Pseudomonas

Planta, 2002 Jun, 215(2), 239 - 47 Epub 2002 Mar 22.
Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance; Li L et al.; Polyphenol oxidases (PPOs; EC 1.10.3.2 or EC 1.14.18.1) catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms and assumed to be involved in plant defense against pests and pathogens . In order to investigate the role of PPO in plant disease resistance, we made transgenic tomato ( Lycopersicon esculentum Mill . cv . Money Maker) plants that overexpressed a potato ( Solanum tuberosum L.) PPO cDNA under control of the cauliflower mosaic virus 35S promoter . The transgenic plants expressed up to 30-fold increases in PPO transcripts and 5- to 10-fold increases in PPO activity and immunodetectable PPO . As expected, these PPO-overexpressing transgenic plants oxidized the endogenous phenolic substrate pool at a higher rate than control plants . Three independent transgenic lines were selected to assess their interaction with the bacterial pathogen Pseudomonas syringae pv . tomato . The PPO-overexpressing tomato plants exhibited a great increase in resistance to P . syringae . Compared with control plants, these transgenic lines showed less severity of disease symptoms, with over 15-fold fewer lesions, and strong inhibition of bacterial growth, with over 100-fold reduction of bacterial population in the infected leaves . These results demonstrate the importance of PPO-mediated phenolic oxidation in restricting plant disease development.

Plant J, 2002 May, 30(4), 467 - 80
The Arabidopsis hrl1 mutation reveals novel overlapping roles for salicylic acid, jasmonic acid and ethylene signalling in cell death and defence against pathogens; Devadas SK et al.; Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) . The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA- and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens . Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death . RNA gel-blot analysis of RNA isolated separately from the lesion+ and the lesion- leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues . Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants . In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration . The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica . Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants . Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex . Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.

Eur J Biochem, 2002 May, 269(10), 2498 - 505
Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans . A pathogen of cultivated mushrooms; Silipo A et al.; The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR) . The sugar backbone was composed of the beta-(1'-->6)-linked d-glucosamine disaccharide 1-phosphate . The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition . The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid {C12:0 (3OH)} in amide linkage and a (R)-3-hydroxydecanoic {C10:0 (3OH)} in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH) . A nonstoichiometric phosphate substitution at position C-4' of the distal 2-deoxy-2-amino-glucose was detected . Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3' . The potential biological meaning of this peculiar lipid A is also discussed.

Mikrobiologiia, 2002 Mar-Apr, 71(2), 240 - 6
{Some characteristics of Pseudomonas syringae pv . maculicola dissociants}; Iakovleva LM et al.; Pseudomonas syringae pv . maculicola dissociants producing colonies of different morphotype were found to possess similar biochemical and serological properties but different virulence to the host plant . The heterogeneous extracellular and intracellular lipopolysaccharide-protein complexes of the dissociants differed in their chemical composition and biological activity towards test plants.

Cancer Res, 2002 May 15, 62(10), 2848 - 55
A recombinant CD7-specific single-chain immunotoxin is a potent inducer of apoptosis in acute leukemic T cells; Peipp M et al.; A recombinant immunotoxin was constructed from the hybridoma antibody TH-69 directed against human CD7, a surface antigen of leukemic T cells . The antibody was subcloned as a single chain Fv (scFv) fragment and genetically linked to a truncated Pseudomonas exotoxin A fragment containing the catalytic domains II and III but lacking the receptor binding domain I . Domain I was replaced by the scFv, thus conferring restricted specificity for CD7-positive cells . The bacterially expressed and purified toxin retained binding specificity for CD7-positive cells . It promoted apoptosis in two CD7-positive cell lines derived from T-lineage acute lymphoblastic leukemias, CEM and Jurkat, but not in the CD7-negative B-lymphoid lines REH, Nalm-6, and SEM . Maximum killing in excess of 95% was reached after 96 h in CEM and Jurkat cells with a single dose of 100 ng/ml . Cells treated with a similarly constructed scFv-exotoxin A immunotoxin against melanoma-associated chondroitin sulfate proteoglycan, an antigen absent from leukemic T cells, remained unaffected . Lysis of target cells occurred via apoptosis as evidenced by staining with Annexin V and specific cleavage of poly(ADP-ribose) polymerase . Approximately 20% of leukemic cells from a patient with CD7-positive acute T-cell leukemia kept in long-term primary culture for 30 cell generations were killed within 96 h after treatment with the toxin . These findings justify further evaluation of the agent in view of potential therapeutic applications.

