulloblastoma cell lines . The sensitivity of medulloblastoma cell lines to IL-13 cytotoxin could be completely eliminated by concurrent incubation with excess of IL-13, but not with IL-2 or IL-4 . CONCLUSION: These studies establish IL-13R, in particular IL-13Ra2, as a medulloblastoma-associated target for IL-13 cytotoxin therapy . IL-13 cytotoxin may be useful for medulloblastoma therapy . Alternatively, IL-13Ra2 may serve as a tumor-specific antigen for active immunotherapy.

Science, 2003 Aug 29, 301(5637), 1230 - 3
Cleavage of Arabidopsis PBS1 by a bacterial type III effector; Shao F et al.; Plant disease-resistance (R) proteins are thought to function as receptors for ligands produced directly or indirectly by pathogen avirulence (Avr) proteins . The biochemical functions of most Avr proteins are unknown, and the mechanisms by which they activate R proteins have not been determined . In Arabidopsis, resistance to Pseudomonas syringae strains expressing AvrPphB requires RPS5, a member of the class of R proteins that have a predicted nucleotide-binding site and leucine-rich repeats, and PBS1, a protein kinase . AvrPphB was found to proteolytically cleave PBS1, and this cleavage was required for RPS5-mediated resistance, which indicates that AvrPphB is detected indirectly via its enzymatic activity.

Mol Microbiol, 2003 Sep, 49(5), 1239 - 51
Pseudomonas syringae pv . tomato DC3000 HopPtoM (CEL ORF3) is important for lesion formation but not growth in tomato and is secreted and translocated by the Hrp type III secretion system in a chaperone-dependent manner; Badel JL et al.; Pseudomonas syringae pv . tomato DC3000 is a pathogen of tomato and Arabidopsis that injects virulence effector proteins into host cells via a type III secretion system (TTSS) . TTSS-deficient mutants have a Hrp- phenotype, that is, they cannot elicit the hypersensitive response (HR) in non-host plants or pathogenesis in host plants . Mutations in effector genes typically have weak virulence phenotypes (apparently due to redundancy), but deletion of six open reading frames (ORF) in the DC3000 conserved effector locus (CEL) reduces parasitic growth and abolishes disease symptoms without affecting function of the TTSS . The inability of the DeltaCEL mutant to cause disease symptoms in tomato was restored by a clone expressing two of the six ORF that had been deleted: CEL ORF3 (HopPtoM) and ORF4 (ShcM) . A DeltahopPtoM::nptII mutant was constructed and found to grow like the wild type in tomato but to be strongly reduced in its production of necrotic lesion symptoms . HopPtoM expression in DC3000 was activated by the HrpL alternative sigma factor, and the protein was secreted by the Hrp TTSS in culture and translocated into Arabidopsis cells by the Hrp TTSS during infection . Secretion and translocation were dependent on ShcM, which was neither secreted nor translocated but, like typical TTSS chaperones, could be shown to interact with HopPtoM, its cognate effector, in yeast two-hybrid experiments . Thus, HopPtoM is a type III effector that, among known plant pathogen effectors, is unusual in making a major contribution to the elicitation of lesion symptoms but not growth in host tomato leaves.

Drug News Perspect, 2000 Sep, 13(7), 395 - 402
IL-4 receptor-directed cytotoxin for therapy of AIDS-associated KS tumors; Puri RK; AIDS-associated Kaposi's sarcoma (AIDS-KS) represents one of the most common malignancies associated with human immunodeficiency virus infection . To target effective therapeutic agents, we have discovered that AIDS-KS cells express high-affinity receptors for interleukin-4 (IL-4), a pleiotropic immune regulatory cytokine . Molecular studies have revealed that AIDS-KS cells express type II IL-4 receptors, in which IL-4 forms a productive complex with primary IL-4 binding protein (IL-4R beta, also known as IL-4R alpha) and a shared subunit between IL-4 and IL-13R systems (IL-13R alpha', also known as IL-13R alpha 1) . A recombinant fusion protein composed of IL-4 and a mutated form of a powerful bacterial toxin called Pseudomonas exotoxin (PE)--the fusion protein is termed IL4(3837)-PE38KDEL or cpIL4-PE--was found to be highly and specifically cytotoxic to AIDS-KS cells in vitro . Normal human immune cells (e.g., resting T and B cells and monocytes) or endothelial cells, a possible precursor of AIDS-KS, expressed low numbers of IL-4R and showed little or no sensitivity to cpIL4-PE . Administration of cpIL4-PE in nude mice with established subcutaneously growing AIDS-KS tumors produced remarkable antitumor activity in a dose-dependent manner with the highest dose exhibiting complete responses without any visible toxicity . KS tumors produced metabolic changes including cachexia, hypoglycemia and lymphopenia, all of which were prevented by cpIL4-PE treatment . These studies indicate that cpIL4-PE is a promising experimental therapeutic agent for treatment of AIDS-KS.

Protein Sci, 2003 Sep, 12(9), 1855 - 64
The structure of Pseudomonas P51 Cl-muconate lactonizing enzyme: co-evolution of structure and dynamics with the dehalogenation function; Kajander T et al.; Bacterial muconate lactonizing enzymes (MLEs) catalyze the conversion of cis,cis-muconate as a part of the beta-ketoadipate pathway, and some MLEs are also able to dehalogenate chlorinated muconates (Cl-MLEs) . The basis for the Cl-MLEs dehalogenating activity is still unclear . To further elucidate the differences between MLEs and Cl-MLEs, we have solved the structure of Pseudomonas P51 Cl-MLE at 1.95 A resolution . Comparison of Pseudomonas MLE and Cl-MLE structures reveals the presence of a large cavity in the Cl-MLEs . The cavity may be related to conformational changes on substrate binding in Cl-MLEs, at Gly52 . Site-directed mutagenesis on Pseudomonas MLE core positions to the equivalent Cl-MLE residues showed that the variant Thr52Gly was rather inactive, whereas the Thr52Gly-Phe103Ser variant had regained part of the activity . These residues form a hydrogen bond in the Cl-MLEs . The Cl-MLE structure, as a result of the Thr-to-Gly change, is more flexible than MLE: As a mobile loop closes over the active site, a conformational change at Gly52 is observed in Cl-MLEs . The loose packing and structural motions in Cl-MLE may be required for the rotation of the lactone ring in the active site necessary for the dehalogenating activity of Cl-MLEs . Furthermore, we also suggest that differences in the active site mobile loop sequence between MLEs and Cl-MLEs result in lower active site polarity in Cl-MLEs, possibly affecting catalysis . These changes could result in slower product release from Cl-MLEs and make it a better enzyme for dehalogenation of substrate.

Zhonghua Yi Xue Za Zhi, 2003 Jul 25, 83(14), 1246 - 50
{Molecular design and construction of IL6D24-PE40KDEL, a novel recombinant interleukin6-pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemias expressing interleukin6 receptors}; Zheng LY et al.; OBJECTIVE: A novel recombinant interleukin6-Pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemic cells highly expressing IL6R has been molecularly designed and constructed in this study . METHODS: Firstly, REDLK at C-terminus of Pseudomonas exotoxin PE40 was replaced with KDEL using point mutagenesis technology . Secondly, a cDNA encoding interleukin-6 devoid of N-terminal 24 amino acids {IL6D24} was fused to 5' terminus of PE40KDEL DNA by the method of gene splicing by overlap extension, which could generate recombinant IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL fusion genes . Thirdly, recombinant fusion genes IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL were ligated into the EcoR I and Sma I cloning sites in the pBV220 plasmids respectively and then transformed into E.coli HB101 cells . The expressed recombinant exotoxin fusion proteins were purified to electrophoresis purity by Mono Q column chromatography . Its selectively killing was tested by the MTS colorimetric method using both U937 and CEM cells lines . RESULTS: Recombinant exotoxin fusion proteins IL6D24-PE40KDEL was expressed as a form of inclusion bodies at higher level of 40% approximately of total proteins in bacterial cells . Western blot showed that the purified products could react specifically with IL6 monoclonal antibody and PEA antiserum, respectively . IL6D24-PE40KDEL was selectively cytotoxic to U937 cells expressing IL6R-positive with ID50 of 250 ng/ml, and but not CEM cells expressing IL6R-negative . CONCLUSIONS: Two novel recombinant interleukin6-Pseudomonas exotoxin fusion proteins IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL have been successfully constructed and they can selectively kill the leukemic cells expressing highly IL6R.

Org Biomol Chem, 2003 Apr 21, 1(8), 1282 - 91
The utility of cyclodextrins in lipase-catalyzed transesterification in organic solvents: enhanced reaction rate and enantioselectivity; Ghanem A; The use of enzymes as valuable catalysts in organic solvents has been well documented . However, some of their features limit their application in organic synthesis, especially the frequently lower enzyme activity under nonaqueous conditions, which constitutes a major drawback in the application of enzymes in organic solvents . In addition, many enzymatic reactions are subject to substrate or product inhibition, leading to a decrease in the reaction rate and enantioselectivity . To overcome these drawbacks and to make enzymes more appealing to organic chemists, we demonstrate the use of cyclodextrins as regulators for the Pseudomonas cepacia lipase (PSL) and macrocyclic additives to enhance the reaction rate and enantioselectivity E in lipase-catalyzed enantioselective transesterification of 1-(2-furyl)ethanol in organic solvents . Both reaction rate and enantioselectivity were significantly enhanced by several orders of magnitude when using co-lyophilized lipase in the presence of cyclodextrins . The effect of cyclodextrin derivatives as well as solvents on the improvement of the reaction parameters has been studied . The observed enhancement was tentatively interpreted in terms of their ability to give a certain flexibility to the enzyme and to form a host-guest complex, thus avoiding product inhibition and leading to enhancement of the reaction rate and enantioselectivity . The effect of cyclodextrin additives on the enzyme morphology has been studied using scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) of the co-lyophilized lipase with cyclodextrins . The ability of cyclodextrins to form a host-guest complex to avoid product inhibition, which leads to the observed enhancement, has been proved by NOESY, COSY, 13C and 1H NMR.

Mar Biotechnol (NY), 2003 Jan-Feb, 5(1), 13 - 9
Structural revision of sulfated polysaccharide B-1 isolated from a marine Pseudomonas species and its cytotoxic activity against human cancer cell lines; Matsuda M et al.; The structure of a sulfated polysaccharide (B-1) isolated and purified from the culture filtrate of marine Pseudomonas sp . WAK-1 was revised to have a repeating unit as follows: -2)-beta-D-Galp(4SO4)(1-4){beta-D-Glcp(1-6)}-beta-D-Galp(3SO4)(1- . B-1 was evaluated for anticancer activity using a human cancer cell line panel coupled with a drug sensitivity database . The average B-1 concentration required for 50% growth inhibition against the panel of 39 cell lines was 63.2 micro g/ml . Among the cancer cell lines tested, high sensitivities to B-1 were observed in central nervous system cancer and lung cancer cell lines . The COMPARE analysis revealed that the differential growth inhibition pattern of B-1 had no significant correlation with those of more than 200 standard compounds, most of which were anticancer drugs and different types of inhibitors . This lack of similarities in the cytotoxic patterns appears to reflect previously unrecognized biological properties of B-1 . It was revealed that B-1 induced apoptosis in U937 cells, as shown by cell morphology and internucleosomal DNA fragmentation.

J Dairy Res, 2003 Aug, 70(3), 293 - 6
Growth kinetics and hydrolytic enzyme production of Pseudomonas spp . isolated from pasteurized milk; Stevenson RG et al.; Psychrotrophs, particularly Pseudomonas spp . are known to be the main determinants of the shelf-life of pasteurized milk and refrigerated raw milk . It is presumed that they mainly cause spoilage through the elaboration of proteinase and lipase enzymes . At the time of this research, under the relevant European Directive, one of the means of determining the quality of pasteurized milk was the pre-incubated count, which involves incubating the milk sample for 5 d at 6 degrees C followed by a plate count . Examination of numerous pre-incubated counts revealed a bimodal rather than a normal distribution indicating that the types of contaminants in pasteurized milk may be as important as their initial concentration . Pseudomonads that gave particularly high (> 5 x 10(6) cfu/ml) and low (< 10(3) cfu/ml) pre-incubated counts were isolated (high and low count isolates respectively) . After the organisms had been subjected to a cold shock no consistent trend between the groups of isolates was detected with respect to lag phase duration . However, the high count isolates consistently had a faster exponential growth rate . Unexpectedly, with the exception of one isolate, the low count isolates produced detectable proteinase and lipase earlier . In addition, with one exception, maximal proteinase and lipase production was observed with the low count isolates . These findings indicate that there is no causal relationship between selective growth advantage and ability to produce proteinase and lipase . It also indicates that the spoilage of pasteurized milk is a complex phenomenon and is worthy of further research.

Biosci Biotechnol Biochem, 2003 Jul, 67(7), 1479 - 84
Effects of high concentrations of inorganic salts on swarming ability in fluorescent pseudomonas strains; Sakai M et al.; We did tests using swarm plates, to examine the effects of various salts and their concentrations on the chemotaxis of fluorescent Pseudomonas strains . As a result, we found that the swarming ability of the Pseudomonas strains was inhibited by high concentrations of Ca2+ . The growth of the strains was not affected at the high concentration of Ca2+, but the cells grown in swarm agar under the condition were extended in the filaments . Most of the cells had reached 10 microm to 40 microm in length . Such cell elongation was not observed with salts other than calcium salts . A significant correlation between the cell elongation and the decrease of swarming ability by the high concentration of Ca2+ was observed.

Plant Physiol, 2003 Aug, 132(4), 2023 - 33
The Arabidopsis NHL3 gene encodes a plasma membrane protein and its overexpression correlates with increased resistance to Pseudomonas syringae pv . tomato DC3000; Varet A et al.; The Arabidopsis genome contains a family of NDR1/HIN1-like (NHL) genes that show homology to the nonrace-specific disease resistance (NDR1) and the tobacco (Nicotiana tabacum) harpin-induced (HIN1) genes . NHL3 is a pathogen-responsive member of this NHL gene family that is potentially involved in defense . In independent transgenic NHL3-overexpressing plant lines, a clear correlation between increased resistance to virulent Pseudomonas syringae pv . tomato DC3000 and enhanced NHL3 transcript levels was seen . These transgenic plants did not show enhanced pathogenesis-related gene expression or reactive oxygen species accumulation . Biochemical and localization experiments were performed to assist elucidation of how NHL3 may confer enhanced disease resistance . Gene constructs expressing amino-terminal c-myc-tagged or carboxyl-terminal hemagglutinin epitope (HA)-tagged NHL3 demonstrated membrane localization in transiently transformed tobacco leaves . Stable Arabidopsis transformants containing the NHL3-HA construct corroborated the findings observed in tobacco . The detected immunoreactive proteins were 10 kD larger than the calculated size and could be partially accounted for by the glycosylation state . However, the expected size was not attained with deglycosylation, suggesting possibly additional posttranslational modification . Detergent treatment, but not chemicals used to strip membrane-associated proteins, could displace the immunoreactive signal from microsomal fractions, showing that NHL3 is tightly membrane associated . Furthermore, immunofluorescence and immunogold labeling, coupled with two-phase partitioning techniques, revealed plasma membrane localization of NHL3-HA . This subcellular localization of NHL3 positions it at an initial contact site to pathogens and may be important in facilitating interception of pathogen-derived signals.

Plant Physiol, 2003 Aug, 132(4), 1901 - 12
Overexpression of the disease resistance gene Pto in tomato induces gene expression changes similar to immune responses in human and fruitfly; Mysore KS et al.; The Pto gene encodes a serine/threonine protein kinase that confers resistance in tomato (Lycopersicon esculentum) to Pseudomonas syringae pv tomato strains that express the type III effector protein AvrPto . Constitutive overexpression of Pto in tomato, in the absence of AvrPto, activates defense responses and confers resistance to several diverse bacterial and fungal plant pathogens . We have used a series of gene discovery and expression profiling methods to examine the effect of Pto overexpression in tomato leaves . Analysis of the tomato expressed sequence tag database and suppression subtractive hybridization identified 600 genes that were potentially differentially expressed in Pto-overexpressing tomato plants compared with a sibling line lacking Pto . By using cDNA microarrays, we verified changes in expression of many of these genes at various time points after inoculation with P . syringae pv tomato (avrPto) of the resistant Pto-overexpressing line and the susceptible sibling line . The combination of these three approaches led to the identification of 223 POR (Pto overexpression responsive) genes . Strikingly, 40% of the genes induced in the Pto-overexpressing plants previously have been shown to be differentially expressed during the human (Homo sapiens) and/or fruitfly (Drosophila melanogaster) immune responses.

Extremophiles, 2003 Aug, 7(4), 335 - 7 Epub 2003 Apr 09.
Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization; Zeng R et al.; An extracellular protease was purified from a deep-sea psychrophilic bacterium strain DY-A which was identified as a Pseudomonas species . The optimal growth and protease-producing temperatures of the strain were all 10 degrees C, and the protease was secreted only at temperatures under 20 degrees C . The enzyme was most active at 40 degrees C and at pH 10.0 . It was inhibited by phenylmethyl sulfonylfluoride and diisopropyl fluorophosphate, indicating that it is a serine protease . Chelators such as EDTA, EGTA, 1,10-phenanthroline and 2,2'-bipyridyl produced a decrease of activity . The enzyme was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and resistant to thiol-containing reducing agents such as dithiotreitol . The enzyme was active towards N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu- p-nitroanilide . The native molecular mass of the enzyme determined by native PAGE and SDS-PAGE was 25 kDa.