Mol Cells, 2002 Apr 30, 13(2), 309 - 14
DNA markers for identification of Pseudomonas syringae pv . actinidiae; Koh YJ et al.; The specific DNA fragment was screened by RAPD analysis of Pseudomonas syringae pv . actinidiae, as well as similar strains that were isolated from kiwifruits . The primer C24 detected a fragment that is specific in P . syringae pv . actinidiae . This fragment was cloned . The pathovar-specific fragment was detected from a Southern blot analysis of the genomic DNAs of P . syringae pv . actinidiae using the cloned fragment as a probe . The sequence size of the cloned fragment was determined as 675 bp . A DNA Database search suggested that the fragment was a novel one . Approximately 9 kb of a single fragment was detected only in the P . syringae pv . actinidiae by a Southern blot analysis of the genomic DNAs of P . syringae pv . actinidiae . Similar strains were also detected with the use of the cloned fragment as a probe . Since the genomic DNAs were digested with HindIII without a cleavage site, the result reveals that the cloned fragment exists on the genome of P . syringae pv . actinidiae as a single copy . A pair of primers that produced a 492 bp single fragment (only in the strains of P . syringae pv . actinidiae) were synthesized, based on the pathovar-specific sequences of the cloned fragment of P . syringae pv . actinidiae . The development of the primers and probe made it possible to diagnose the bacterial canker infection from leaves or trunks of kiwifruit trees before any symptom appeared on the tree.

Int J Colorectal Dis, 2002 Mar, 17(2), 77 - 84
The in vitro anti-inflammatory effects of recombinant anti-CD25 immunotoxin on lamina propria T cells of patients with inflammatory bowel disease are not sufficient to cure experimental colitis in mice; Pfister K et al.; BACKGROUND AND AIMS: In chronic inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis an aberrant mucosal immune regulation is observed accompanied by upregulation of proinflammatory cytokines . Lamina propria T cells of inflamed mucosa have an activated phenotype characterized by increased expression of surface markers such as CD25 . We therefore determined the anti-inflammatory effect of a recombinant immunotoxin consisting of an anti-CD25 single chain variable fragment (scFv) fused to a deletion mutant of Pseudomonas exotoxin A {RFT5(scFv)ETA'} on isolated lamina propria lymphocytes of patients with IBD and in the murine model of trinitrobenzene sulfonic acid (TNBS) induced colitis . PATIENTS AND/METHODS: Lamina propria lymphocytes of 25 patients with IBD and 19 control patients were stimulated in absence or presence of RFT5(scFv)ETA' . Interferon-gamma production was determined in the supernatant by ELISA and the induction of apoptosis by flow cytometry after propidium iodide staining . BALB/c mice received TNBS intrarectally and were treated with RFT5(scFv)ETA' . RESULTS: In vitro the administration of RFT5(scFv)ETA' significantly reduced interferon-gamma production and increased apoptosis in lamina propria lymphocytes isolated of inflamed mucosa . However, this contrainflammatory regulation did not result in gain of weight or increased life span in experimental colitis in vivo . CONCLUSION: In addition to the downregulation of the proinflammatory cytokine in vitro, RFT5(scFv)ETA' induced neither a direct nor a bystander effect in an in vivo model of colitis . Therefore our data do not support potential therapeutic implications of targeting CD25 by RFT5(scFv)ETA' in chronic IBD.

Biochim Biophys Acta, 2002 May 20, 1597(1), 60 - 6
Fluorescent inhibitors reveal solvent-dependent micropolarity in the lipid binding sites of lipases; Oskolkova OV et al.; Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase-inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis . Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active site of the enzyme in different solvents . The respective compounds are hexyl and methyl dimethylamino-naphthalenecarbonylethylmercaptoethoxy-phosphonates . The hexyl phosphonate derivative was reacted with lipases from Rhizopus oryzae (ROL), Chromobacterium viscosum (CVL), and Pseudomonas cepacia (PCL) . The resulting lipid-protein complexes were characterized in solution with respect to water penetration into the lipid binding site and the associated conformational changes of the proteins as a consequence of solvent polarity changes . We found that the accessibility of the lipid-binding site in all lipases studied was lowest in water . It was much higher when the protein was dissolved in aqueous ethanol . These biophysical effects may contribute to the previously observed dramatic changes of enzyme functions such as activity and stereoselectivity depending on the respective solvents.

Biomacromolecules, 2002 May-Jun, 3(3), 525 - 30
Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol); Li S et al.; Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst . The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction . Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type . Films were prepared by casting dichloromethane solutions of the polymers on a glass plate . Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase . Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone).

Biosci Biotechnol Biochem, 2002 Mar, 66(3), 543 - 8
Purification and characterization of pyridoxal 4-dehydrogenase from Aureobacterium luteolum; Trongpanich Y et al.; A pyridoxal dehydrogenase was purified to homogeneity from Aureobacterium luteolum, which can use pyridoxine as a carbon and nitrogen source, and characterized . The enzyme was a dimeric protein with a subunit molecular weight of 38,000 . It had several properties distinct from those of the partially purified enzyme from Pseudomonas MA-1 . The optimum pH (8.0-8.5) was 0.8-1.3 lower than that of the Pseudomonas enzyme . The Aureobacterium enzyme showed much higher and lower affinities for NAD+ (Km, 0.140 +/- 0.008 mM) and pyridoxal (0.473 +/- 0.109 mM), respectively, than those of the Pseudomonas enzyme . The Aureobacterium enzyme could use NADP+ as a substrate: the reactivity was 6.5% of NAD+ . The enzyme was much more tolerant to metal-chelating agents . Irreversibility of the enzymatic reaction was shared by the two enzymes . No aldehyde dehydrogenase showed similarity to the amino-terminal amino acid sequence of the enzyme.

J Bacteriol, 2002 Jun, 184(11), 3146 - 9
Methylation of inorganic and organic selenium by the bacterial thiopurine methyltransferase; Ranjard L et al.; Escherichia coli cells expressing the tpm gene encoding the bacterial thiopurine methyltransferase (bTPMT) are shown to methylate selenite and (methyl)selenocysteine into dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) . E . coli cells expressing tpm from a gene library cosmid clone (harboring a Pseudomonas syringae insert of about 20 kb) also methylated selenate into DMSe and DMDSe . bTPMT is the first methyltransferase shown to be involved in the methylation of these selenium derivatives.

J Bacteriol, 2002 Jun, 184(11), 2914 - 24
Identification and physical characterization of the HbpR binding sites of the hbpC and hbpD promoters; Tropel D et al.; Pseudomonas azelaica HBP1 can use 2-hydroxybiphenyl (2-HBP) and 2,2'-dihydroxybiphenyl as sole carbon and energy sources by means of the hbp regulon . This regulon is composed of three genes, hbpCA and hbpD, coding for enzymes of a meta-cleavage pathway and the hbpR gene, which codes for a XylR/DmpR-type transcription regulator . It was previously shown that HbpR activates transcription from two sigma(54)-dependent promoters, P(hbpC) and P(hbpD), in the presence of 2-HBP . In this study, by using gel mobility shift assays with a purified fusion protein containing calmodulin binding protein (CBP) and HbpR, we detected two binding regions for HbpR in P(hbpC) and one binding region in P(hbpD) . DNase I footprints of the proximal binding region of P(hbpC) and of the binding region in P(hbpD) showed that CBP-HbpR protected a region composed of two inverted repeat sequences which were homologous to the binding sites identified for XylR . Unlike the situation in the XylR/P(u) system, we observed simultaneous binding of CBP-HbpR on the two upstream activating sequences (UASs) . Fragments with only one UAS did not show an interaction with HbpR, indicating that both pairs of UASs are needed for HbpR binding . The addition of both ATP and 2-HBP increased the DNA binding affinity of HbpR . These results showed for the first time that, for regulators of the XylR/DmpR type, the effector positively affects the recruitment of the regulatory protein on the enhancer DNA.