J Biol Chem, 2003 Oct 31, 278(44), 43709 - 16 Epub 2003 Aug 07.
The structure of 4-hydroxybenzoyl-CoA thioesterase from arthrobacter sp . strain SU; Thoden JB et al.; The 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a thioesterase . Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp . strain SU . The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called "hot dog fold" composed of six strands of anti-parallel beta-sheet flanked on one side by a rather long alpha-helix . The dimers come together to form the tetramer with their alpha-helices facing outwards . This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their alpha-helices projecting toward the interfacial region . In the Arthrobacter thioesterase, each of the four active sites is formed by three of the subunits of the tetramer . On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter thioesterase . Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme . Although these two bacterial thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different.

Appl Environ Microbiol, 2003 Aug, 69(8), 4979 - 82
Analysis of the argK-tox gene cluster in nontoxigenic strains of Pseudomonas syringae pv . phaseolicola; Gonzalez AI et al.; The analysis of 46 isolates obtained directly from different and distant common bean fields from the northwestern part of Spain revealed that they do not produce phaseolotoxin . The isolates were classified as race 5, and their analysis revealed that they do not carry the argK-tox gene cluster involved in the biosynthesis of the phaseolotoxin.

Isr Med Assoc J, 2003 Jul, 5(7), 491 - 5
Cystic fibrosis in adults: a changing scene; Blau H et al.; BACKGROUND: Cystic fibrosis is no longer a terminal illness of childhood and mean survival is now over 30 years . Adult patients with atypical CF are increasingly being diagnosed . In Israel, all patients are still followed in pediatric centers . OBJECTIVES: To describe our experience with adult CF, stressing the importance of adult-related health and psychosocial issues . METHODS: Twenty-five CF patients aged 20-50 years, constituting 44% of the 57 patients followed at our center, were analyzed for pulmonary and extrapulmonary features and management . RESULTS: Nineteen were diagnosed as children and 6 as adults . Nineteen were pancreatic-insufficient and 6 were pancreatic-sufficient, including 5 diagnosed as adults . Pulmonary status was usually stable, with forced expiratory volume in 1 second 66.3 +/- 21% (mean +/- SD) and no difference between pancreatic-sufficient and insufficient patients . The latter had more hemoptysis, Pseudomonas infection, intestinal obstruction, liver disease and diabetes . Two patients died of malignancy and two of advanced lung disease . A majority received continuous inhaled and oral antibiotics, bronchodilators, Dnase, physiotherapy and periodic home intravenous antibiotics . Psychosocial functioning was excellent: 60% were employed, 36% were married and 40% had children (none with CF) . Patients diagnosed as adults had mild multisystem disease or isolated severe lung disease . CONCLUSIONS: CF adults generally have a good quality of life . Advances in understanding the CF defect and a plethora of new treatment modalities bode well for the future . Patients must be maintained in optimal condition to reap the benefits, and there is an urgent necessity for adult physicians to develop expertise in CF.

Mikrobiologiia, 2003 May-Jun, 72(3), 407 - 13
{Bacterial chemotaxis to naphthalene}; Zaval'skii LIu et al.; The chemotaxis of two pseudomonads, P . putida AZ (Naph+) and P . putida AZ (Naph-), differing in the ability to metabolize naphthalene was studied by the known capillary method of Adler and the densitometric method devised in our laboratory . The migration of P . putida AZ (Naph+) cells toward increasing levels of naphthalene was accompanied by the formation of a migrating front of converted naphthalene . P . putida AZ (Naph-) cells, too, exhibited positive chemotaxis to naphthalene, but they did not form the front of converted naphthalene . The analysis of experimental data in terms of a kinetic model of bacterial chemotaxis showed that the densitometric method is a potential tool for studying bacterial chemotaxis to hydrophobic organic substances.

J Biol Chem, 2003 Oct 31, 278(44), 43373 - 83 Epub 2003 Aug 04.
p-Coumaroylnoradrenaline, a novel plant metabolite implicated in tomato defense against pathogens; Von Roepenack-Lahaye E et al.; The Avr9 peptide elicitor from the fungus Cladosporium fulvum, the bacterial pathogen Pseudomonas syringae pathovar tomato carrying the avirulence gene avrPto (Pst (avrPto)), and the organophosphorous insecticide fenitrothion induce resistance-related responses in tomato lines carrying the Cf-9, Pto, and Fen genes, respectively . These responses were associated with synthesis of p-coumaroyloctopamine and p-coumaroylnoradrenaline, a novel compound for plants . In susceptible near isogenic tomato lines (Cf-0, pto, fen) and wounded tomato leaves, the levels of these compounds were reduced or undetectable . The elevated levels of p-coumaroyloctopamine and p-coumaroylnoradrenaline were accompanied by elevated mRNA levels of genes encoding phenylalanine ammonia lyase, p-coumarate CoA ligase, and hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT), enzymes that are involved in the hydroxycinnamic acid amide biosynthesis . Southern hybridization indicated that THT is encoded by a multigene family in tomato . Four different THT full-length cDNAs were derived by reverse transcriptase-PCR using degenerate primers based on potato and tobacco THT sequences . Transcripts for all four homologs were present in unchallenged tomato leaves, but only tomTHT1-3 was highly expressed following challenge with Pst (avrPto) . Furthermore, tomTHT1-3 showed a more substantial and rapid induction in the incompatible interaction than in the compatible interaction . The cDNAs tomTHT1-3, tomTHT7-1, and tomTHT7-8 encoded proteins with a high degree of amino acid sequence homology, although the recombinant proteins had different preferences for octopamine and noradrenaline . The fourth cDNA, tomTHT1-4, directed synthesis of a truncated enzymatically inactive protein due to the presence of a premature stop codon.

Biochemistry, 2003 Aug 12, 42(31), 9316 - 23
Importance of hydrophobic and polar residues in ligand binding in the family 15 carbohydrate-binding module from Cellvibrio japonicus Xyn10C; Pell G et al.; Modular glycoside hydrolases that degrade the plant cell wall often contain noncatalytic carbohydrate-binding modules (CBMs) that interact with specific polysaccharides within this complex macromolecule . CBMs, by bringing the appended catalytic module into intimate and prolonged association with the substrate, increase the rate at which these enzymes are able to hydrolyze glycosidic bonds . Recently, the crystal structure of the family 15 CBM (CBM15) from Cellvibrio japonicus (formerly Pseudomonas cellulosa) Xyn10C was determined in complex with the ligand xylopentaose . In this report we have used a rational design approach, informed by the crystal structure of the CBM15-ligand complex, to probe the importance of hydrophobic stacking interactions and both direct and water-mediated hydrogen bonds in the binding of this protein to xylan and xylohexaose . The data show that replacing either Trp 171 or Trp 186, which stack against xylose residues n and n + 2 in xylopentaose, with alanine abolished ligand binding . Similarly, replacing Asn 106, Gln 171, and Gln 217, which make direct hydrogen bonds with xylopentaose, with alanine greatly reduced the affinity of the protein for its saccharide ligands . By contrast, disrupting water-mediated hydrogen bonds between CBM15 and xylopentaose by introducing the mutations S108A, Q167A, Q221A, and K223A had little effect on the affinity of the protein for xylan or xylohexaose . These data indicate that CBM15 binds xylan and xylooligosaccharides via the same interactions and provide clear evidence that direct hydrogen bonds are a key determinant of affinity in a type B CBM . The generic importance of these data is discussed.

Cell Mol Biol (Noisy-le-grand), 2003 Jun, 49(4), 621 - 6
Component analysis and growth process of nasopharyngeal calculus as revealed by Fourier transform infrared (FT-IR) spectroscopy; Ogawa T et al.; A quite rare case of nasopharyngeal calculus in a woman in her twenties associated with the nasal discharge of pseudomonas infection was reported . As the substance was irregularly large in size, we extracted it partially by piecemeal resection using forceps and also by cracking technique using the holmium yttrium-aluminum-garnet (YAG) laser, under saline irrigation and stereotactic microscopic navigator (SMN) system under endoscopic observation . The substance was firmly fixed to the pharyngeal tonsil bed . The final extract was a small piece of singly folded bandage, which is probably the focal background for calculus formation . In a cross section of calculus specimen removed during surgery, Fourier transform infrared (FT-IR) analysis revealed that a) signal ratio of methylene group (organic substance) to amide I (protein) was 21.6% at the nasal cavity side, gradually decreased toward nasal mucous membrane showing approximate 50%, b) signal ratio of amide I to P04(3-) (inorganic substance) ranged between 17.7% and 26.7% at the different sites and inside the calculus, the protein content was approximate 1/5 of the inorganic substance, and c) signal ratio of the methylene group to amide I at the nasal cavity site showed that their contents were almost equal . The quantity of the organic substance was estimated at approximate 1/2 quantity of the protein at both the central part and the part contacted with the mucous membrane . From these results, it seems that throughout the course of calculus growth, both inorganic substance and protein remain almost constant inside the calculus, while organic substance is released from the internal part of the calculus being probably formed at an early stage.

An Acad Bras Cienc, 2003 Jun, 75(2), 157 - 62 Epub 2003 Jul 31.
Evaluation of the catalytic activity of lipases immobilized on chrysotile for esterification; Silva JE et al.; In the present work, the ester synthesis in organic media catalyzed by lipases immobilized on chrysotile was studied . Lipases of different sources (Mucor javanicus, Pseudomonas cepacia, Rhizopus oryzae, Aspergillus niger and Candida rugosa) were immobilized on chrysotile, an inexpensive magnesium silicate, and used for esterification of hexanoic, octanoic and lauric acid with methanol, ethanol, 1-butanol and 1-octanol at 25 C in hexane as solvent . The best results were obtained with Mucor javanicus lipase and lauric acid giving yields of 62-97% of ester.

Biophys J, 2003 Aug, 85(2), 1165 - 75
3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering; Occhipinti E et al.; Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000 . Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B) . The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions . The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure . Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site . With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry . Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168) . We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site . Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS) . An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves . The results clearly support a tetrameric structure . The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles . The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.

Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 284 - 90 Epub 2003 Feb 26.
A novel catabolic activity of Pseudomonas veronii in biotransformation of pentachlorophenol; Nam IH et al.; Pseudomonas veronii PH-05, a bacterial strain capable of transforming pentachlorophenol (PCP) to a metabolic intermediate, was isolated by selective enrichment of soil samples from a timber storage yard . Strain PH-05 was shown to be able to grow using PCP as the sole source of carbon and energy . GC-MS analysis showed that the metabolic intermediate was tetrachlorocatechol, which inhibited the growth of this strain . The formation of tetrachlorocatechol during biotransformation was monitored, and its inhibitory effect on growth of strain PH-05 was analyzed at a range of concentrations . The catabolic activity of the isolated strain differs from that of other PCP-degrading bacteria, which metabolize PCP through a chlorinated hydroquinone intermediate.

Am J Physiol Lung Cell Mol Physiol, 2003 Nov, 285(5), L1099 - 105 Epub 2003 Jul 25.
Pulmonary cytochrome P-450 2J4 is reduced in a rat model of acute Pseudomonas pneumonia; Yaghi A et al.; We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are depressed in microsomes prepared from lungs of rats with acute Pseudomonas pneumonia . We also showed a potential role for cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) in contractile responses of both normal pulmonary arteries and pulmonary arteries from rats with pneumonia . The CYP2J subfamily enzymes (endogenous source of EETs and HETEs) are constitutively expressed in human and rat lungs where they are localized in vascular smooth muscle and endothelium . The purpose of this study was to determine if CYP2J proteins are modified in pneumonia . Pseudomonas organisms were injected via a tracheostomy in the lungs of rats . Later (44 h), lungs were frozen, and microsomes were prepared from pneumonia and control rat lung homogenates . Lung microsomal proteins were then immunoblotted with anti-CYP2B1/2B2, anti-CYP4A, anti-CYP2J9pep2 (which reacts with rat CYP2J3), anti-CYP2J6pep1 (which reacts with rat CYP2J4), anti-CYP2J2pep4, or anti-CYP2J2pep3 (both of which react with all known CYP2J isozymes) . Western blotting revealed a prominent 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia compared with control lung microsomes . The CYP2B bands (51-52 kDa) were less prominent and not different between pneumonia and control lungs . CYP4A proteins (20-HETE sources) were not detected in rat lung microsomes . Therefore, rat lung contains a protein with immunological characteristics similar to CYP2J4, and this CYP is reduced after pneumonia . We speculate that CYP2J (but not CYP2B) enzymes and their AA metabolic products (EETs) are involved in the modulation of pulmonary vascular tone in pneumonia in rats.

Biotechnol Lett, 2003 Feb, 25(3), 199 - 203
Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae; Bassett CL et al.; Transformation efficiencies as high as 10(7) transformants microg(-1) DNA have been previously reported for pseudomonads using electroporation protocols established for E . coli with plasmid DNAs prepared from methylation proficient E . coli hosts . We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E . coli methods . Transformation efficiencies of 10(7) or higher were obtained with DNA recovered from initial P . syringae transformation or with DNA prepared from methylation deficient E . coli . Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.

Plant Cell Physiol, 2003 Jul, 44(7), 750 - 7
The rapid induction of glutathione S-transferases AtGSTF2 and AtGSTF6 by avirulent Pseudomonas syringae is the result of combined salicylic acid and ethylene signaling; Lieberherr D et al.; The expression of two members of the glutathione S-transferase (GST) multigene family was studied in Arabidopsis plants inoculated with an avirulent strain of Pseudomonas syringae pv . tomato (Pst) . Accumulation of AtGSTF2 and AtGSTF6 transcripts started 4 and 2 h after inoculation, respectively, and clearly preceded the induction of the pathogenesis-related PR-1 gene . The aim of this work was to find the reason for the faster induction of the two GSTs compared with classical salicylic acid (SA)-regulated PR-proteins . Expression studies in Pst-inoculated SA-signaling mutants NahG and npr1 revealed that induction of both GSTs was SA-dependent and partially NPR1-independent . The induction of AtGSTF2 by Pst was also strongly repressed in the ethylene insensitive etr1 mutant . Both GSTs were induced by low amounts of SA (0.1 mM) and ethylene (0.1 ppm) while PR-1 gene expression was unaffected by ethylene . Interestingly, ethylene was about 50-fold less effective in NahG compared with wild-type plants thus suggesting a potentiation effect of SA on ethylene-induced accumulation of AtGST transcripts . Increased AtGST expression in plants inoculated with Pst correlated with increased production of SA and ethylene . However, the initial phase of AtGSTF6 induction was independent of SA- and ethylene-signaling . The jasmonate (JA)-insensitive mutant jar1 showed normal induction kinetics for both GSTs . Our data support the hypothesis that full expression of the pathogen-induced AtGSTF2 and, to a lesser extent AtGSTF6, is the result of combined SA- and ethylene-signaling and that early AtGSTF6 expression depends on additional unknown signaling mechanisms.

Protein Expr Purif, 2003 Aug, 30(2), 171 - 8
Cloning, heterologous expression, renaturation, and characterization of a cold-adapted esterase with unique primary structure from a psychrotroph Pseudomonas sp . strain B11-1; Suzuki T et al.; A gene coding for an esterase (PsEst1, 1911bp in length) of the psychrotrophic bacterium Pseudomonas sp . B11-1 isolated from Alaskan soil was cloned and sequenced . The deduced amino acid sequence revealed a protein of 637 amino acid residues with a molecular mass of 69 kDa . Although the expression product, PsEst1, showed no appreciable sequence similarity (less than 15% identity) to any known proteins with the established biochemical functions, it is expected to be related to the alpha/beta hydrolase superfamily because it shared sequence motifs that have been identified with this superfamily . For example, a unique 'nucleophilic elbow' motif, -Gly(36)-Asp-Ser-Leu-Asn(40)-, was identified, and Ser(38) was predicted to constitute a catalytic triad with Asp(162) and His(303) . PsEst1 was overexpressed using a T7 RNA polymerase transcription (pET21a) system in the Escherichia coli BL21(DE3) cells as an inclusion body . A soluble denatured form of the enzyme was purified to homogeneity in the presence of 8M urea, and the catalytically active form of the enzyme could be obtained by subsequent removal of urea by dialysis, where the addition of 0.1% Triton X-100 was essential for the efficient renaturation of the enzyme . To our knowledge, this was the first example of the successful renaturation of the recombinant cold-adapted enzyme . The enzyme efficiently hydrolyzed vinyl and aryl esters with the C4-C6 acyl chain . The activation energy of the enzymatic p-nitrophenyl butyrate hydrolysis (20.1 kcal/mol at 10 degrees C) was significantly lower than the value (79.9 kcal/mol) of the mesophilic lipase . It was observed that the K(m) values for p-nitrophenyl butyrate in the growth temperature range of strain B11-1 (5-15 degrees C) were lower than those at higher temperatures.

Arch Microbiol, 2003 Oct, 180(4), 264 - 71 Epub 2003 Jul 23.
Role of GacA, LasI, RhlI, Ppk, PsrA, Vfr and ClpXP in the regulation of the stationary-phase sigma factor rpoS/RpoS in Pseudomonas; Bertani I et al.; RpoS is the stationary phase sigma factor responsible for increased transcription of a set of genes when bacterial cells enter stationary phase and under stress conditions . In Escherichia coli, RpoS expression is modulated at the level of transcription, translation, and post-translational stability whereas in Pseudomonas, previous studies have implicated four genetic loci ( psrA, gacA, lasI and rhlI) involved in rpoS transcription . In this report, the transcription, translation and proteins profiles of rpoS/RpoS were analyzed in response to growth phase of knockout genomic mutants in the P . aeruginosa transcriptional regulatory loci psrA, gacA, vfr, and in the las and rhl quorum-sensing systems . Gene expression and protein profiles were also analyzed in the ppk genomic mutant . This gene is responsible for the biosynthesis of polyphosphate, an alarmone involved in the regulation of RpoS accumulation in E . coli . Finally, the role of the ClpXP protease in RpoS regulation was also studied; in E . coli, this protease has been shown to rapidly degrade RpoS during exponential growth . These studies confirm the significant role of PsrA in rpoS transcription during the late-exponential and stationary growth phases, the probable role of Vfr in transcriptional repression during exponential phase, and the function of the ClpXP protease in RpoS degradation during exponential phase . GacA/GacS, the quorum-sensing systems, and the polyphosphate alarmone molecule were not significant in rpoS/RpoS regulation . These results demonstrate important similarities and differences with the regulation of this sigma factor in E . coli and in Pseudomonas.