Microbiol Res, 2002, 157(2), 93 - 102
Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii, cause of brown blotch disease of the cultivated mushroom Agaricus bisporus; Munsch P et al.; Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized . Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329 . These 16 isolates of P . tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates . Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping . Ribotyping differentiated P . tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes . A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P . tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2) . Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T . Sequence determination and analysis of the internally transcribed spacer region ITSI for P . tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars . It is concluded that considerable genotypic differences exist among Finnish isolates of P . tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies.

Pediatr Pulmonol, 2002 Jun, 33(6), 483 - 91
Predictors of deterioration of lung function in cystic fibrosis; Schaedel C et al.; The severity of lung disease in cystic fibrosis (CF) may be related to the type of mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and to environmental and immunological factors . Since pulmonary disease is the main determinant of morbidity and mortality in CF, it is important to identify factors that can explain and predict this variation . The aim of this longitudinal study of the whole Swedish CF population over age 7 years was to correlate genetic and clinical data with the rate of decline in pulmonary function . The statistical analysis was performed using the mixed model regression method, supplemented with calculation of relative risks for severe lung disease in age cohorts.The severity of pulmonary disease was to some extent predicted by CFTR genotype . Furthermore, the present investigation is the first long-term study showing a significantly more rapid deterioration of lung function in patients with concomitant diabetes mellitus . Besides diabetes mellitus, pancreatic insufficiency and chronic Pseudomonas colonization were found to be negative predictors of pulmonary function . In contrast to several other reports, we found no significant differences in lung function between genders . Patients with pancreatic sufficiency have no or only a slight decline of lung function with age once treatment is started, but an early diagnosis in this group is desirable .

Plant J, 2002 May, 30(3), 361 - 71
The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes; Ballvora A et al.; Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide . New, more virulent P . infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties . R genes (for single-gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato . The molecular basis of single-gene and quantitative resistance to late blight is unknown . We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach . The R1 gene is member of a gene family . It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa . The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine-rich repeat domain . The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato . R1 is located within a hot spot for pathogen resistance on potato chromosome V . In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.

Trends Plant Sci, 2002 May, 7(5), 210 - 6
Priming in plant-pathogen interactions; Conrath U et al.; Plants can acquire enhanced resistance to pathogens after treatment with necrotizing attackers, nonpathogenic root-colonizing pseudomonads, salicylic acid, beta-aminobutyric acid and many other natural or synthetic compounds . The induced resistance is often associated with an enhanced capacity to mobilize infection-induced cellular defence responses - a process called 'priming' . Although the phenomenon has been known for years, most progress in our understanding of priming has been made only recently . These studies show that priming often depends on the induced disease resistance key regulator NPR1 (also known as NIM1 or SAI1) and that priming has a major effect on the regulation of cellular plant defence responses.

J Biol Chem, 2002 Jul 12, 277(28), 25096 - 105 Epub 2002 May 01.
Isolation of cyanophycin-degrading bacteria, cloning and characterization of an extracellular cyanophycinase gene (cphE) from Pseudomonas anguilliseptica strain BI . The cphE gene from P . anguilliseptica BI encodes a cyanophycinhydrolyzing enzyme; Obst M et al.; Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated . One isolate was taxonomically affiliated as Pseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described . CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix . The mature form of the inducible enzyme consisted of one type of subunit with M(r) = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed . Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (beta-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry . The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression in Escherichia coli, and its nucleotide sequence was determined . The enzyme exhibited only 27-28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria . Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue . In addition, the strong inhibition of the enzyme by Pefabloc((R)) and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases . Quantitative analysis on the release of beta-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 45 - 50
{Nucleotide sequence and protein sequence analysis of GL-7-ACA acylase from Pseudomonas sp . 130}; Mao X et al.; The nucleotide sequence and N-, C-terminal amino acid sequences of alpha,beta-subunit of glutaryl 7-ACA acylase C130 from Pseudomonas sp . 130 were determined . The alignment of the acylase C130 with the other acylases shows that it has high homology with the acylases from Pseudomonas sp . GK16 and C427, but low homology with the others . There is large difference in the N-terminal of alpha-subunit, while the N-terminal of beta-subunit has significant conservation.