Drug News Perspect, 2000 Dec, 13(10), 599 - 605
Preclinical studies with IL-13PE38QQR for therapy of malignant glioma; Joshi BH et al.; To develop novel therapeutic agents for the treatment of brain tumors, we have been investigating the expression of unique tumor-associated receptors or antigens on the tumor cell surface . About six years ago, we discovered that human solid tumor cell lines, including human malignant glioma, express high- to intermediate-affinity receptors (R) for a Th2 cell-derived cytokine, interleukin-13 (IL-13) . Analysis of the subunit composition of IL-13R in primary explants of malignant glioma cells has demonstrated that IL-13R is composed of three different chains (IL-13R alpha 1, IL-13R alpha 2 and IL-4R alpha, also known as IL-13R alpha', alpha and IL-4R beta, respectively) and that IL-13R alpha 2 chain is overexpressed on these cells . Normal brain tissues express IL-13R alpha 1 and IL-4R alpha chains, but show only marginal expression of IL-13R alpha 2 chain . Thus IL-13R alpha 2 chain appears to be overexpressed on glioma cells and may serve as a novel tumor biomarker or a target for receptor-directed therapeutic agents for brain tumors . To target IL-13 receptors, we have produced a recombinant fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (PE) . This cytotoxin, termed IL-13PE38QQR or IL-13 cytotoxin, is highly and specifically cytotoxic to a spectrum of human glioma cell lines . In preclinical models of human glioblastoma tumors growing subcutaneously in immunodeficient mice, IL-13 cytotoxin has been found to have remarkable antitumor activity . The data that emerged from these studies reveal that localized or systemic administration of IL-13 cytotoxin can produce nontoxic drug levels and that IL-13 cytotoxin is potently effective against established glioblastoma tumors . On the basis of these and other preclinical studies, we have begun a phase I clinical trial using IL-13PE38QQR for therapy of recurrent malignant glioma.

Arch Dis Child, 2003 Aug, 88(8), 715 - 8
Comparison of lung deposition of colomycin using the HaloLite and the Pari LC Plus nebulisers in patients with cystic fibrosis; Byrne NM et al.; AIM: To examine the effectiveness of delivery of nebulised colistin by the HaloLite nebuliser compared to the Pari LC Plus in patients with cystic fibrosis . METHODS: Randomised crossover trial of 15 patients aged >6 years . Inhalation of one mega unit of colistin in 3 ml diluent, labelled with technetium-99m DTPA, was used to assess lung deposition . The Pari was nebulised to dryness and one button press of the HaloLite was completed . Following a seven day washout period, patients inhaled colistin twice daily for seven days through the first device . Sputum specimens were analysed for colistin levels and pseudomonas load . This procedure was repeated with the alternative device . RESULTS: Lung uptake of radiolabelled colistin was significantly higher with the Pari . However, lung uptake calculated as a percentage of the amount of drug used was significantly higher for the HaloLite . Time to nebulise was significantly shorter with the HaloLite . Sputum levels of colistin were higher following use of the Pari; this was close to significance . CONCLUSION: The manufacturer's recommended dosages for nebulising antibiotics with a HaloLite result in a lower delivery than patients receive when using a Pari nebuliser . The concept of adaptive aerosol delivery has several theoretical advantages but the recommended doses for the HaloLite need to be modified in order to improve effectiveness.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10552 - 7 Epub 2003 Jul 21.
Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants; Schmelz EA et al.; Phytohormones regulate the protective responses of plants against both biotic and abiotic stresses by means of synergistic or antagonistic actions referred to as signaling crosstalk . A bottleneck in crosstalk research is the quantification of numerous interacting phytohormones and regulators . The chemical analysis of salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid is typically achieved by using separate and complex methodologies . Moreover, pathogen-produced phytohormone mimics, such as the phytotoxin coronatine (COR), have not been directly quantified in plant tissues . We address these problems by using a simple preparation and a GC-MS-based metabolic profiling approach . Plant tissue is extracted in aqueous 1-propanol and mixed with dichloromethane . Carboxylic acids present in the organic layer are methylated by using trimethylsilyldiazomethane; analytes are volatilized under heat, collected on a polymeric absorbent, and eluted with solvent into a sample vial . Analytes are separated by using gas chromatography and quantified by using chemical-ionization mass spectrometry that produces predominantly {M+H}+ parent ions . We use this technique to examine levels of COR, phytohormones, and volatile organic compounds in model systems, including Arabidopsis thaliana during infection with Pseudomonas syringae pv . tomato DC3000, corn (Zea mays) under herbivory by corn earworm (Helicoverpa zea), tobacco (Nicotiana tabacum) after mechanical damage, and tomato (Lycopersicon esculentum) during drought stress . Numerous complex changes induced by pathogen infection, including the accumulation of COR, salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid illustrate the potential and simplicity of this approach in quantifying signaling crosstalk interactions that occur at the level of synthesis and accumulation.

J Bacteriol, 2003 Aug, 185(15), 4530 - 8
Unusual integrase gene expression on the clc genomic island in Pseudomonas sp . strain B13; Sentchilo V et al.; An unusual type of gene expression from an integrase promoter was found in cultures of the bacterium Pseudomonas sp . strain B13 . The promoter controls expression of the intB13 integrase gene, which is present near the right end of a 105-kb conjugative genomic island (the clc element) encoding catabolism of aromatic compounds . The enzymatic activity of integrase IntB13 is essential for site-specific integration of the clc element into the bacterial host's chromosome . By creating transcription fusions between the intB13 promoter and the gfp gene, we showed that integrase expression in strain B13 was inducible under stationary-phase conditions but, strangely, occurred in only a small proportion of individual bacterial cells rather than equally in the whole population . Integrase expression was significantly stimulated by growing cultures on 3-chlorobenzoate . High cell density, heat shock, osmotic shock, UV irradiation, and treatment with alcohol did not result in measurable integrase expression . The occurrence of the excised form of the clc element and an increase in the rates of clc element transfer in conjugation experiments correlated with the observed induction of the intB13'-gfp fusion in stationary phase and in the presence of 3-chlorobenzoate . This suggested that activation of the intB13 promoter is the first step in stimulation of clc transfer . To our knowledge, this is the first report of a chlorinated compound's stimulating horizontal transfer of the genes encoding its very metabolism.

Biochemistry, 2003 Jul 22, 42(28), 8387 - 93
Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily; Schmidt DM et al.; The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction . The molecular pathway for natural divergent evolution of function in the superfamily is unknown . We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp . P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction . These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms . A comparison of the structures of AEE and the OSBS from E . coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments . Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E . coli OSBS auxotroph and have measurable levels of OSBS activity . The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction . The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines . The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.

Plant Physiol, 2003 Jul, 132(3), 1370 - 81
Regulation of Arabidopsis COPINE 1 gene expression in response to pathogens and abiotic stimuli; Jambunathan N et al.; The copines are a widely distributed class of calcium-dependent, phospholipid-binding proteins of undetermined biological function . Mutation of the Arabidopsis CPN1 (COPINE 1) gene causes a humidity-sensitive lesion mimic phenotype with increased resistance to a bacterial and an oomyceteous pathogen, constitutive pathogenesis-related gene expression, and an accelerated hypersensitive cell death defense response . Here, we show that the disease resistance phenotype of the cpn1-1 mutant was also temperature sensitive, demonstrate increased CPN1 gene transcript accumulation in wild-type plants under low-humidity conditions, and present a detailed analysis of CPN1 gene transcript accumulation in response to bacterial pathogens . In wild-type plants, CPN1 transcript accumulation was rapidly, locally, and transiently induced by both avirulent and virulent strains of Pseudomonas syringae pv tomato bacteria . However, induction of CPN1 transcript accumulation by avirulent bacteria was much faster and stronger than that induced by virulent bacteria . Bacterial induction of CPN1 transcript accumulation was dependent on a functional type III bacterial protein secretion system . In planta expression of the avrRpt2 avirulence gene was sufficient to trigger rapid CPN1 transcript accumulation . CPN1 transcript accumulation was induced by salicylic acid treatment but was not observed during lesion formation in the lesion mimic mutants lsd1 and lsd5 . These results are consistent with CPN1 playing a role in plant disease resistance responses, possibly as a suppressor of defense responses including the hypersensitive cell death defense response . The results also suggest that CPN1 may represent a link between plant disease resistance and plant acclimation to low-humidity and low-temperature conditions.

Biomacromolecules, 2003 Jul-Aug, 4(4), 1092 - 7
Fourier transform infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate; Randriamahefa S et al.; Poly(hydroxyalkanoates) PHAs are synthesized by many bacteria as inclusion bodies and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials . A method based on FT-IR was developed to carry out rapid qualitative and quantitative analysis of PHAs in Pseudomonas, when they were grown on sodium octanoate . Using absorbance of the ester band of PHAs, a rapid method was reported to distinguish PHB and PHO and to determine polymer content in intact bacteria . Relative areas in which the C=O area was normalized to the area of the peak representing the amid group (1656 cm(-1)) characteristic of bacteria were calibrated to the polymer content which was determined after solvent extraction . Polymer contents vary from 0% to 53% and depend on the nature of the bacteria . Among 27 strains of Pseudomonas belonging to the rRNA homology group I, a very low amount of bacteria were able to produce PHB . The majority of strains were able to produce a copolymer, PHO, in which the major constituent unit is 3-hydroxyoctanoate . The FT-IR results were further confirmed by gas chromatography analysis after methanolysis of polymer, but FT-IR method requires less preparation of sample than gas chromatography and it is very useful for screening a large variety of Pseudomonas.

J Chem Ecol, 2003 May, 29(5), 1159 - 65
Effect of green manure on the incidence of cyanogenic Pseudomonas strains in hop garden soils; Paszkowski WL et al.; Incidence of cyanogenic Pseudomonas strains in hop garden soils in relation to the kind of fertilization was studied . Incidence differed with respect to the fertilization treatment and the age of the plantation . Amendment of soil with rye and with white mustard as green manures limited the number of cyanogenic Pseudomonas strains relative to farmyard manures and NPK fertilization . Among all fertilization treatments, cyanogenic Pseudomonas spp . strains had lowest populations in soils amended with white mustard.

Acta Paediatr, 2003 Jun, 92(6), 688 - 93
Decreased bone mineral density in normal-growing patients with cystic fibrosis; Gronowitz E et al.; AIM: To study bone mineral density (BMD) in normal-growing patients with cystic fibrosis (CF) and its relation to clinical and biochemical markers of nutrition and lung function . METHODS: Seventy consecutive patients aged 6-49 y with CF were investigated using dual X-ray absorptiometry and the findings related to anthropometric data . Energy intake was calculated and basal metabolic rate and serum values for calcium, phosphorus, calcitonin and 25(OH) calcidiol measured . Working capacity, lung function and pseudomonas colonization were determined as parameters of physical fitness and severity of pulmonary disease . RESULTS: The average z-score of BMD was decreased in the lumbar spine in both children and adults, being -0.7 +/- 1.0 and -0.5 +/- 1.0, respectively, as was the femoral neck BMD z-score, being -0.3 +/- 0.9 and -1.1 +/- 1.0 for children and adults, respectively . BMD was correlated to lung function and working capacity, but not to anthropometric data at multiple regression analysis compensating for age and calcitonin . No correlation was found with energy intake, basal metabolic rate or biochemical markers, with the exception of calcitonin . CONCLUSION: BMD z-scores were significantly lower than those in the normal population despite normal anthropometry . Osteoporosis was rare . The strongest correlation was found with lung function . Our data indicate that BMD at all ages might be a sensitive indicator of the general status of patients with CF.

Curr Opin Biotechnol, 2003 Jun, 14(3), 248 - 54
Genomic islands and the evolution of catabolic pathways in bacteria; van der Meer JR et al.; Genes for the degradation of organic pollutants have usually been allocated to plasmid DNAs in bacteria or considered non-mobile when detected in the chromosome . New discoveries have shown that catabolic genes can also be part of so-called integrative and conjugative elements (ICElands), a group of mobile DNA elements also known as genomic islands and conjugative transposons . One such ICEland is the clc element for chlorobenzoate and chlorocatechol degradation in Pseudomonas sp . strain B13 . Genome comparisons and genetic data on integrase functioning reveal that the clc element and several other unclassified ICElands belong to a group of elements with conserved features . The clc element is unique among them in carrying the genetic information for several degradation pathways, whereas the others give evidence for pathogenicity functions . Many more such elements may exist, bridging the gap between pathogenicity and degradation functions.

Plant J, 2003 Jul, 35(2), 206 - 18
The lower cell density of leaf parenchyma in the Arabidopsis thaliana mutant lcd1-1 is associated with increased sensitivity to ozone and virulent Pseudomonas syringae; Barth C et al.; Under optimal growth conditions (120 micro mol photons m-2 sec-1 photosynthetically active radiation (PAR), 16-h photoperiod), the recessive ozone-sensitive Arabidopsis thaliana L . Heynh . mutant lcd1-1 exhibits a pale phenotype compared to the wild type . Confocal and multiphoton microscopy revealed that the paleness of lcd1-1 is because of a lower cell density in the leaf palisade parenchyma, resulting in decreased chlorophyll content . When exposed to ozone, lcd1-1 leaves become paler and contain an increased amount of the lipid peroxidation product malondialdehyde compared to the wild type, suggesting that lcd1-1 suffers from elevated levels of reactive oxygen species (ROS) generated in the apoplast . Infection of leaves with virulent Pseudomonas syringae reveals higher bacterial growth as well as lower pathogenesis-related protein 1 (PR-1) and PR-5 expression in lcd1-1 than in the wild type . When the wild type and lcd1-1 are exposed to short-term high-light stress, leaves do not bleach in lcd1-1 and potential activities of photosystems I (PSI) and II (PSII) decrease to a similar extent in both the genotypes, indicating that the photosynthetic apparatus is not affected by lcd1-1 mutation . The LCD1 gene, found to contain a nonsense mutation in the mutant, has been identified . It is located at the bottom of chromosome 2 of the Arabidopsis genome . However, the function of the protein encoded by LCD1 is not yet known . We hypothesize that LCD1 plays a role in normal leaf development, and that the increased sensitivity to ozone and virulent P . syringae is a secondary effect that presumably results from the lower-cell-density phenotype in lcd1-1.

Mol Plant Microbe Interact, 2003 Jul, 16(7), 588 - 99
Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant; Nandi A et al.; Salicylic acid (SA), ethylene, and jasmonic acid (JA) are important signaling molecules in plant defense to biotic stress . An intricate signaling network involving SA, ethylene, and JA fine tunes plant defense responses . SA-dependent defense responses in Arabidopsis thaliana are mediated through NPR1-dependent and -independent mechanisms . We have previously shown that activation of an NPR1-independent defense mechanism confers enhanced disease resistance and constitutive expression of the pathogenesis-related (PR) genes in the Arabidopsis ssi1 mutant . In addition, the ssi1 mutant constitutively expresses the defensin gene PDF1.2 . Moreover, SA is required for the ssi1-conferred constitutive expression of PDF1.2 in addition to PR genes . Hence, the ssi1 mutant appears to target a step common to SA- and ethylene- or JA-regulated defense pathways . In the present study, we show that, in addition to SA, ethylene and JA signaling also are required for the ssi1-conferred constitutive expression of PDF1.2 and the NPR1-independent expression of PR-1 . Furthermore, the ethylene-insensitive ein2 and JA-insensitive jar1 mutants enhance susceptibility of ssi1 plants to the necrotrophic fungus Botrytis cinerea . However, defects in either the ethylene- or JA-signaling pathways do not compromise ssi1-conferred resistance to the bacterial pathogen Pseudomonas synringae pv . maculicola and the oomycete pathogen Peronospora parasitica . Interestingly, ssi1 exhibits a marginal increase in the levels of ethylene and JA, suggesting that low endogenous levels of these phytohormones are sufficient to activate expression of defense genes . Taken together, our results indicate that although cross talk in ssi1 renders expression of ethylene- or JA-responsive defense genes sensitive to SA and vice versa, it does not affect downstream signaling leading to resistance.

Mol Genet Genomics, 2003 Aug, 269(5), 583 - 91 Epub 2003 Jul 01.
Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen, but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana; Sharma PC et al.; Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens . We isolated cDNAs for these MAPKs (NbWIPKand NbSIPK) from Nicotiana benthamiana . The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen (Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N . benthamiana . Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P . cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.

J Bacteriol, 2003 Jul, 185(14), 4195 - 203
Probing the role of divalent metal ions in a bacterial psychrophilic metalloprotease: binding studies of an enzyme in the crystalline state by x-ray crystallography; Ravaud S et al.; The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity . Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease . Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites . Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator . Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme . Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted approximately 4, 1.0, and 1.6 A, respectively . Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions . A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions.

Plant Cell Rep, 2003 Jun, 21(10), 1027 - 34 Epub 2003 Apr 26.
Characterization of salicylic acid-induced genes in Chinese cabbage; Park YS et al.; Salicylic acid is a messenger molecule in the activation of defense responses in plants . In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp . pekinensis) by subtractive hybridization . Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein . The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein . The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase . Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain . The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences . These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole . Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv . tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage . None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.