Appl Environ Microbiol, 2002 May, 68(5), 2600 - 4
Role of glucose in enhancing the temperature-dependent growth inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp; Samelis J et al.; Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25 degrees C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp . found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition . The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested . The pathogen grew better as the temperature increased from 10 to 15 and 25 degrees C and grew better in pure culture than in mixed cultures . Pseudomonas sp . inhibited E . coli O157:H7 in cocultures with glucose at 10 degrees C, while at 15 degrees C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period . Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15 degrees C and, irrespective of glucose, at 25 degrees C . These results indicate that glucose enhances the growth inhibition of E . coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (< or = 15 degrees C) temperatures.

Appl Environ Microbiol, 2002 May, 68(5), 2368 - 75
Transformation of isopropylamine to L-alaninol by Pseudomonas sp . strain KIE171 involves N-glutamylated intermediates; de Azevedo Wasch SI et al.; Pseudomonas sp . strain KIE171 was able to grow with isopropylamine or L-alaninol {S-(+)-2-amino-1-propanol} as the sole carbon source, but not with D-alaninol . To investigate the hypothesis that L-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and L-alaninol were isolated . Whereas mutant KIE171-BI transformed isopropylamine to L-alaninol, mutant KIE171-BII failed to do so . The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced . Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified . Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG . The next step, the formation of gamma-glutamyl-isopropylamide from isopropylamine, ATP, and L-glutamate, was shown to be catalyzed by IpuC, a gamma-glutamylamide synthetase . gamma-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding gamma-glutamyl-L-alaninol {gamma(L-glutamyl)-L-hydroxy-isopropylamide} . Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate L-alaninol and to regenerate L-glutamate . No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters . Presumably, L-alaninol is oxidized by an alcohol dehydrogenase to yield L-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde . Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either L-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.

Appl Environ Microbiol, 2002 May, 68(5), 2179 - 87
Genes from Pseudomonas sp . strain BS involved in the conversion of L-2-amino-Delta(2)-thiazolin-4-carbonic acid to L-cysteine; Shiba T et al.; DL-2-amino-Delta(2)-thiazolin-4-carbonic acid (DL-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry . We cloned a DNA fragment containing the genes involved in the conversion of L-ATC to L-cysteine from Pseudomonas sp . strain BS . The introduction of this DNA fragment into Escherichia coli cells enabled them to convert L-ATC to cysteine via N-carbamyl-L-cysteine (L-NCC) as an intermediate . The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has L-cysteine synthetic activity . The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the L-cysteine synthesis from DL-ATC . These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method . The functions of these gene products were examined using extracts of E . coli cells carrying deletion derivatives of pTK10 . The results indicate that atcB and atcC are involved in the conversion of L-ATC to L-NCC and the conversion of L-NCC to cysteine, respectively . atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening . AtcC is highly homologous with L-N-carbamoylases . Since both enzymes can only catalyze the L-specific conversion from L-ATC to L-NCC or L-NCC to L-cysteine, it is thought that atcB and atcC encode L-ATC hydrolase and N-carbamyl-L-cysteine amidohydrolase, respectively.

J Biol Chem, 2002 Jun 28, 277(26), 23414 - 9 Epub 2002 Apr 24.
Trifluoroethanol increases the stability of Delta(5)-3-ketosteroid isomerase . 15N NMR relaxation studies; Yun S et al.; In the equilibrium unfolding process of Delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni by urea, it was observed that the enzyme stability increases by 2.5 kcal/mol in the presence of 5% trifluoroethanol (TFE) . To elucidate the increased enzyme stability by TFE, the backbone dynamics of Delta(5)-3-ketosteroid isomerase were studied in the presence and absence of 5% TFE by (15)N NMR relaxation measurements, and the motional parameters (S(2), tau(e), and R(ex)) were extracted from the relaxation data using the model-free formalism . The presence of 5% TFE causes little change or a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in the dimer interface region . However, the majority of the residues exhibit reduced order parameters in the presence of 5% TFE, indicating that high frequency (pico- to nanosecond) motions are generally enhanced by TFE . The results suggest that the entropy can be an important factor for the enzyme stability, and the increase in entropy by TFE is partially responsible for the increased stability of Delta(5)-3-ketosteroid isomerase.

Genetics, 2002 Apr, 160(4), 1661 - 71
Isolation and characterization of broad-spectrum disease-resistant Arabidopsis mutants; Maleck K et al.; To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc) . After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M(2) plants for constitutive expression of the reporter gene in vivo . From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death . We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms . Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants . The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

Plant Cell, 2002 Apr, 14(4), 817 - 31
Tomato transcription factors pti4, pti5, and pti6 activate defense responses when expressed in Arabidopsis; Gu YQ et al.; The Pti4, Pti5, and Pti6 proteins from tomato were identified based on their interaction with the product of the Pto disease resistance gene, a Ser-Thr protein kinase . They belong to the ethylene-response factor (ERF) family of plant-unique transcription factors and bind specifically to the GCC-box cis element present in the promoters of many pathogenesis-related (PR) genes . Here, we show that these tomato ERFs are localized to the nucleus and function in vivo as transcription activators that regulate the expression of GCC box-containing PR genes . Expression of Pti4, Pti5, or Pti6 in Arabidopsis activated the expression of the salicylic acid-regulated genes PR1 and PR2 . Expression of jasmonic acid- and ethylene-regulated genes, such as PR3, PR4, PDF1.2, and Thi2.1, was affected differently by each of the three tomato ERFs, with Arabidopsis-Pti4 plants having very high levels of PDF1.2 transcripts . Exogenous application of salicylic acid to Arabidopsis-Pti4 plants suppressed the increased expression of PDF1.2 but further stimulated PR1 expression . Arabidopsis plants expressing Pti4 displayed increased resistance to the fungal pathogen Erysiphe orontii and increased tolerance to the bacterial pathogen Pseudomonas syringae pv tomato . These results indicate that Pti4, Pti5, and Pti6 activate the expression of a wide array of PR genes and play important and distinct roles in plant defense.

Plant J, 2002 Apr, 30(1), 61 - 70
The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease; Pilloff RK et al.; We describe the characterization of a novel gain-of-function Arabidopsis mutant, dll1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection . Despite the constitutive expression of defense-related responses, dll1 is unable to suppress the growth of virulent pathogens . However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses . The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants . This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease . Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death . Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process.

Mol Microbiol, 2002 Apr, 44(1), 73 - 88
Identification of Pseudomonas syringae pv . tomato genes induced during infection of Arabidopsis thaliana; Boch J et al.; Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants . Many of these genes should be induced when the bacteria come in contact with plant tissue . We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv . tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions . Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes . In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors . Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes . Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue . Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P . syringae . Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis.

Fiziol Zh, 2001, 47(6), 55 - 7
{The protective effect of anti-Pseudomonas blood preparations}; Nazarchuk LV et al.; Human blood antipseudomonas preparation activity has been studied on the model of Ps . Aeruginosa etiology sepsis in white not pedigree mice . It has been found out that antipseudomonas plasma and antipseudomonas immunoglobulin with antibody tire 1:80 possess marked therapeutic properties . The obtained results of the experimental studies are the grounds for clinical studies carrying out with the use of blood antipseudomonas preparations in combined therapy of the patients with diseases of Ps . Aeruginosa etiology.

J Appl Physiol, 2002 May, 92(5), 2169 - 76
Characterization of LPS-induced lung inflammation in cftr-/- mice and the effect of docosahexaenoic acid; Freedman SD et al.; The mechanism by which Pseudomonas causes excessive inflammation in the cystic fibrosis lung is unclear . We have reported that arachidonic acid is increased and docosahexaenoic acid (DHA) decreased in lung, pancreas, and ileum from cftr-/- mice . Oral DHA corrected this defect and reversed the pathology . To determine which mediators regulate inflammation in lungs from cftr-/- mice and whether inhibition occurs with DHA, cftr-/- and wild-type (WT) mice were exposed to aerosolized Pseudomonas lipopolysaccharide (LPS) . After 2 days of LPS, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2, and KC levels in bronchoalveolar lavage fluid were increased in cftr-/- compared with WT mice and not suppressed by pretreatment with oral DHA . Neutrophil levels were not different between cftr-/- and WT mice . After 3 days of aerosolized LPS, neutrophil concentration, TNF-alpha, and the eicosanoids 6-keto-PGF1alpha, PGF2alpha, PGE2, and thromboxane B2 were all increased in bronchoalveolar lavage fluid from cftr-/- mice compared with WT controls . Oral DHA had no significant effect on TNF-alpha levels in cftr-/- mice . In contrast, neutrophils and eicosanoids were decreased in cftr-/- but not in WT mice treated with DHA, indicating that the effects of DHA on these inflammatory parameters may be related to correction of the membrane lipid defect.