Vet Microbiol, 2003 Jul 30, 94(4), 325 - 33
Existence of two O-serotypes in the fish pathogen Pseudomonas anguilliseptica; Lopez-Romalde S et al.; The serological characteristics of a group of 32 Pseudomonas anguilliseptica strains isolated in Spain from seabream (Sparus aurata) and turbot (Scophthalmus maximus) were compared with a total of 18 collection strains of this bacterial species with different geographical and host origin . The employment of different techniques, including slide agglutination, microagglutination and dot blot, allowed us to establish two serological groups, one comprising practically all the eel isolates, and the other including the majority of isolates from other fish species . The study of the lipopolysaccharides (LPS) and outer membrane proteins (OMP) corroborated these results, indicating that the serological differences among strains are due to the somatic antigen and not to antigenic determinants of protein nature . Therefore, a serological scheme of two "O" serotypes is proposed for P . anguilliseptica . The results obtained will be of importance for epidemiological studies as well as for the design of adequate vaccine formulations.

Mol Microbiol, 2003 Jul, 49(2), 389 - 400
A translocated protein tyrosine phosphatase of Pseudomonas syringae pv . tomato DC3000 modulates plant defence response to infection; Bretz JR et al.; Pseudomonas syringae strains translocate effector proteins into host cells via the hrp-encoded type III protein secretion system (TTSS) to facilitate pathogenesis in susceptible plants . However, the mechanisms by which pathogenesis is favoured by these effectors are not well understood . Individual strains express multiple effectors with apparently distinct activities that are co-ordinately regulated by the alternative sigma factor HrpL . Genes for several effectors were identified in the P . syringae pv . tomato DC3000 genome using a promoter trap assay to identify HrpL-dependent promoters . In addition to orthologues of avrPphE and hrpW, an unusual allele of avrPphD was detected that carried an IS52 insertion . Using this avrPphD::IS52 allele as a probe, a wild-type allele of avrPphD, hopPtoD1, and a chimeric homologue were identified in the DC3000 genome . This chimeric homologue, identified as HopPtoD2 in the annotated DC3000 genome, consisted of an amino terminal secretion domain similar to that of AvrPphD fused to a potential protein tyrosine phosphatase domain . Culture filtrates of strains expressing HopPtoD2 were able to dephosphorylate pNPP and two phosphotyrosine peptides . HopPtoD2 was shown to be translocated into Arabidopsis thaliana cells via the hrp-encoded TTSS . A DeltahopPtoD2 mutant of DC3000 exhibited strongly reduced virulence in Arabidopsis thaliana . Ectopic expression of hopPtoD2 in P . syringae Psy61 that lacks a native hopPtoD2 orthologue delayed the development of several defence-associated responses including programmed cell death, active oxygen production and transcription of the pathogenesis-related gene PR1 . The results indicate that HopPtoD2 is a translocated effector with protein tyrosine phosphatase activity that modulates plant defence responses.

Mol Microbiol, 2003 Jul, 49(2), 377 - 87
The Pseudomonas syringae type III-secreted protein HopPtoD2 possesses protein tyrosine phosphatase activity and suppresses programmed cell death in plants; Espinosa A et al.; The bacterial plant pathogen Pseudomonas syringae possesses a type III protein secretion system that delivers many virulence proteins into plant cells . A subset of these proteins (called Avr proteins) is recognized by the plant's innate immune system and triggers defences . One defence-associated response is the hypersensitive response (HR), a programmed cell death (PCD) of plant tissue . We have previously identified HopPtoD2 as a type III secreted protein from P . s . pv . tomato DC3000 . Sequence analysis revealed that an N-terminal domain shared homology with AvrPphD and a C-terminal domain was similar to protein tyrosine phosphatases (PTPs) . We demonstrated that purified HopPtoD2 possessed PTP activity and this activity required a conserved catalytic Cys residue (Cys(378)) . Interestingly, HopPtoD2 was capable of suppressing the HR elicited by an avirulent P . syringae strain on Nicotiana benthamiana . HopPtoD2 derivatives that lacked Cys(378) no longer suppressed the HR indicating that HR suppression required PTP activity . A constitutively active MAPK kinase, called NtMEK2DD, is capable of eliciting an HR-like cell death when transiently expressed in tobacco . When NtMEK2DD and HopPtoD2 were co-delivered into plant cells, the HR was suppressed indicating that HopPtoD2 acts downstream of NtMEK2DD . DC3000 hopPtoD2 mutants were slightly reduced in their ability to multiply in planta and displayed an enhanced ability to elicit an HR . The identification of HopPtoD2 as a PTP and a PCD suppressor suggests that the inactivation of MAPK pathways is a virulence strategy utilized by bacterial plant pathogens.

Plant Mol Biol, 2003 May, 52(1), 143 - 59
Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains; Reddy VS et al.; Ca2+ and calmodulin (CaM), a key Ca2+ sensor in all eukaryotes, have been implicated in defense responses in plants . To elucidate the role of Ca2+ and CaM in defense signaling, we used 35S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv . tabaci . Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated . The PICBP fusion proteins bound 35S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca2+ whereas EGTA, a Ca2+ chelator, abolished binding, confirming that PICBP binds CaM in a Ca2+-dependent manner . Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified . The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats . The PICBP gene is differentially expressed in tissues with the highest expression in stem . The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv . tomato carrying avrRpm1 . Furthermore, PICBP is constitutively expressed in the Arabidopsis accelerated cell death2-2 mutant . The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains . In addition, the hrp1 mutant of Pseudomonas syringae pv . tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean . Our data suggest a role for PICBP in Ca2+-mediated defense signaling and cell-death . Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca2+-dependent CaM-binding domains.

Mol Microbiol, 2003 Jul, 49(1), 93 - 104
Characterization of two alternative promoters for integrase expression in the clc genomic island of Pseudomonas sp . strain B13; Sentchilo V et al.; The clc genomic island is a 105 kb integrative and conjugative element (ICE) in Pseudomonas sp . strain B13, which encodes metabolism of 3-chlorocatechol . The clc island is integrated in a tRNAGly gene, but can excise and form a circular intermediate in which both ends are connected . The integrase gene (intB13) of the clc genomic island is located at the right end, 202 bp from the junction site facing inwards . Fragments upstream of intB13 in the circular form and in the integrated form were fused to a promoterless gfp gene for Green Fluorescent Protein and introduced in monocopy onto the chromosome of strain B13 . Quantitative GFP fluorescence measurements in individual cells of the different B13-derivatives revealed that the circular form fragment contained a strong constitutive promoter (Pcirc) driving intB13 expression in all cells . By using primer extension Pcirc could be mapped near the left end of the clc element and Pcirc can therefore only control intB13 expression when left and right ends are connected as in the circular form . Expression from intB13 upstream fragments from the integrated clc element was weaker than that from Pcirc and only occurred in maximally 15% of individual cells in a culture . A promoter (Pint) could be roughly mapped in this region by using reverse-transcription PCR and by successively shortening the fragment from the 5' end . Transposon mutants in cloned left end sequences of the clc element were selected which had lost the activation potential on the Pint promoter and those which resulted in overexpression of GFP from Pint . The DNA sequence of the region of the transposon insertions pointed to a relatively well conserved area among various other genomic islands . The activator mutants mapped in an open reading frame (ORF) encoding a 175 amino acid protein without any significant similarity to functionally characterized proteins in the databases.

Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8577 - 82 Epub 2003 Jun 19.
A Pseudomonas syringae type III effector suppresses cell wall-based extracellular defense in susceptible Arabidopsis plants; Hauck P et al.; Bacterial effector proteins secreted through the type III secretion system (TTSS) play a crucial role in causing plant and human diseases . Although the ability of type III effectors to trigger defense responses in resistant plants is well understood, the disease-promoting functions of type III effectors in susceptible plants are largely enigmatic . Previous microscopic studies suggest that in susceptible plants the TTSS of plant-pathogenic bacteria transports suppressors of a cell wall-based plant defense activated by the TTSS-defective hrp mutant bacteria . However, the identity of such suppressors has remained elusive . We discovered that the Pseudomonas syringae TTSS down-regulated the expression of a set of Arabidopsis genes encoding putatively secreted cell wall and defense proteins in a salicylic acid-independent manner . Transgenic expression of AvrPto repressed a similar set of host genes, compromised defense-related callose deposition in the host cell wall, and permitted substantial multiplication of an hrp mutant . AvrPto is therefore one of the long postulated suppressors of an salicylic acid-independent, cell wall-based defense that is aimed at hrp mutant bacteria.

J Bacteriol, 2003 Jul, 185(13), 3718 - 25
Repression of phenazine antibiotic production in Pseudomonas aureofaciens strain 30-84 by RpeA; Whistler CA et al.; Pseudomonas aureofaciens strain 30-84 is a biological control bacterium that utilizes a two-component GacS/GacA regulatory system interconnected with the PhzR/PhzI quorum sensing system to positively regulate biosynthesis of phenazine antibiotics that contribute to its association with plant hosts . To date, no negative regulators of phenazine production have been identified, nor has the role of repression been studied . Here we describe a novel repressor of secondary metabolism in P . aureofaciens strain 30-84, RpeA, whose deduced amino acid sequence is similar to those of a group of putative two-component regulatory systems of unknown function found in several animal and plant-pathogenic bacteria . In minimal medium where phenazine production is very low, inactivation of the rpeA gene enhanced phenazine biosynthetic gene expression and increased phenazine production but did not increase quorum sensing signal accumulation . Furthermore, RpeA functioned to block phenazine biosynthetic gene transcription in minimal medium even when quorum-sensing signals were at a level that was sufficient for induction of phenazine gene expression in rich medium . Additionally, in the absence of rpeA, the quorum sensor PhzR was not required for phenazine production . Although repression plays a critical role in phenazine regulation, the rpeA mutation could not bypass the requirement for a functional GacS/GacA system, demonstrating that activation is required even in the absence of the RpeA repressor . This study reinforces that multiple signals, including nutrition and population density, are integrated to control the appropriate expression of phenazine antibiotics.

Cancer Res, 2003 Jun 15, 63(12), 3257 - 62
Specific tumoricidal activity of a secreted proapoptotic protein consisting of HER2 antibody and constitutively active caspase-3; Jia LT et al.; In this study, a novel approach to antitumor therapy was devised by generating a chimeric tumor-targeted killer protein, referred to as immunocasp-3, that comprises a single-chain anti-erbB2/HER2 antibody with a NH(2)-terminal signal sequence, a Pseudomonas exotoxin A translocation domain, and a constitutively active caspase-3 molecule . In principle, cells transfected with the immunocasp-3 gene would express and secrete the chimeric protein, which then binds to HER2-overexpressing tumor cells . Subsequent cleavage of the constitutively active capase-3 domain from the immunocasp-3 molecule and its release from internalized vesicles would lead to apoptotic tumor cell death . To test this strategy, we transduced human lymphoma Jurkat cells with a chimeric immunocasp-3 gene expression vector and showed that they not only expressed and secreted the fusion protein but also selectively killed tumor cells overexpressing HER2 in vitro . i.v . injection of the transduced Jurkat cells led to tumor regression in a mouse xenograft model because of continuous secretion of immunocasp-3 by the transduced cells . The growth of HER2-positive tumor cells in this model was inhibited by i.m . as well as intratumor injection of immunocasp-3 expression plasmid DNA, indicating that the immunocasp-3 molecules secreted by transfected cells have systematic antitumor activity . We conclude that the immunocasp-3 molecule, combining the properties of a tumor-specific antibody with the proapoptotic activity of a caspase, has potent and selective antitumor activity, either as cell-based therapy or as a DNA vaccine . These findings provide a compelling rationale for therapeutic protocols designed for erbB2/HER2-positive tumors.

Phytochemistry, 2003 Jul, 63(5), 505 - 15
Vanillin; Walton NJ et al.; Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an important flavour and aroma molecule, but is also of interest because of its biogenetic relationship to the phenylpropanoid pathway and to other molecules of physiological significance, notably salicylate . Recent progress towards characterisation of the biosynthesis of vanillin is reviewed . In Vanilla, there is some evidence that the route to vanillin-beta-D-glucoside may proceed from 4-coumaric acid via 4-hydroxybenzaldehyde, with glucoside formation occurring not necessarily as the final step, and possibly with the involvement of 4-hydroxybenzyl alcohol beta-D-glucoside tartrate bis-esters as "shunt" metabolites . This appears to be given tentative support by the recent partial characterisation of a 4-hydroxybenzaldehyde synthase from Vanilla . On the other hand, a well-characterised, CoA-dependent, non-oxidative chain-shortening mechanism to produce vanillin from ferulic acid, occurring as part of a pathway of hydroxycinnamate degradation in Pseudomonas, may not be representative of hydroxycinnamate chain-shortening mechanism(s) occurring in Vanilla and other plants . Nevertheless, by expression of the Pseudomonas enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), attempts have been made to introduce a direct capacity for vanillin formation into model plants by diversion of the phenylpropanoid pathway . The results obtained have emphasised the obstacles to achieving the desired oxidation level (aldehyde) and ring substitution (4-hydroxy-3-methoxyphenyl), even when a substantial metabolic diversion is successfully achieved . Finally, the significance of the latest biosynthetic and biotechnological developments is reviewed briefly in relation to authentication of vanillin.

Plant Physiol, 2003 Jun, 132(2), 840 - 7
The pattern of systemic acquired resistance induction within the Arabidopsis rosette in relation to the pattern of translocation; Kiefer IW et al.; Local leaf infections by a necrogenic pathogen can lead to systemic acquired resistance (SAR) in untreated leaves . We reasoned that, whatever the nature of the long-distance signal, if it is transported in the phloem, the pattern of SAR induced within the plant by treatment of a single leaf should match the pattern of translocation out of that leaf . The source-sink relationships (orthostichies) in the Arabidopsis rosette were established with {14C}Suc or phloem-mobile 3-aminotriazole at herbicidal concentrations . SAR was activated by infiltrating a single Columbia leaf with Pseudomonas syringae pv maculicola DC3000/avrRPM1, which causes a hypersensitive response . The pattern of SAR in the rosette was monitored by assessing the growth of wild-type DC3000 and by measuring the SAR markers salicylic acid and PR1 transcripts . Although the orthostichy of a single leaf was clearly limited to a row of vertically aligned leaves, SAR and SAR markers were also found outside the orthostichy . This indicates that, whatever the nature of the long-distance signal from the treated leaf to the upper responding leaves, its transport is either not limited exclusively to the phloem or the minor proportion of translocate that is not confined to the orthostichy contains enough of the SAR systemic signal to set in motion events leading to the establishment of the SAR state in the upper leaves.

Plant Physiol, 2003 Jun, 132(2), 606 - 17 Epub 2003 May 15.
Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling; van Wees SC et al.; All tested accessions of Arabidopsis are resistant to the fungal pathogen Alternaria brassicicola . Resistance is compromised by pad3 or coi1 mutations, suggesting that it requires the Arabidopsis phytoalexin camalexin and jasmonic acid (JA)-dependent signaling, respectively . This contrasts with most well-studied Arabidopsis pathogens, which are controlled by salicylic acid-dependent responses and do not benefit from absence of camalexin or JA . Here, mutants with defects in camalexin synthesis (pad1, pad2, pad3, and pad5) or in JA signaling (pad1, coi1) were found to be more susceptible than wild type . Mutants with defects in salicylic acid (pad4 and sid2) or ethylene (ein2) signaling remained resistant . Plant responses to A . brassicicola were characterized using expression profiling . Plants showed dramatic gene expression changes within 12 h, persisting at 24 and 36 h . Wild-type and pad3 plants responded similarly, suggesting that pad3 does not have a major effect on signaling . The response of coi1 plants was quite different . Of the 645 genes induced by A . brassicicola in wild-type and pad3 plants, 265 required COI1 for full expression . It is likely that some of the COI1-dependent genes are important for resistance to A . brassicicola . Responses to A . brassicicola were compared with responses to Pseudomonas syringae infection . Despite the fact that these pathogens are limited by different defense responses, approximately 50% of the induced genes were induced in response to both pathogens . Among these, requirements for COI1 were consistent after infection by either pathogen, suggesting that the regulatory effect of COI1 is similar regardless of the initial stimulus.

FEBS Lett, 2003 Jun 19, 545(2-3), 188 - 92
Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family; Shanklin J et al.; Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases . No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains . To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB . These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation . These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7622 - 5 Epub 2003 Jun 11.
Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase; Farver O et al.; Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water . Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have been studied and found to be dominated by pronounced interactions between the c and the d1 hemes . The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity . The results constitute a prime example of intraprotein control of the electron-transfer rates by allosteric interactions.

J Am Chem Soc, 2003 Jun 18, 125(24), 7200 - 8
A strategy for the NMR characterization of type II copper(II) proteins: the case of the copper trafficking protein CopC from Pseudomonas Syringae; Arnesano F et al.; CopC from Pseudomonas syringae was found to be a protein capable of binding both Cu(I) and Cu(II) at two different sites . The solution structure of the apo protein is available, and structural information has been obtained on the Cu(I) bound form . We attempt here to set the limits for the determination of the solution structure of a Cu(II) protein, such as the Cu(II) bound form of CopC, in which the Cu(II) ion takes a type II coordination . The electron relaxation time is estimated from NMRD measurements to be 3 ns which leads to a correlation time for the nuclear spin-electron spin dipolar interaction of 2 ns . This information allowed us to tailor the NMR experiments and to fully exploit purely heteronuclear spectroscopy to assign as many signals as possible . In this way, 37 (13)C and 11 (15)N signals that completely escape detection with conventional approaches were assigned . Paramagnetic based structural constraints were obtained by measuring paramagnetic longitudinal relaxation enhancements (rho(para)) which allowed us to precisely locate the copper ion within the protein frame . Pseudocontact shifts (pcs's) were also used as constraints for 83 (1)H and 18 (13)C nuclei . With them, together with other standard structural constraints, a structure is obtained (and submitted to PDB) where information is only missing in a sphere with a 6 A radius from the copper ion . If we borrow information from EXAFS data, which show evidence of two copper coordinated histidines, then His 1 and His 91 are unambiguously identified as copper ligands . EXAFS data indicate two more light donor atoms (O/N) which could be from Asp 27 and Glu 89, whereas the NMRD data indicate the presence of a semicoordinated water molecule at 2.8 A (Cu-O distance) roughly orthogonal to the plane identified by the other four ligands . This represents the most extensively characterized structure of a type II Cu(II) protein obtained employing the most advanced NMR methods and with the aid of EXAFS data . The knowledge of the location of the Cu(II) in the protein is important for the copper transfer mechanism.

Chest, 2003 Jun, 123(6), 2130 - 9
Does deficiency of arylsulfatase B have a role in cystic fibrosis?
Tobacman JK.
Cystic fibrosis (CF) is associated with mutation and abnormal function of the cystic fibrosis transmembrane conductance regulator (CFTR) that affects cellular chloride transport . Clinically, CF of the lung is associated with excessive accumulation of secretions, including the sulfated glycosaminoglycans, chondroitin sulfate and dermatan sulfate (DS), both of which contain sulfated N-acetylgalactosamine residues . The sulfatase enzymes, which are a highly conserved group of enzymes with high specificity for designated sulfate groups, include arylsulfatase B, a lysosomal enzyme . Arylsulfatase B, also known as N-acetyl galactosamine 4-sulfatase, can degrade DS and chondroitin-4 sulfate . Previously reported data demonstrated diminished activity of arylsulfatase B in lymphoid cell lines of patients with CF compared to normal control subjects . Frequent infections with Pseudomonas, a sulfatase-producing organism, occur in patients with CF, whereas infections with Mycobacterium tuberculosis, which lacks sulfatase activity, are infrequent . Additional investigation to determine if diminished function of arylsulfatase B is a consistent finding in cells of patients with CF may be informative, and may help to correlate the molecular, biochemical, and clinical characteristics of CF.

Mol Plant Microbe Interact, 2003 Jun, 16(6), 495 - 507
Novel exchangeable effector loci associated with the Pseudomonas syringae hrp pathogenicity island: evidence for integron-like assembly from transposed gene cassettes; Charity JC et al.; Pseudomonas syringae strains use a type III secretion system (TTSS) to translocate effector proteins that assist in the parasitism of host plant cells . Some genes for effector proteins are clustered in the exchangeable effector locus (EEL) associated with the hrp pathogenicity island . A polymerase chain reaction-based screen was developed to amplify the EEL from P . syringae strains . Of the 86 strains screened, the EEL was successfully amplified from 30 predominately North American P . syringae pv . syringae strains using hrpK and queA-derived primers and from an additional three strains using hrpL and queA-derived primers . Among the amplified EEL, ten distinct types of EEL were identified that could be classified into six families distinguishable by genetic composition, but other types of EEL may be present in strains isolated in other geographical regions . No linkage with the host range of the source strain was apparent . Gene cassettes carrying conserved flanking, coding, and intergenic sequences, present in different combinations, were identified in the characterized EEL . Six new alleles of known effectors were identified that differed from the homolog in sequence, size, or both of the gene . One of these apparently novel effector proteins, HopPsyB, retained a strongly conserved amino terminus similar to that of HopPsyA, but other regions of the two polypeptides were only weakly similar . hopPsyB was expressed from an apparent operon that included hrpK and a shcA homolog, shcB . Escherichia coli MC4100 expressing the hrp TTSS, ShcB, and HopPsyB elicited the hypersensitive response (HR) in tobacco, consistent with effector production . Indicative of translocation as an effector, P . syringae pv . tomato DC3000 expressing a HopPsyB':'AvrRpt2 fusion elicited the HR in RPS2+ Arabidopsis thaliana . P . syringae pv . tomato DC3000 carrying HopPsyB exhibited slightly enhanced virulence in several Brassica spp . These results are consistent with the hypotheses that the EEL is a source of disparate effectors functioning in pathogenicity of P . syringae strains and that it evolved independently of the hrp pathogenicity island central conserved region, most likely through integron-like assembly of transposed gene cassettes.

Biotechnol Prog, 2003 May-Jun, 19(3), 734 - 8
Small RNA sequences are readily stabilized by inclusion in a carrier rRNA; D'Souza LM et al.; This laboratory previously showed that an RNA derived from 5S ribosomal RNA could be used as a carrier to harbor a nucleic acid "tag" for monitoring genetically engineered or naturally occurring bacteria . The prototype system expressed a specific tagged RNA that was stable and accumulated to high levels . For such a system to be useful there should, however, be little limitation on the sequence composition and length of the insert . To test these limitations, a collection of insertion sequences were created and introduced into the artificial 5S rRNA cassette . This library consisted of random 13- and 50-base oligonucleotides that were inserted into the carrier RNA . We report here that essentially all of the insert-containing RNAs are stable and accumulate to detectable levels . Tagged RNAs were produced by both plasmid-borne and chromosomally integrated expression systems in E . coli and several Pseudomonas strains without obvious effect on the host cell . It is anticipated that in addition to its intended use in environmental monitoring, this system can be used for in vivo selection of useful artificial RNAs . Because the carrier lends stability to the RNAs, the system may also be useful in RNA production.

Appl Environ Microbiol, 2003 Jun, 69(6), 3653 - 7
On the origins of cyanuric acid hydrolase: purification, substrates, and prevalence of AtzD from Pseudomonas sp . strain ADP; Fruchey I et al.; Cyanuric acid hydrolase (AtzD) from Pseudomonas sp . strain ADP was purified to homogeneity . Of 22 cyclic amides and triazine compounds tested, only cyanuric acid and N-methylisocyanuric acid were substrates . Other cyclic amidases were found not to hydrolyze cyanuric acid . Ten bacteria that use cyanuric acid as a sole nitrogen source for growth were found to contain either atzD or trzD, but not both genes.

Biotechnol Bioeng, 2003 Aug 5, 83(3), 274 - 81
Lipase-catalyzed ethanolysis of fish oils: multi-response kinetics; Torres CF et al.; The kinetics of the lipase-catalyzed (Pseudomonas cepacia) ethanolysis of fish oil has been studied in a batch reactor using menhaden oil, tuna oil, and acylglycerol mixtures derived from menhaden oil . Multi-response models derived from a generalized Michaelis-Menten mechanism were developed to describe the rates of formation of ethyl esters of the primary fatty acids present in the precursor oil . A first-order model for deactivation of the lipase was fit simultaneously to one of the data sets .

Burns, 2003 Jun, 29(4), 381 - 4
Proliferative "crescentic" glomerulonephritis in a burned patient; Deveci M et al.; Acute renal failure is one of the major complications of burn and it is accompanied by a high mortality rate . However, acute glomerulonephritis due to major burn have not been reported in burn literature . We report a case of crescentic glomerulonephritis which began at 27 days postburn . In this case glomerulonephritis may be due to infection probably pseudomonas or enterococus sepsis . We also felt that imipenem may be contributed the formation of glomerulonephritis.

Transgenic Res, 2003 Jun, 12(3), 293 - 304
Monitoring the spread of recombinant DNA from field plots with transgenic sugar beet plants by PCR and natural transformation of Pseudomonas stutzeri; Meier P et al.; Previous studies had shown that recombinant DNA can be detected for several months in soil after the deposition of litter from transgenic (tg) plants . Here we show by PCR monitoring of field releases of tg sugar beet plants that during the growth of the plants the soil close to the plants and also plant material contains recombinant DNA, in the form of extracellular molecules . Surprisingly, the monitoring also revealed the presence of tg DNA in many field plots (30-70%) in which tg plants were never grown . These studies and the further monitoring during other tg sugar beet release experiments by PCR and a novel bioassay (measuring the transforming potential of recombinant DNA for Pseudomonas stutzeri) indicated that recombinant DNA was only detectable in the surface soil of field plots and their vicinity where flowering of the tg beet plants was allowed . Recombinant DNA was found in soil at a distance of 50 m from pollen-producing plants surrounded by a strip with hemp plants as a containment regime . It is concluded that recombinant DNA is deposited in soil during the growth of tg sugar beets and that a major mechanism of recombinant DNA spread in the environment is the dispersal of pollen which allows recombinant DNA to persist in the field plot for at least a year.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 953 - 4 Epub 2003 Apr 25.
Crystallization and preliminary X-ray crystallographic analysis of d-phenylglycine aminotransferase from Pseudomonas stutzeri ST201; Kongsaeree P et al.; d-Phenylglycine aminotransferase (d-PhgAT) catalyzes the reversible transamination of d-phenylglycine to l-glutamate with 2-oxoglutarate as the amino-group acceptor . Crystals of substrate-free Pseudomonas stutzeri d-PhgAT bound to the cofactor pyridoxal-5'-phosphate (PLP) were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant . The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 75.155, c = 147.554 A . The asymmetric unit contains one molecule of d-PhgAT and has a solvent content of 50.0% . A complete native X-ray diffraction data set was collected from a single crystal at 100 K to a resolution of 2.3 A.

Plant Mol Biol, 2003 Apr, 51(6), 913 - 24
Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpin(pss)-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS); Dayakar BV et al.; The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection . Harpin(pss), an elicitor from the bacterium Pseudomonas syringae pv . syringae, induces a HR in non-host plants . Previously, we reported an amphipathic protein from sweet pepper interfering with harpin(pss)-mediated HR . In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper . This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity . Recombinant PFLP obtained from Escherichia coli was able to significantly increase active oxygen species (AOS) generation when mixed with harpin(pss) in tobacco suspension cells . It also showed enhanced HR when co-infiltrated with harpin(pss) in tobacco leaves . We used a transgenic tobacco suspension cells system that constitutively expresses the Pflp gene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin(pss)-mediated hypersensitive cell death in vivo . In response to harpin(pss), suspension cells derived from Pflp transgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type . AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl3) were used to study the involvement of AOS in harpin(pss)-induced cell death . Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent . Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells . It is possible that PFLP can change the redox state of the cell upon harpin(pss) inoculation to increase AOS generation and hypersensitive cell death . Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.

BMC Bioinformatics . 2003 May 22;4(1):19.
Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences; Purohit HJ et al.; BACKGROUND: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions . We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases . Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus . RESULTS: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene . Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified . The sub-sequences between the repeating patterns yielded a continuous region of 495 bases . The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern . A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns . Nine patterns in this stretch showed nearly 70% specificity to the target genus . These patterns were further used to obtain a signature that is highly specific to Pseudomonas . The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment . CONCLUSIONS: The developed approach was successfully applied to genus Pseudomonas . It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

Rev Gastroenterol Peru, 2003 Jan-Mar, 23(1), 17 - 21
{Percutaneous drainage of hepatic pyogenic abscess: management efficacy}; Bazan Portocarrero S et al.; OBJECTIVE: To show the success of percutaneous drainage combined with an antibiotic therapy in the management of hepatic pyogenic abscess . EQUIPMENT AND METHODS: Health histories of 24 patients diagnosed with hepatic pyogenic abscess were evaluated in the Unit of Vascular and Intervention Radiology (URVI) of the Eduardo Rebagliatti Martins Hospital and were checked, during the time period beginning in January 2001 and ending in June 2002 . 23 patients underwent percutaneous drainage, guided by echography . RESULTS: A total of 36 abscesses were found, with an average diameter of 6,78 cm (3-18cm); the most common location was on the right (78%) . In 37,5% of the patients, the cause of the abscess could not be determined; in 33,3%, the cause was determined after surgical intervention, primarily cholecystectomy (12,5%) . Pseudomona (12,5%) was the species most found in cultivation . Only 28 abscesses were drained percutaneously . On average, drainage lasted 15,8 days, and there was an average of 3,6 controls per patient . There was 89,30% overall success for the procedure with three documented errors . CONCLUSION: Percutaneous drainage in conjunction with proper antibiotic coverage is efficient in the management of hepatic pyogenic collections, and its use must be generalized.

Biochem Biophys Res Commun, 2003 Jun 13, 305(4), 862 - 8
Oxygen-evolving enhancer protein 2 is phosphorylated by glycine-rich protein 3/wall-associated kinase 1 in Arabidopsis; Yang EJ et al.; The Arabidopsis wall-associated receptor kinase, WAK1, is a member of WAK family that links the plasma membrane to the extracellular matrix . A glycine-rich secreted protein, AtGRP-3, was previously shown to regulate WAK1 functions through binding to the extracellular domain of WAK1 . In this study, we sought to determine the downstream molecules of the AtGRP-3/WAK1 signaling pathway, by using two-dimensional gel electrophoresis combined with Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) . We report here that a chloroplast protein, oxygen-evolving enhancer protein 2 (OEE2), specifically interacts with the cytoplasmic kinase domain of WAK1 and becomes phosphorylated in an AtGRP-3-dependent manner . The phosphorylation of OEE2 is also induced in Arabidopsis by treatment with avirulent Pseudomonas syringae . Taken together, these results suggest that OEE2 activity is regulated by AtGRP-3/WAK1.

Protein Expr Purif, 2003 Jun, 29(2), 244 - 51
Expression of Pseudomonas stutzeri Zobell cytochrome c-551 and its H47A variant in Escherichia coli; Miller GT et al.; The nirM gene encoding cytochrome c-551 from Pseudomonas stutzeri Zobell (PZ) has been expressed in Escherichia coli at levels higher than those previously reported but only under strict anaerobic growth conditions . Expression yields for wild-type cytochrome in this study typically reached 0.6 micromol per liter of saturated E . coli culture (5.5mg/L) . Culture conditions investigated are compared to obtained c-551 expression levels; the results may lead to a greater understanding of the challenges encountered when expressing c-type hemoproteins in E . coli . The nirM gene was mutated to produce a histidine-47-alanine mutation of c-551 that been heterologously expressed in E . coli using optimum culture conditions and had its physiochemical properties compared to those of the wild-type protein . In PZ, the histidine-47 residue is part of a conserved hydrogen-bonding network located at the bottom of the heme crevice that also involves tryptophan-56 and a heme propionate . Ionization events within this network are experimentally demonstrated to modulate c-551 oxidation-reduction potential and its observed dependence on pH around neutrality . The redox potential of the mutant cytochrome still displays pH-dependence; however, the midpoint potential is approximately 25mV lower with respect to wild-type c-551 at neutral pH while the pK at which the heme propionate (HP-17) ionizes is lowered by 1.3 pH units . Temperature and chemical denaturant studies also show that loss of the hydrogen-bond-donating imidazole leads to a large decrease in c-551 tertiary stability.

J Ind Microbiol Biotechnol, 2003 May, 30(5), 322 - 6 Epub 2003 May 22.
Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases; Solaiman DK; Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes ( phaC1(Pre), phaC2(Pre)) responsible for the production of intracellular medium-chain-length (mcl-)PHAs . Sequence analysis showed that the putative gene-products of these genes contain a conserved alpha/beta-hydrolase fold in the carboxy-terminal half of the proteins . Hybrid genes pha7 and pha8 were constructed by exchanging the alpha/beta-hydrolase-fold coding portions of phaC1(Pre) and phaC2(Pre) at the 3' terminal . When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73-75% beta-hydroxydecanoate (beta-HD) and 25-27% beta-hydroxyoctanoate (beta-HO) . Deletion mutants, Delta pha7 and Delta pha8, were isolated during the PCR-based construction of pha7 and pha8, respectively . Cells harboring these mutants produced PHAs containing 55-60 mol% beta-HD and 40-45 mol% beta-HO . These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.

J Biochem (Tokyo), 2003 Jan, 133(1), 139 - 45
Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase gene; Takase K et al.; Type II synthase (PhaC1(Ps)) for polyhydroxyalkanoate (PHA) from Pseudomonas sp . 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1(Ps) in terms of its ability to produce poly(3-hydroxybutyrate) {P(3HB)} in recombinant Escherichia coli . Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified . Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library . As a result, five single mutants {S325C (T) and Q481M (K, R)} gave rise to highly enhanced P(3HB) synthesis . Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481 . It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases . The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 536 - 43 Epub 2003 May 21.
Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans; Solaiman DK et al.; The phaZ gene of Pseudomonas resinovorans codes for a poly(hydroxyalkanoates) (PHA) depolymerase . Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination . A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZ(FOAC001) (239 amino-acid residues) and PhaZ(FOAC002) (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively . In vivo PHA degradation data indicated that PhaZ(FOAC001) might still retain a partial PHA depolymerization activity, while PhaZ(FOAC002) is completely devoid of this function . The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days . A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source . The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of beta-hydroxyoctanoate and higher beta-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition . While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up . These phaZ mutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.

Plant Physiol, 2003 May, 132(1), 343 - 51
The expression of the t-SNARE AtSNAP33 is induced by pathogens and mechanical stimulation; Wick P et al.; The fusion of vesicles in the secretory pathway involves the interaction of t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) on the target membrane and v-SNAREs on the vesicle membrane . AtSNAP33 is an Arabidopsis homolog of the neuronal t-SNARE SNAP-25 involved in exocytosis and is localized at the cell plate and at the plasma membrane . In this paper, the expression of AtSNAP33 was analyzed after different biotic and abiotic stresses . The expression of AtSNAP33 increased after inoculation with the pathogens Plectosporium tabacinum and virulent and avirulent forms of Peronospora parasitica and Pseudomonas syringae pv tomato . The expression of PR1 transcripts encoding the secreted pathogenesis-related protein 1 also increased after inoculation with these pathogens and the expression of AtSNAP33 preceded or occurred at the same time as the expression of PR1 . AtSNAP33 was also expressed in npr1 plants that do not express PR1 after pathogen inoculation as well as in cpr1 plants that overexpress PR1 in the absence of a pathogen . The level of AtSNAP33 decreased slightly in leaves inoculated with P . parasitica in the NahG plants, and eds5 and sid2 mutants that are unable to accumulate salicylic acid (SA) after pathogen inoculation, indicating a partial dependence on SA . AtSNAP33 was also expressed in systemic noninoculated leaves of plants inoculated with P . syringae . In contrast to the situation in infected leaves, the expression of AtSNAP33 in systemic leaves was fully SA dependent . Thus, the expression of AtSNAP33 after pathogen attack is regulated by SA-dependent and SA-independent pathways . Mechanical stimulation also led to an increase of AtSNAP33 transcripts.