J Mol Evol, 2002 Apr, 54(4), 437 - 57
A phylogenomic study of the OCTase genes in Pseudomonas syringae pathovars: the horizontal transfer of the argK-tox cluster and the evolutionary history of OCTase genes on their genomes; Sawada H et al.; Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity . Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al . (1999) showed that the ancestor of P . syringae had diverged into at least three monophyletic groups during its evolution . Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome . The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P . syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements . Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK-tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P . syringae pathovars (pv . actinidiae and pv . phaseolicola) from bacterial species distantly related to P . syringae and that its acquisition was quite recent (i.e., after the ancestor of P . syringae diverged into the respective pathovars) . Furthermore, the results of a detailed analysis of argK {an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene}, which is present within the argK-tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P . aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P . syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK-tox cluster has horizontally transferred onto the genomes of pv . actinidiae and pv . phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars . Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P . syringae.

Cell, 2002 Mar 22, 108(6), 743 - 54
RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis; Mackey D et al.; In Arabidopsis, RPM1 confers resistance against Pseudomonas syringae expressing either of two sequence unrelated type III effectors, AvrRpm1 or AvrB . An RPM1-interacting protein (RIN4) coimmunoprecipitates from plant cell extracts with AvrB, AvrRpm1, or RPM1 . Reduction of RIN4 protein levels inhibits both the hypersensitive response and the restriction of pathogen growth controlled by RPM1 . RIN4 reduction causes diminution of RPM1 . RIN4 reduction results in heightened resistance to virulent Peronospora parasitica and P . syringae, and ectopic defense gene expression . Thus, RIN4 positively regulates RPM1-mediated resistance yet is, formally, a negative regulator of basal defense responses . AvrRpm1 and AvrB induce RIN4 phosphorylation . This may enhance RIN4 activity as a negative regulator of plant defense, facilitating pathogen growth . RPM1 may "guard" against pathogens that use AvrRpm1 and AvrB to manipulate RIN4 activity.

Mol Plant Microbe Interact, 2002 Mar, 15(3), 281 - 91
Functional analyses of the Pto resistance gene family in tomato and the identification of a minor resistance determinant in a susceptible haplotype; Chang JH et al.; Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv . tomato expressing avrPto . The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases . In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system . The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophosphorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf . LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction . The interaction with AvrPto was unique to Pto in the yeast two-hybrid system . LescPth2 autophosphorylated in vitro as a fusion protein . LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro . Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana . LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf . Consequently, some homologs could potentially encode functional resistance proteins . LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a 'susceptible' haplotype encoding a minor recognition determinant.

Plant Physiol, 2002 Apr, 128(4), 1313 - 22
Identification of Arabidopsis ethylene-responsive element binding factors with distinct induction kinetics after pathogen infection; Onate-Sanchez L et al.; Ethylene-responsive element binding factors (ERF) proteins are plant-specific transcription factors, many of which have been linked to stress responses . We have identified four Arabidopsis ERF genes whose expression was specifically induced by avirulent and virulent strains of the bacterial pathogen Pseudomonas syringae pv tomato, with overlapping but distinct induction kinetics . However, a delay in ERF mRNA accumulation after infection with the virulent strain was observed when compared with the avirulent strain . The induction of ERF gene expression in most cases preceded the mRNA accumulation of a basic chitinase gene, a potential downstream target for one or more of these ERFs . The expression of the ERF genes was examined among different Arabidopsis tissues, in response to the signaling molecules ethylene, methyl jasmonate, and salicylic acid (SA), and in Arabidopsis mutants with decreased or enhanced susceptibility to pathogens, and significant differences were observed . For example, in seedlings, some of the ERF genes were not induced by SA in the wild-type but were SA responsive in the pad4-1 mutant, suggesting that PAD4-1, which acts upstream of SA accumulation, is also involved in repressing the SA-induced expression of specific ERF genes . The four ERF proteins were shown to contain transcriptional activation domains . These results suggest that transcriptional activation cascades involving ERF proteins may be important for plant defense to pathogen attack and that some ERF family members could be involved in the cross-talk between SA- and jasmonic acid-signaling pathways.