Appl Environ Microbiol, 2003 May, 69(5), 2936 - 41
Identification of an emergent and atypical Pseudomonas viridiflava lineage causing bacteriosis in plants of agronomic importance in a Spanish region; Gonzalez AJ et al.; Pseudomonas strains with an atypical LOPAT profile (where LOPAT is a series of determinative tests: L, levan production; O, oxidase production; P, pectinolitic activity; A, arginine dihydrolase production; and T, tobacco hypersensibility) can be regarded as emergent pathogens in the Principality of Asturias (Spain), where they have been causing, since 1999, severe damage in at least three taxonomically unrelated orchard plants of agronomic importance: common bean (Phaseolus vulgaris), kiwifruit (Actinidia deliciosa), and lettuce (Lactuca sativa) . These strains are mainly differentiated by production of yellowish mucoid material in hypersucrose medium, used for the levan test, and by a variable pectinolytic activity on different potato varieties . The atypical organisms were identified as Pseudomonas viridiflava based on their 16S rRNA sequences . Among them a certain intraspecies genetic heterogeneity was detected by randomly amplified polymorphic DNA (RAPD) typing . To differentiate between isolates of P . viridiflava and Pseudomonas syringae pathovars, a 16S ribosomal DNA restriction fragment length polymorphism method employing the restriction endonucleases SacI and HinfI was developed . This could be used as a means of reliable species determination after the usual phenotypical characterization, which includes the LOPAT tests.

Appl Environ Microbiol, 2003 May, 69(5), 2786 - 93
Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups; Nadeau LJ et al.; Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds . The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism) . The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer . The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp . strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism . GC-MS analysis of the reaction products formed in H(2)(18)O did not indicate any (18)O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R . eutropha JMP134 . During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp . strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was (18)O labeled . The other hydroxyl group in the product must have come from the substrate . The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol . The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups.

Appl Environ Microbiol, 2003 May, 69(5), 2707 - 11
Characterization of the 4-hydroxybenzoyl-coenzyme A thioesterase from Arthrobacter sp . strain SU; Zhuang Z et al.; The Arthrobacter sp . strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA) . The pathway operon contains the genes fcbA, fcbB, and fcbC (A . Schmitz, K . H . Gartemann, J . Fiedler, E . Grund, and R . Eichenlaub, Appl . Environ . Microbiol . 58:4068-4071, 1992) . Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known . We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein . A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates . Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k(cat) of 6.7 s(-1) and a K(m) of 1.2 micro M . The k(cat) pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10 . The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks . A large number of sequence homologues of unknown function were identified . On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.

Annu Rev Phytopathol, 2003, 41, 117 - 53 Epub 2003 Apr 29.
Regulation of antibiotic production in root-colonizing Peudomonas spp . and relevance for biological control of plant disease; Haas D et al.; Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants . The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics . Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots . Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide . In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level . Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway . One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

J Bacteriol, 2003 May, 185(10), 3210 - 3
Characterization of the chlorate reductase from Pseudomonas chloritidismutans; Wolterink AF et al.; A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans . Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P . chloritidismutans reduced only chlorate and bromate . Differences were also found in N-terminal sequences, molecular weight, and subunit composition . Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases.

J Biotechnol, 2003 May 8, 102(3), 301 - 6
Construction of a bacterial biosensor for styrene; Alonso S et al.; A new bacterial biosensor for styrene has been developed and characterized . A translational fusion of the lacZ gene to the sty promoter of Pseudomonas sp . strain Y2 has been inserted into miniTn5 . Transposition of the recombinant transposon to the chromosome of Pseudomonas sp . strain Y2 resulted in a whole-cell biosensor able to detect and degrade styrene . In this biosensor, the endogenous StyS/StyR system detects the presence of styrene and turns on the expression of the exogenous reporter gene from the transferred construction . Other compounds such as toluene, epoxystyrene, phenylacetaldehyde and 2-phenylethanol also induced expression of beta-galactosidase although quantitative differences in their effect are clearly detected . Non-inducing compounds affect differently the sensitivity to inducing compounds when present in a mixture.

Biosci Biotechnol Biochem, 2003 Feb, 67(2), 322 - 8
N-cyanomethyl-2-chloroisonicotinamide induces systemic acquired resistance in arabidopsis without salicylic acid accumulation; Yasuda M et al.; Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway . N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco . To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways . Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv . tomato DC3000 . NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant . NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants . Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.

Biosci Biotechnol Biochem, 2003 Feb, 67(2), 300 - 7
Expression, purification, and characterization of 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase from carbazole-degrader Pseudomonas resinovorans strain CA10; Iwata K et al.; The two-subunit meta-cleavage enzyme, 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarBaBb), from the carbazole degrader Pseudomonas resinovorans strain CA10 was purified to homogeneity from an Escherichia coli strain carrying the expression vector pUCA503, in which two copies of the carBaBb genes are tandemly linked . SDS-PAGE and gel filtration showed that CarB was a alpha2beta2-heterotetrameric enzyme with subunit molecular masses of approximately 10,000 for CarBa and 29,000 for CarBb . The optimum pH for activity was 8.5 and that of temperature was 35 degrees C . The CarB enzyme had a Km of 14 microM and a kcat/Km of 0.25 microM(-1) s(-1) for 2'-aminobiphenyl-2,3-diol, and the catalytic activities for biphenyl-type catecholic substrates were higher than those for monoaromatic catechol derivatives . The enzyme was originally isolated as a meta-cleavage enzyme for 2'-aminobiphenyl-2,3-diol involved in carbazole degradation, but the enzyme was highly specific for 2,3-dihydroxybiphenyl.

Plasmid, 2003 Mar, 49(2), 106 - 17
RpoN (sigma(54)) is required for plasmid-encoded coronatine biosynthesis in Pseudomonas syringae; Alarcon-Chaidez FJ et al.; The plant pathogen Pseudomonas syringae pv . glycinea PG4180 produces coronatine (COR), a phytotoxin which functions as a virulence factor in bacterial blight of soybeans . The COR biosynthetic gene cluster in PG4180 is borne on a 90-kb plasmid named p4180A . Although pathway-specific regulatory genes for COR have been identified, global regulatory genes for COR production in PG4180 remain undefined . In the present study, we evaluated the role of rpoN, which encodes sigma(54), in the virulence of strain PG4180 . A rpoN mutant of PG4180, designated PG4180.K2, was unable to grow in M9 minimal medium; however, the addition of exogenous glutamate, glutamine or aspartate to M9 medium enabled PG4180.K2 to grow in vitro . PG4180.K2 could not induce disease symptoms or multiply in soybean plants and was defective in COR production and cor gene expression . Furthermore, PG4180.K2 was impaired in transcription of hrpL, an alternate sigma factor that mediates expression of genes in the type III secretion system of P . syringae . PG4180.K2 transconjugants with a wild-type copy of rpoN were complemented for hrpL and cor gene expression, COR biosynthesis, and growth in vitro . Our results indicate that rpoN is required for growth and the expression of both chromosomal and plasmid-encoded virulence factors in P . syringae pv . glycinea PG4180.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 221 - 6
Two types of ion channel formation of tolaasin, a Pseudomonas peptide toxin; Cho KH et al.; Tolaasin is a peptide toxin produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms . Two types of ion channels were identified by the incorporation of tolaasin into lipid bilayer . The slope conductance of type 1 channel measured in the buffer containing 100 mM KCl was 150 pS with a linear current vs . voltage relationship . The type 2 tolaasin channel had two subconductance states of 300 and 500 pS . Both channels were inhibited by Zn(2+) . Ion channel formations of tolaasin were concentration-dependent and single channel currents were successfully obtained at 0.6 unit tolaasin, 15.9 nM . The type 1 channel was obtained more frequently than the type 2 channel and the ratio of their appearance was approximately 4:1, respectively.

Microbiology, 2003 May, 149(Pt 5), 1127 - 38
Alginate gene expression by Pseudomonas syringae pv . tomato DC3000 in host and non-host plants; Keith RC et al.; Pseudomonas syringae produces the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid . Although alginate has been isolated from plants infected by P . syringae, the signals and timing of alginate gene expression in planta have not been described . In this study, an algD : : uidA transcriptional fusion, designated pDCalgDP, was constructed and used to monitor alginate gene expression in host and non-host plants inoculated with P . syringae pv . tomato DC3000 . When leaves of susceptible collard plants were spray-inoculated with DC3000(pDCalgDP), algD was activated within 72 h post-inoculation (p.i.) and was associated with the development of water-soaked lesions . In leaves of the susceptible tomato cv . Rio Grande-PtoS, algD activity was lower than in collard and was not associated with water-soaking . The expression of algD was also monitored in leaves of tomato cv . Rio Grande-PtoR, which is resistant to P . syringae pv . tomato DC3000 . Within 12 h p.i., a microscopic hypersensitive response (micro-HR) was observed in Rio Grande-PtoR leaves spray-inoculated with P . syringae pv . tomato DC3000(pDCalgDP) . As the HR progressed, histochemical staining indicated that individual bacterial cells on the surface of resistant tomato leaves were expressing algD . These results indicate that algD is expressed in both susceptible (e.g . collard, tomato) and resistant (Rio Grande-PtoR) host plants . The expression of algD in an incompatible host-pathogen interaction was further explored by monitoring transcriptional activity in leaves of tobacco, which is not a host for P . syringae pv . tomato . In tobacco inoculated with DC3000(pDCalgDP), an HR was evident within 12 h p.i., and algD expression was evident within 8-12 h p.i . However, when tobacco was inoculated with an hrcC mutant of DC3000, the HR did not occur and algD expression was substantially lower . These results suggest that signals that precede the HR may stimulate alginate gene expression in P . syringae . Histochemical staining with nitro blue tetrazolium indicated that the superoxide anion () is a signal for algD activation in planta . This study indicates that algD is expressed when P . syringae attempts to colonize both susceptible and resistant plant hosts.

Rev Laryngol Otol Rhinol (Bord), 2002, 123(4), 225 - 30
{Necrotizing external otitis in children in Abidjan (Ivory Coast)}; Akre EE et al.; Necrotizing external otitis (NEO) is a fulminant Pseudomonas infection of the external auditory canal affecting mainly elderly diabetic patients . Since the early descriptions, many authors have related cases of NEO in non diabetic patients . We report eight cases of NEO in young children . They are less than two years old, they are undernourished and non diabetics . We get a good evolution in spite of our modest therapeutic ways . Emphasis is placed on efficiency of local remedy with colistine.

Arch Virol, 2003 May, 148(5), 1017 - 26
Activation of defense-related gene expression and systemic acquired resistance in cucumber mosaic virus-infected tobacco plants expressing the mammalian 2'5'oligoadenylate system; Honda A et al.; Tobacco plants expressing the mammalian 2'5'oligoadenylate system (2-5A system) exhibit resistance to cucumber mosaic virus (CMV) . Here, to characterize the molecular aspect of the resistance to CMV in 2-5A system-expressing tobaccos, the activation of defense-related genes and systemic acquired resistance (SAR) as the markers for the hypersensitive resistance (HR), were elucidated . Northern hybridization analysis indicated that the expression of four pathogenesis-related (PR) protein genes and five HR-related genes were induced in CMV-infected tobaccos expressing 2-5A system . Furthermore, the induction of SAR against Pseudomonas syringae pv . tabaci as second challenge, was observed on CMV-inoculated tobaccos expressing 2-5A system . These results suggested that the resistance to CMV in tobacco expressing 2-5A system is associated with the establishment of an HR-like response.

Surgery, 2003 Apr, 133(4), 404 - 10
CD8+ T-cell mediated tumor protection by Pseudomonas exotoxin fused to ovalbumin in C57BL/6 mice; Becerra JC et al.; BACKGROUND: Pseudomonas exotoxin (PE) is a 66 kDa bacterial toxin that is able to bind to mammalian cells, undergo receptor mediated endocytosis, and translocate its C-terminal catalytic domain into the cytosol . We investigated whether PE could be used in vivo to deliver CD8+ T-cell epitopes to the MHC-class I antigen presentation pathway to trigger a specific cytotoxic T-lymphocyte (CTL) response . METHODS: Amino acid 553 of PE was deleted to eliminate toxin catalytic activity, and amino acids 204-386 of ovalbumin were fused near the nontoxic PE C-terminus to produce PE(D)-OVA200 . Mice were vaccinated with 100 microg of PE(D)-OVA200 3 times at 21 day intervals . Splenocytes were harvested 1 week later, and stimulated in vitro with ovalbumin expressing EG7 murine thymoma cells . In vivo tumor protection experiments were performed by vaccinating groups of mice as before, followed by a lethal dose of ovalbumin expressing tumor cells (MO5) injected subcutaneously . RESULTS: Splenocytes from PE(D)-OVA200 vaccinated mice lysed (51)Cr labeled EG7 cells but not the untransfected EL4 parent cell line, whereas splenocytes from mice immunized with PBS, PE(D), or ovalbumin were unable to lyse EG7 cells . Cytotoxicity in vitro was mediated by CD8+ T-cells . PE(D)-OVA200 vaccinated mice survived (88%) a lethal subcutaneous challenge of ovalbumin expressing MO5 cells . Depletion of CD8+ cells from PE(D)-OVA200 vaccinated mice abolished this protection, indicating that this cell population is required for tumor rejection in vivo . CONCLUSIONS: Our results indicate that PE(D) may be used as a vehicle to stimulate a protective CTL response to heterologous antigen in vivo.

Mymensingh Med J, 2003 Jan, 12(1), 48 - 50
Increasing trend of ciprofloxacin resistance amongst common bacterial isolates at Mymensingh Medical College and Hospital; Hossain MA et al.; Ciprofloxacin resistance among common bacterial pathogen comprising Esch.coli, Staph.aureus and Pseudomonas spp . isolated from different clinical samples of Mymensingh Medical College Hospital during the periods of September, 1999 to March, 2001 and September, 2001 to August, 2002 were recorded . Values of two periods were compared and increased rate of ciprofloxacin resistance were noted in every bacterial species, e.g . 32.0% in Esch.coli, 8.7% in Staph.aureus and 5.1% in Pseudomonas spp . It was suggested to be aware and careful regarding use of ciprofloxacin in clinical practice so as to limit emergence of bacterial strains resistance towards the drug.

Mol Genet Genomics, 2003 Apr, 269(1), 21 - 30 Epub 2003 Feb 13.
The DeltafliD mutant of Pseudomonas syringae pv . tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells; Shimizu R et al.; To investigate the role of flagella and monomer flagellin in the interaction between Pseudomonas syringae pv . tabaci and plants, non-polar fliC and fliD mutants were produced . The ORFs for fliC and fliD are deleted in the DeltafliC and DeltafliD mutants, respectively . Both mutants lost all flagella and were non-motile . The DeltafliC mutant did not produce flagellin, whereas the DeltafliD mutant, which lacks the HAP2 protein, secreted large amounts of monomer flagellin into the culture medium . Inoculation of non-host tomato leaves with wild-type P . syringae pv . tabaci or the DeltafliD mutant induced a hypersensitive reaction (HR), whereas the DeltafliC mutant propagated and caused characteristic symptom-like changes . In tomato cells in suspension culture, wild-type P . syringae pv . tabaci induced slight, visible HR-like changes . The DeltafliC mutant did not induce HR, but the DeltafliD mutant induced a remarkably strong HR . Expression of the hsr203J gene was rapidly and strongly induced by inoculation with the DeltafliD mutant, compared to inoculation with wild-type P . syringae pv . tabaci . Furthermore, introduction of the fliC gene into the DeltafliC mutant restored motility and HR-inducing ability in tomato . These results, together with our previous study, suggest that the flagellin monomer of pv . tabaci acts as a strong elicitor to induce HR-associated cell death in non-host tomato cells.

Planta, 2003 Sep, 217(5), 767 - 75 Epub 2003 Apr 24.
Differential volatile emissions and salicylic acid levels from tobacco plants in response to different strains of Pseudomonas syringae; Huang J et al.; Pathogen-induced plant responses include changes in both volatile and non-volatile secondary metabolites . To characterize the role of bacterial pathogenesis in plant volatile emissions, tobacco plants, Nicotiana tabacum L . K326, were inoculated with virulent, avirulent, and mutant strains of Pseudomonas syringae . Volatile compounds released by pathogen-inoculated tobacco plants were collected, identified, and quantified . Tobacco plants infected with the avirulent strains P . syringae pv . maculicola ES4326 (Psm ES4326) or pv . tomato DC3000 (Pst DC3000), emitted quantitatively different, but qualitatively similar volatile blends of (E)-beta-ocimene, linalool, methyl salicylate (MeSA), indole, caryophyllene, beta-elemene, alpha-farnesene, and two unidentified sesquiterpenes . Plants treated with the hrcC mutant of Pst DC3000 (hrcC, deficient in the type-III secretion system) released low levels of many of the same volatile compounds as in Psm ES4326- or Pst DC3000-infected plants, with the exception of MeSA, which occurred only in trace amounts . Interaction of the virulent pathogen P . syringae pv . tabaci (Pstb), with tobacco plants resulted in a different volatile blend, consisting of MeSA and two unidentified sesquiterpenes . Overall, maximum volatile emissions occurred within 36 h post-inoculation in all the treatments except for the Pstb infection that produced peak volatile emissions about 60 h post-inoculation . (E)-beta-Ocimene was released in a diurnal pattern with the greatest emissions during the day and reduced emissions at night . Both avirulent strains, Psm ES4326 and Pst DC3000, induced accumulation of free salicylic acid (SA) within 6 h after inoculation and conjugated SA within 60 h and 36 h respectively . In contrast, SA inductions by the virulent strain Pstb occurred much later and conjugated SA increased slowly for a longer period of time, while the hrcC mutant strain did not trigger free and conjugated SA accumulations in amounts significantly different from control plants . Jasmonic acid, known to induce plant volatile emissions, was not produced in significantly higher levels in inoculated plants compared to the control plants in any treatments, indicating that induced volatile emissions from tobacco plants in response to P . syringae are not linked to changes in jasmonic acid.

Am J Pathol, 2003 May, 162(5), 1475 - 86
Impact of interleukin-13 responsiveness on the synthetic and proliferative properties of Th1- and Th2-type pulmonary granuloma fibroblasts; Jakubzick C et al.; Interleukin-13 (IL-13) has emerged as a major cytokine mediator of fibroblast activation and pulmonary fibrosis . Normal (from noninflamed lung), Th1-type (induced by the pulmonary embolization of purified peptide derivative-coated beads in mice sensitized to purified peptide derivative), and Th2-type (induced by the pulmonary embolization of Schistosoma mansoni egg antigen-coated beads in mice sensitized with S . mansoni eggs) primary fibroblast cell lines all exhibited constitutive gene expression of two receptor chains that bind and signal IL-13-mediated cellular events: IL-4Ralpha and IL-13Ralpha1 . However, all three fibroblast cell lines exhibited divergent synthetic and proliferative responses to the exogenous addition of either recombinant IL-13 or a chimeric protein comprised of IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), which targets and kills IL-13 receptor overexpressing cells . The exogenous addition of IL-13 to Th1-type and Th2-type fibroblast cultures significantly increased the cellular expression of IL-13Ralpha2, which may function as an IL-13 decoy receptor . After a 24-hour exposure to IL-13, the total collagen generation and cellular proliferation by Th2-type fibroblasts were significantly higher than that observed in similar numbers of normal and Th1-type fibroblasts . In addition IL13-PE, which binds with highest affinity to IL-13Ralpha2, exhibited down-regulatory effects on proliferation and matrix generation expression by Th1- and Th2-type, but not normal, fibroblasts . Thus, these data demonstrate that fibroblasts derived from murine pulmonary granulomas exhibit divergent expression of functional IL-13 receptor and this expression dictates the responsiveness and susceptibility to recombinant IL-13 and IL-13 immunotoxin, respectively.

Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5016 - 21 Epub 2003 Apr 17.
Targeting therapeutics to an exposed and conserved binding element of the HIV-1 fusion protein; Root MJ et al.; There is an urgent need for new drugs that can kill HIV type 1 (HIV-1)-infected cells . HIV-1 glycoprotein Env, which promotes viral membrane fusion through receptor-mediated conformational changes, is an attractive target for such agents because it is expressed on the surface of both virions and infected cells . Unfortunately, conserved binding elements on this protein frequently are buried under a canopy of flexible, glycosylated peptide loops or exposed only transiently during the fusion process . Here, we investigate the exposure of the C-terminal region of the Env ectodomain outside the context of membrane fusion . This binding element is the target of the 5-Helix protein, a designed entry inhibitor that disrupts conformational changes in Env subunit gp41, essential for the fusion process . We show that 5-Helix is capable of interacting with HIV-1 Env in a receptor-independent fashion and that a chimeric 5-Helix/Pseudomonas exotoxin protein recognizes cells expressing Env from a broad spectrum of HIV-1 strains including primary isolates from clades B, D, E, G, and H . This recombinant toxin selectively kills HIV-1-infected cells and blocks spreading infection while still maintaining potent inhibitory activity against membrane fusion . Our results demonstrate that the C-terminal region of the gp41 ectodomain is an accessible target on HIV-1-infected cells for the development of antiviral therapeutics and neutralizing antibodies.

Physiol Plant, 2003 May, 118(1), 138 - 146
Differential expression of the LePS2 phosphatase gene family in response to phosphate availability, pathogen infection and during development; Stenzel I et al.; In this study, we report the cloning of the three-member LePS2 gene family of acid phosphatases via subtractive screening of a cDNA library of Pi-starved cultivated tomato cells (Lycopersicon esculentum Mill . cv . Lukullus) . As members of the plant Pi-starvation response, LePS2 genes were tightly regulated in cultivated cells and tomato seedlings by Pi availability . The LePS2 enzymes which are most likely expressed in the cytoplasma could be involved in processes that are accompanied by degradation of phosphorylated organic substrates . Independently from exogenous phosphate supply LePS2 expression was detected in tomato endosperm during germination . LePS2 genes were differentially induced after infection with the bacterial pathogen Pseudomonas syringae and in the early stages of flower development . Using RT-PCR it was found that the gene LePS2B was the most abundant transcript in phosphate-depleted cells, but a reduced expression was determined in floral buds and it was not found during pathogen interaction . In this respect, it is interesting that the promoter sequences of the LePS2 genes are also divergent . LePS2 gene products may have functions in developmental processes which are restricted to distinct plant tissues or cell types.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2002, 67(2), 209 - 16
An integrated control of Pythium root rot of greenhouse tomato; Tu JC; Pythium root rot caused by Pythium aphanidermatum is one of the most important diseases of greenhouse tomatoes . Hydroponic culture exacerbates the problem . Both nutrient film technique (NFT) and recirculating growing systems pose a challenge in the control of this disease, because the pathogen, especially the zoospores, can spread easily in the recirculating solution to the whole growing system . Fortunately, hydroponically grown plants are easier to manipulate than soil grown plants, proper manipulation of root environments can lead to excellent disease control . This paper reports the development of an effective integrated control measure for pythium root rot of tomato by integrating pH, bioagent, and ultra-violet irradiation in a specific manner . This integrated control consists of three operations: a) before transplanting, the UV system is connected to sterilize the recirculating solution using 100 mJcm-2; b) after transplanting, the nutrient solution is delivered at pH 5.0 regime for five weeks followed by adjusting pH to 5.8 to 6.2 regime for one week; and c) bacterial bioagent, such as Pseudomonas is introduced into the root zone at 100 mL per plant at 10(8) bacteria mL-1 or added to the nutrient solution to arrive at 10(6) bacteria mL-1 in the solution . This report also discusses the advantages and limitations of this measure in the control of pythium root rot.

Biochemistry, 2003 Apr 15, 42(14), 4084 - 93
Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation; Kim JK et al.; Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC 3.5.1.11) is a member of N-terminal nucleophile (Ntn) hydrolases . The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide . The activation of precursor GCA consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide . We have determined the crystal structures of the recombinant selenomethionyl native and S170A mutant precursor from Pseudomonas sp . strain GK16 . Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip . Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack . The activation results in building up a catalytic triad composed of Ser170/His192/Glu624 . However, the triad is not linked to the usual hydroxyl but the free alpha-amino group of the N-terminal serine residue of the native GCA . Mutagenesis and structural data support the notion that the stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N --> O acyl shift . The autoproteolytic activation mechanism for GCA is described.

Front Biosci, 2003 May 01, 8, s472 - 83
On the mechanism of solute uptake in Pseudomonas; Tamber S et al.; Pseudomonas species have over 300 known and putative nutrient uptake systems enabling them to metabolize a large number of organic compounds, and thus inhabit many diverse ecological niches . The outer membrane of these organisms acts as a semi-permeable barrier, excluding many classes of potentially toxic molecules from the cell . Nutrients use specialized water-filled channels called porins to traverse this barrier . Entry into the cell is mediated by one of four classes of cytoplasmic membrane transporters: glycerol facilitators, phosphotransferase systems, primary active transporters, and secondary active transporters . The class of transporter used is dependent on the environmental conditions, as well as the type and concentration of solute . The recent advances in elucidating the structures and functional mechanisms of these uptake systems will be discussed in this review.

Bioresour Technol, 2003 Sep, 89(2), 177 - 83
Removal of phenanthrene from soil by co-cultures of bacteria and fungi pregrown on sugarcane bagasse pith; Chavez-Gomez B et al.; Sixteen co-cultures composed of four bacteria and four fungi grown on sugarcane bagasse pith were tested for phenanthrene degradation in soil . The four bacteria were identified as Pseudomonas aeruginose, Ralstonia pickettii, Pseudomonas sp . and Pseudomonas cepacea . The four fungi were identified as: Penicillium sp., Trichoderma viride, Alternaria tenuis and Aspergillus terrus that were previously isolated from different hydrocarbon-contaminated soils . Fungi had a statistically significant positive (0.0001<p) effect on phenanthrene removal, that ranged from 35% to 50% and bacteria removed the compound by an order of 20% . Co-cultures B . cepacea-Penicillium sp., R . pickettii-Penicillium sp., and P . aeruginose-Penicillium sp . exhibited synergism for phenanthrene removal, reaching 72.84+/-3.85%, 73.61+/-6.38% and 69.47+/-4.91%; in 18 days, respectively.

J Ind Microbiol Biotechnol, 2003 May, 30(5), 271 - 8 Epub 2003 Apr 15.
The role of active-site residues in naphthalene dioxygenase; Parales RE; The three-component naphthalene dioxygenase enzyme system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp . strain NCIB 9816-4 . A member of a large family of bacterial Rieske non-heme iron oxygenases, naphthalene dioxygenase is known to oxidize over 60 different aromatic compounds, and many of the products are enantiomerically pure . The crystal structure of the oxygenase component revealed the enzyme to be an alpha(3)beta(3) hexamer and identified the amino acids located near the active site . Site-directed mutagenesis studies have identified the residues involved in electron transfer and those responsible for controlling the regioselectivity and enantioselectivity of the enzyme . The results of these studies suggest that naphthalene dioxygenase can be engineered to catalyze a new and extended range of useful reactions.

Traffic, 2003 Apr, 4(4), 232 - 42
A class of mutant CHO cells resistant to cholera toxin rapidly degrades the catalytic polypeptide of cholera toxin and exhibits increased endoplasmic reticulum-associated degradation; Teter K et al.; After binding to the eukaryotic cell surface, cholera toxin undergoes retrograde transport to the endoplasmic reticulum . The catalytic A1 polypeptide of cholera toxin (CTA1) then crosses the endoplasmic reticulum membrane and enters the cytosol in a process that may involve the quality control mechanism known as endoplasmic reticulum-associated degradation . Other toxins such as Pseudomonas exotoxin A and ricin are also thought to exploit endoplasmic reticulum-associated degradation for entry into the cytosol . To test this model, we mutagenized Chinese hamster ovary cells and selected clones that survived a prolonged coincubation with Pseudomonas exotoxin A and ricin . These lethal endoplasmic reticulum-translocating toxins bind different surface receptors and target different cytosolic substrates, so resistance to both would likely result from disruption of a shared trafficking or translocation event . Here we characterize two Pseudomonas exotoxin A/ricin-resistant clones that exhibited increased endoplasmic reticulum-associated degradation . Both clones acquired the following unselected traits: (i) resistance to cholera toxin; (ii) increased degradation of an endoplasmic reticulum-localized CTA1 construct; (iii) increased degradation of an established endoplasmic reticulum-associated degradation substrate, the Z variant of alpha1-antitrypsin (alpha1AT-Z); and (iv) reduced secretion of both alpha1AT-Z and the transport-competent protein alpha1AT-M . Proteosome inhibition partially rescued the alpha1AT-M secretion deficiencies . However, the mutant clones did not exhibit increased proteosomal activity against cytosolic proteins, including a second CTA1 construct that was expressed in the cytosol rather than in the endoplasmic reticulum . These results suggested that accelerated endoplasmic reticulum-associated degradation in the mutant clones produced a cholera toxin/Pseudomonas exotoxin A/ricin-resistant phenotype by increasing the coupling efficiency between toxin translocation and toxin degradation.

Mol Ecol, 2003 May, 12(5), 1125 - 35
Negative cross-talk between salicylate- and jasmonate-mediated pathways in the Wassilewskija ecotype of Arabidopsis thaliana; Traw MB et al.; Plants often respond to attack by insect herbivores and necrotrophic pathogens with induction of jasmonate-dependent resistance traits, but respond to attack by biotrophic pathogens with induction of salicylate-dependent resistance traits . To assess the degree to which the jasmonate- and salicylate-dependent pathways interact, we compared pathogenesis-related protein activity and bacterial performance in four mutant Arabidopsis thaliana lines relative to their wild-type backgrounds . We found that two salicylate-dependent pathway mutants (cep1, nim1-1) exhibited strong effects on the growth of the generalist biotrophic pathogen, Pseudomonas syringae pv . tomato, whereas two jasmonate-dependent pathway mutants (fad3-2fad7-2fad8, jar1-1) did not . Leaf peroxidase and exochitinase activity were negatively correlated with bacterial growth, whereas leaf polyphenol oxidase activity and trypsin inhibitor concentration were not . Interestingly, leaf total glucosinolate concentration was positively correlated with bacterial growth . In the same experiment, we also found that application of jasmonic acid generally increased leaf peroxidase activity and trypsin inhibitor concentration in the mutant lines . However, the cep1 mutant, shown previously to overexpress salicylic acid, exhibited no detectable biological or chemical responses to jasmonic acid, suggesting that high levels of salicylic acid may have inhibited a plant response . In a second experiment, we compared the effect of jasmonic acid and/or salicylic acid on two ecotypes of A . thaliana . Application of salicylic acid to the Wassilewskija ecotype decreased bacterial growth . However, this effect was not observed when both salicylic acid and jasmonic acid were applied, suggesting that jasmonic acid negated the beneficial effect of salicylic acid . Collectively, our results confirm that the salicylate-dependent pathway is more important than the jasmonate-dependent pathway in determining growth of P . syringae pv . tomato in A . thaliana, and suggest important negative interactions between these two major defensive pathways in the Wassilewskija ecotype . In contrast, the Columbia ecotype exhibited little evidence of negative interactions between the two pathways, suggesting intraspecific variability in how these pathways interact in A . thaliana.

J Gen Appl Microbiol, 2003 Feb, 49(1), 1 - 19
The unique aromatic catabolic genes in sphingomonads degrading polycyclic aromatic hydrocarbons (PAHs); Pinyakong O et al.; Many members of the sphingomonad genus isolated from different geological areas can degrade a wide variety of polycyclic aromatic hydrocarbons (PAHs) and related compounds . These sphingomonads such as Sphingobium yanoikuyae strain B1, Novosphingobium aromaticivorans strain F199, and Sphingobium sp . strain P2 have been found to possess a unique group of genes for aromatic degradation, which are distantly related with those in pseudomonads and other genera reported so far both in sequence homology and gene organization . Genes for aromatics degradation in these sphingomonads are complexly arranged; the genes necessary for one degradation pathway are scattered through several clusters . These aromatic catabolic gene clusters seem to be conserved among many other sphingomonads such as Sphingobium yanoikuyae strain Q1, Sphingomonas paucimobilis strain TNE12, S . paucimobilis strain EPA505, Sphingobium agrestis strain HV3, and Sphingomonas chungbukensis strain DJ77 . Furthermore, some genes for naphthalenesulfonate degradation found in Sphingomonas xenophaga strain BN6 also share a high sequence homology with their homologues found in these sphingomonads . On the other hand, protocatechuic catabolic gene clusters found in fluorene-degrading Sphingomonas sp . strain LB126 appear to be more closely related with those previously found in lignin-degrading S . paucimobilis SYK-6 than the genes in this group of sphingomonads . This review summarizes the information on the distribution of these strains and relationships among their aromatic catabolic genes.

Mycorrhiza, 2003 Apr, 13(2), 85 - 91 Epub 2003 Feb 06.
A mycorrhiza helper bacterium enhances ectomycorrhizal and endomycorrhizal symbiosis of Australian Acacia species; Duponnois R et al.; The aims of this study were to test the effects of a mycorrhiza helper bacterium (MHB), Pseudomonas monteilii strain HR13 on the mycorrhization of (1) an Australian Acacia, A . holosericea, by several ectomycorrhizal fungi or one endomycorrhizal fungus Glomus intraradices, and (2) several Australian Acacia species by Pisolithus alba strain IR100 under glasshouse conditions . Bacterial inoculant HR13 significantly promoted ectomycorrhizal colonization for all the Acacia species, from 45.8% ( A . mangium) to 70.3% ( A . auriculiformis) . A stimulating effect of HR13 on the ectomycorrhizal establishment was recorded with all the fungal isolates (strains of Pisolithus and Scleroderma) . The same effect of bacteria on the frequency of endomycorrhizal colonization of A . holosericea seedlings by G . intraradices with vesicles and hyphae frequencies was recorded . The stimulation of saprophytic fungal growth by MHB is usually the main mechanism that could explain this bacterial effect on mycorrhizal establishment . MHB could stimulate the production of phenolic compounds such as hypaphorine and increase the aggressiveness of the fungal symbiont . However, no significant effect of MHB on fungal growth was recorded with Scleroderma isolates under axenic conditions but positive bacterial effects were observed with Pisolithus strains . From a practical viewpoint, it appears that MHB could stimulate the mycorrhizal colonization of Australian Acacia species with ectomycorrhizal or endomycorrhizal fungi, and could also facilitate controlled mycorrhization in nursery practices where Acacia species are grown for forestation purposes.

Lett Appl Microbiol, 2003, 36(5), 259 - 62
Fluorescent Pseudomonas mainly produce the dihydro form of pyoverdine at low specific growth rate; Jacques P et al.; AIMS: To analyse the influence of cell growth rate and iron concentration on the production of pyoverdines (PVDs) and of their reduced dihydro forms by three fluorescent Pseudomonas strains (P . putida BTP16, P . fluorescens BTP7 and P . aeruginosa 7NSK2) . METHODS: PVD and dihydropyoverdine (DHPVD) productions were determined by LC ESI-MS and spectrophotometry during batch and chemostat culture at different dilution rates . SIGNIFICANCE: The relatively high PVD-to-DHPVD ratio (0.57) observed in pH-controlled batch cultures suggested that a base-catalysed chemical oxidation of the dihydroform is not the prime mechanism involved in generating PVDs . Interestingly, in chemostat cultures the PVD-to-DHPVD ratio was significantly reduced at low specific growth rate . Our results suggest that the oxidation of DHPVD to PVD is catalysed by an iron-dependent enzymatic reaction rather than a chemical oxidation.

Microbiology, 2003 Apr, 149(Pt 4), 895 - 901
DNA restriction is a barrier to natural transformation in Pseudomonas stutzeri JM300; Berndt C et al.; Natural transformation is a mechanism for intra- and interspecific transfer of chromosomal DNA in Pseudomonas stutzeri . During this process a single strand derived from duplex DNA is transported into the cytoplasm and recombined with resident DNA . By electroporation, which introduces duplex DNA into cells, 100-fold lower transformation frequencies of P . stutzeri JM300 were observed with shuttle vector or broad-host-range plasmid DNA when the plasmids had replicated in Escherichia coli and not in P . stutzeri JM300 . Moreover, the natural transformation with cloned chromosomal P . stutzeri JM300 DNA was reduced about 40-fold when the DNA had not been propagated in P . stutzeri JM300 but in E . coli . Restriction was also active during natural transformation by single-stranded DNA . Restriction during natural transformation and electroporation was abolished in mutants isolated from mutagenized JM300 cells after applying a multiple plasmid electroporation strategy for the enrichment of restriction-defective strains . The mutants had retained the ability for DNA modification . The P . stutzeri strain ATCC 17587 was found to have no restriction-modification system as seen in JM300 . It is discussed whether restriction during natural transformation acts at presynaptic or postsynaptic stages of transforming DNA . Restriction as a barrier to transformation apparently contributes to sexual isolation and therefore may promote speciation in the highly diverse species P . stutzeri.

Chemosphere, 2003 Jan, 50(4), 537 - 43
Metabolic pathways of polychlorinated biphenyls degradation by Pseudomonas sp . 2; Komancova M et al.; Polychlorinated biphenyls (PCBs) included with the commercial mixture Delor 103 were degraded by immobilized cells of aerobic bacterial strain Pseudomonas sp . 2 . The ability of the strain to metabolise selected tri- and tetrachlorobiphenyls, and the site of primary attack of the biphenyl skeleton were investigated . It was observed that the amount of residual PCBs was 1-48% of the original PCBs after three weeks of incubation . Identified metabolites indicate that the used bacterial strain attacks the biphenyl skeleton at the 2,3- and 3,4-positions, and it is also able to dehalogenate PCBs . Metabolic pathways of degradation of individual congeners were proposed . Transformation of 2,4- and 2,5-dichlorobenzoic acids by Pseudomonas sp . 2 was also observed.

Environ Toxicol Chem, 2003 Apr, 22(4), 730 - 5
Degradation of carbazole, dibenzothiophene, and dibenzofuran at low temperature by Pseudomonas sp . strain C3211; Jensen AM et al.; Pseudomonas sp . strain C3211 was isolated from a temperate climate soil contaminated with creosote . This strain was able to degrade carbazole, dibenzothiophene and dibenzofuran at 10 degrees C with acetone as a co-substrate . When dibenzothiophene was degraded by strain C3211, an orange compound, which absorbed at 472 nm, accumulated in the medium . Degradation of dibenzofuran was followed by accumulation of a yellowish compound, absorbing at 462 nm . The temperature optimum of strain C3211 for degradation of dibenzothiophene and dibenzofuran was at 20 to 21 degrees C, while the maximum temperature for degradation was at 27 degrees C . Both compounds were degraded at 4 degrees C . Degradation at 10 degrees C was faster than degradation at 25 degrees C . This indicates that strain C3211 is adapted to life at low temperatures.

Zhonghua Zhong Liu Za Zhi, 2003 Jan, 25(1), 36 - 8
{Anti-CD19-liposomes encapsulated domain III of pseudomonas exotoxin in the targeting of human B lymphoma}; Ma J et al.; OBJECTIVE: To evaluate the targeting ability and cytotoxicity of domain III of pseudomonas exotoxin encapsulated in anti-CD19-immunoliposomes to lymphoma cells in vitro and in vivo . METHODS: Binding ability of anti-CD19 immunoliposomes to B lymphoma cells was detected by binding assay . MTT assay was used to detect the cytotoxicity of free domain III and domain III encapsulated in anti-CD19-immunoliposomes against B lymphoma cells . An in vivo therapeutic study of domain III formulated in immunoliposomes on human B lymphoma was detected in a murine model . RESULTS: The cytotoxicity of free domain III disappeared when domain I and II were deleted . When domain III was encapsulated into anti-CD19-immunoliposomes, the cytotoxicity of immunoliposomes against tumor cells were significantly increased . Treatment, using this formulation, of mice inoculated with B lymphoma could enhance the survival time . CONCLUSION: Anti-CD19-immunoliposomes, as drug carriers, can specifically recognize B lymphoma cells and deliver non-toxic domain III into the tumor cells . This formulation of domain III might be an effective anti-tumor agent.

Appl Environ Microbiol, 2003 Apr, 69(4), 2139 - 52
Gene encoding the hydrolase for the product of the meta-cleavage reaction in testosterone degradation by Comamonas testosteroni; Horinouchi M et al.; In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M . Horinouchi et al., Microbiology 147:3367-3375, 2001) . Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction . We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone . TesD exhibited ca . 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp . The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color . High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction . The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid . 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD . Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD.

Diabet Med, 2003 Mar, 20(3), 242 - 4
An unusual cause of diabetic ketoacidosis and fulminant septicaemia; Hassanein MM et al.; BACKGROUND: Diabetic ketoacidosis (DKA) is a common medical emergency . Resistant and recurrent DKA can be due to underlying infection, and a detailed travel history may be important in determining the cause in such cases . We report here a case of unusual DKA and fulminant septicaemia in a Caucasian male with Type 1 diabetes 2 years after returning from living in Thailand . CASE REPORT: A 39-year-old Caucasian male was diagnosed with Type 1 diabetes whilst working in Thailand where he also subsequently developed a cavitating lung lesion diagnosed locally as pulmonary tuberculosis . Two years after returning to the UK he was admitted with DKA and septicaemia . Despite correction of his DKA his condition deteriorated and he developed a fluid collection anterior to the left hip on computed tomography scanning . Blood and fluid aspirate cultures confirmed a diagnosis of melioidosis, a rare fulminant septicaemia in the UK, but endemic in South-east Asia and tropical Australia . Full recovery followed changing antibiotics to intravenous ceftazidime with no relapse 3 years after acute episode . CONCLUSIONS: Physicians as well as microbiologists should consider melioidosis in anyone presenting with septicaemia and/or resistant DKA, especially if the history includes travel to endemic areas or if the cultures suggest Pseudomonas-like organism . With increasing international travel, it is crucial to remember that good travel history could be life-saving in some cases of septicaemia.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 557 - 64
Gelatin blends with alginate: gels for lipase immobilization and purification; Fadnavis NW et al.; Blends of natural polysaccharide sodium alginate (5%) with gelatin (3%) cross-linked with glutaraldehyde provide beads with excellent compressive strength (8 x 10(4) Pa) and regular structure on treatment with calcium chloride . Lipases from porcine pancreas, Pseudomonas cepacia, and Candida rugosa were immobilized in such a blend with excellent efficiency . The immobilized enzymes were stable and were reused several times without significant loss of enzyme activity both in aqueous and reverse micellar media . The beads were functionalized with succinic anhydride to obtain beads with extra carboxylic acid groups . These functionalized beads were then successfully used for 7.4-fold purification of crude porcine pancreatic lipase in a simple operation of protein binding at pH 5 and release at pH 8.5.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 303 - 11
Lipase-mediated deacetylation and oligomerization of lactonic sophorolipids; Hu Y et al.; The direct enzymatic polymerization of lactonic sophorolipids (SLs) was investigated with four lipases, including porcine pancreatic lipase (PPL), immobilized Mucor miehei lipase (MML), lyophilized Candida antarctica lipase (Fraction B, CAL-B), and lyophilized Pseudomonas sp . lipase (PSL) . Several organic solvents, covering a wide range of polarity, were compared for suitability as the reaction medium . Isopropyl ether and toluene were found most effective . According to the quantification and structure identification by HPLC and LC-MS, the reaction proceeded with the formation of monoacetylated lactonic SLs and the subsequent conversion of the intermediates to oligomers and polymers, presumably through ring-opening polymerization . Temperature was found to have significant effects on the reaction . Both the conversion of reactant SLs and the subsequent formation of oligomers and polymers from the intermediates were faster at 60 degrees C than at 50 degrees C . The substrate selectivity among the three dominant reactant SLs also differed with the temperature . The conversion rate increased with the ring size of the lactones at 60 degrees C, but it decreased with the size at 50 degrees C.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 298 - 302
Effect of reaction parameters on synthesis of citronellyl methacrylate by lipase-catalyzed transesterification; Athawale V et al.; The methacrylate ester of citronellol was synthesized using various lipases as catalyst . The effect of different reaction parameters such as amount of lipase, solvent, temperature, and acylating agent on the conversion of citronellol to citronellyl methacrylate was studied . Methyl methacrylate, vinyl methacrylate, and 2,3-butanedione mono-oxime methacrylate were used as acylating agents . Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Pseudomonas cepacia lipase (Amano-PS) were used as biocatalysts . Diisopropyl ether (DIPE) was found to be the most suitable solvent . The stereoselectivity of CRL in transesterification of (+/-)-citronellol was tested for the optimized reaction parameters.

Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 735 - 9
{Synthesis of partial glycerol caprates by using lipase in nonaqueous media}; Xia YM et al.; Enzymatic esterification by reacting caparic acid with glycerol in solvent-free system was studied . Lipases from Pseudomonas fluoresces(PFL), Mucor miehei(MML) and Candida antarictica(CAL) possessed good catalytic activity . The optimal reaction conditions to convert capric acid with CAL are: 60 degrees C, 20-100 u of CAL per gram capric acid, 12% (W/W) of initial water content in glycerol . CAL does not express its 1,3-specificity in final product . Mechanical fraying denatured CAL partly . 96.4% of catalytic activity of CAL recovered after 5 batches of reaction . Extraction with sodium carbonate solution can decrease acid value of product from 9.8 mg KOH/g to 0.68 mg KOH/g . Applying the enzymatic esterification in open system, under vacuum or dehydrating with molecular sieves all dehydrate effectively . Molar ratio of reactants does not influence the total conversion of capric acid but influences the yield of monoglyceride . With certain protocols, the reaction period could be shortened dramatically; conversion of capric acid reached 96.9% in 5 h.

J Bacteriol, 2003 Apr, 185(8), 2592 - 602
Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P . syringae pv . syringae B728a; Deng WL et al.; Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system . P . syringae pv . syringae B728a, which causes brown spot of bean, is a particularly well-studied strain . The type III secretion system in P . syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL) . The EELs of P . syringae pv . syringae B728a, P . syringae strain 61, and P . syringae pv . tomato DC3000 differ in size and effector gene composition; the EEL of P . syringae pv . syringae B728a is the largest and most complex . The three putative effector proteins encoded by the P . syringae pv . syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture . Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P . syringae pv . tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant . Deletion of the P . syringae pv . syringae B728a EEL strongly reduced virulence in host bean leaves . EELs from nine additional strains representing nine P . syringae pathovars were isolated and sequenced . Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common . Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.

Best Pract Res Clin Haematol, 2003 Mar, 16(1), 117 - 33
Immunobiological treatments of hairy-cell leukaemia; Kreitman RJ et al.; Hairy cell leukaemia (HCL) is extremely responsive to purine analogue theropy developed during the early 1990s, but some patients have emerged with resistance to purine analogues . For these patients, as well as for those with primarily refractory HCL, new treatments are necessary . Several new therapeutic options have been developed for the salvage treatment of HCL . These include recombinant immunotoxins and unlabelled monoclonal antibodies (mAbs) . Recombinant immunotoxins are chimeric proteins in which the Fv portion of a mAb is fused to a 38 kDa fragment of Pseudomonas exotoxin A . Two recombinant immunotoxins, BL22 and LMB-2, targeting CD22 and CD25, respectively, have demonstrated efficacy in patients with HCL resistant to purine analogues . BL22 was reported to induce complete remissions (CRs) in the majority of patients with cladribine-resistant HCL; its clinical efficacy and safety profile are currently being further defined . The unlabelled mAb rituximab has also been reported to induce responses in the majority of HCL patients treated, and several CRs have been observed.

Curr Opin Mol Ther, 2003 Feb, 5(1), 44 - 51
Recombinant toxins for the treatment of cancer; Kreitman RJ; Recombinant toxins are proteins made by genetic engineering consisting of a toxin fused to a ligand which binds selectively to a target cell . Recombinant toxins used for cancer treatment generally contain either a growth factor or a recombinant fragment of a monoclonal antibody fused to a truncated bacterial toxin, derived either from Pseudomonas exotoxin or from diphtheria toxin . One recombinant toxin containing human interleukin (IL)-2 and truncated diphtheria toxin (DAB389-IL-2, denileukin diftitox or Ontak; Seragen Inc) is approved for clinical use in advanced stage cutaneous T-cell lymphoma . Recombinant toxins containing truncated Pseudomonas exotoxin and fragments of anti-CD22 and anti-CD25 monoclonal antibodies have induced remissions in chemotherapy-resistant hematological malignancies, particularly hairy cell leukemia, and are currently undergoing experimental testing . The number of approved recombinant toxins for the treatment of cancer is expected to increase in the coming years.

Plant Cell Physiol, 2003 Mar, 44(3), 342 - 9
Post-translational modification of flagellin determines the specificity of HR induction; Taguchi F et al.; Flagellin, a constituent of the flagellar filament, is a potent elicitor of hypersensitive cell death in plant cells . Flagellins of Pseudomonas syringae pvs . glycinea and tomato induce hypersensitive cell death in their non-host tobacco plants, whereas those of P . syringae pv . tabaci do not remarkably induce it in its host tobacco plants . However, the deduced amino acid sequences of flagellins from pvs . tabaci and glycinea are identical, indicating that post-translational modification of flagellins plays an important role in determining hypersensitive reaction (HR)-inducibility . To investigate genetically the role of modification of flagellin in HR-induction, biological and phytopathological phenotypes of a flagella-defective Delta fliC mutant and Delta fliC mutants complemented by the introduction of the flagellin gene (fliC) from different pathovars of P . syringae were investigated . The Delta fliC mutant of pv . tabaci lost flagella, motility, the ability to induce HR cell death in non-host tomato cells and virulence toward host tobacco plants, whereas all pv . tabaci complemented by the introduction of the fliC gene of pvs . tabaci, glycinea or tomato recovered all the abilities that the Delta fliC mutant had lost . These results indicate that post-translational modification of flagellins is strongly correlated with the ability to cause HR cell death.

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 741 - 3 Epub 2003 Mar 25.
Crystallization and preliminary X-ray analysis of native and selenomethionine 2-hydroxybiphenyl 3-monooxygenase; Meyer A et al.; 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 was produced in Escherichia coli both as native and SeMet-labelled protein . The two enzymes were purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method . The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 108.6, b = 196.8, c = 79.3 A, beta = 97.7 degrees for the native protein and a = 108.3, b = 196.8, c = 79.0 A, beta = 97.8 degrees for SeMet HbpA . Crystal-packing considerations led to the assumption of two HbpA subunits per asymmetric unit, which corresponds to a V(M) value of 3.3 A(3) Da(-1) and a solvent content of 62% . The crystals were radiation-sensitive and only had a lifespan of about 120 s when exposed to synchrotron radiation on an undulator beamline . To obtain complete data sets, data were collected from 23 native and 26 derivative crystals . The high-resolution limit was 2.0 A for native and 2.25 A for SeMet HbpA.

Mol Cancer Ther, 2003 Mar, 2(3), 245 - 54
Interleukin-4 receptor-targeted cytotoxin therapy of androgen-dependent and -independent prostate carcinoma in xenograft models; Husain SR et al.; Prostate cancer is the most commonly diagnosed solid tumors in United States men . Survival with advanced prostate cancer is dismal because of a lack of effective treatments . Overexpression of interleukin 4 receptors (IL-4R) on prostate carcinoma cells makes them suitable targets for the interleukin 4 (IL-4) fused Pseudomonas exotoxin, IL-4 cytotoxin (IL4-CTx) . Androgen-dependent (LNCaP) and -independent (DU145) human prostate cancer cell lines overexpress IL-4Rs and are exquisitely sensitive to IL4-CTx . Using LNCaP and DU145 cell lines, IC(50) values of 4.5 +/- 2.0 and 6.5 +/- 0.5 ng/ml, respectively, were obtained for IL4-CTx in protein synthesis inhibition assays . Primary cultures established from prostate tumor biopsies were equally sensitive to the cytotoxic effects of IL4-CTx . Reverse transcription-PCR analysis, although not quantitative, indicated the presence of mRNA for IL-4Ralpha, a primary subunit of the IL-4R receptor complex in prostate carcinoma cell lines, primary cultures, benign prostatic hyperplasia, and prostate carcinoma tissues . Immunohistochemistry studies revealed the presence of IL-4R in benign prostatic hyperplasia and prostate carcinomas . Five daily (QD) injections of IL4-CTx (100 micro g/kg) administered i.v., i.p., or intratumoral (i.t.) caused several complete responses in nude mice with s.c . DU145 and LNCaP tumors . i.t . injections of IL4-CTx elicited tumor regression in a dose-dependent manner with complete responses occurring in 100% of the animals when treated with IL4-CTx (500 micro g/kg) given five QD injections . Administration of IL4-CTx i.t . (500 micro g/kg) either 10 times QD or six injections on alternate days elicited complete responses in 40% of mice with DU145 tumors that were three times larger (67 mm(2)) on initiation of treatments . IL4-CTx appeared to be well tolerated . On the basis of these results, combining i.t . injections of IL4-CTx with systemic administration may provide an effective strategy for treating patients with advanced, refractory prostate cancer.

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