Clin Cancer Res, 2002 Apr, 8(4), 995 - 1002
Improved cytotoxic activity toward cell lines and fresh leukemia cells of a mutant anti-CD22 immunotoxin obtained by antibody phage display; Salvatore G et al.; Recombinant immunotoxins are fusion proteins composed of the Fv domains of antibodies fused to bacterial or plant toxins that are being developed for the targeted therapy of cancer . RFB4 (Fv)-Pseudomonas exotoxin 38 (PE38) is an immunotoxin that targets CD22 expressed on B cells and B-cell malignancies . A disulfide-stabilized form of RFB4 (Fv)-PE38 is being evaluated in a Phase I clinical trial . The aim of the present study was to improve the activity of RFB4 (Fv)-PE38 to more effectively treat patients with leukemias and lymphomas . To increase the affinity of RFB4 (Fv), we used the techniques of phage display and hot spot mutagenesis . We identified mutational hot spot sequences in heavy chain complementary determining region 3 (V(H) CDR3) and randomized these in a phage display library . Mutant phages were panned on CD22-positive Daudi cells . A variety of mutant Fvs were obtained, and the corresponding immunotoxins were prepared . Several mutant immunotoxins with increased binding affinity and cytotoxic activity were obtained . The most active immunotoxin contained amino acid residues Thr-His-Trp (THW) in place of Ser-Ser-Tyr (SSY) at positions 100, 100A, and 100B of the Fv and had an affinity improved from 85 nM to 6 nM . The THW mutant had a 5- to 10-fold increase in activity on various CD22-positive cell lines and was up to 50 times more cytotoxic to cells from patients with chronic lymphocytic leukemia and hairy-cell leukemia.

J Microbiol Methods, 2002 Jun, 50(1), 97 - 100
A filtration, incubation and staining reactor including a new protocol for FISH; Poschen L et al.; A newly developed device for performing fluorescence in situ hybridization (FISH) is described . An adapted procedure was compared with two typical FISH protocols . Tests were performed with Pseudomonas cells and the gene probe EUB338 . With the novel procedure, we obtained a better recovery of cells and less variability in results.

J Am Chem Soc, 2002 Apr 17, 124(15), 3814 - 5
Decay of the transient Cu(B)-CO complex is accompanied by formation of the heme Fe-CO complex of cytochrome cbb(3)-CO at ambient temperature: evidence from time-resolved Fourier transform infrared spectroscopy; Stavrakis S et al.; Time-resolved step-scan Fourier infrared spectroscopy has been used to study the CO-bound cbb(3)-type cytochrome c oxidase from Pseudomonas stutzeri at room temperature . We observe a single band in the FTIR spectrum at 1956 cm(-1) (beta-form) . The time-resolved data indicate that upon photolysis, CO is transferred from heme b(3) (nu(CO) = 1956 cm(-1)) to CuB (nu(CO) = 2064 cm(-1)) . The decay of the 2065 cm(-1) peak (t(1/2) = 120 +/- 16 ms) and the development of the 1956 cm(-1) peak (t(1/2) = 144 +/- 8 ms ) suggest that formation of the Fe-CO complex is concurrent with the decay of the CuB-CO complex . The intensity ratio of the Fe-CO/CuB-CO (2.15) remains constant for all data points, and thus we conclude that no fraction of CO escapes the binuclear center at 293 K.

Structure (Camb), 2002 Apr, 10(4), 547 - 56
The structural basis for catalysis and specificity of the Pseudomonas cellulosa alpha-glucuronidase, GlcA67A; Nurizzo D et al.; Alpha-glucuronidases, components of an ensemble of enzymes central to the recycling of photosynthetic biomass, remove the alpha-1,2 linked 4-O-methyl glucuronic acid from xylans . The structure of the alpha-glucuronidase, GlcA67A, from Pseudomonas cellulosa reveals three domains, the central of which is a (beta/alpha)(8) barrel housing the catalytic apparatus . Complexes of the enzyme with the individual reaction products, either xylobiose or glucuronic acid, and the ternary complex of both glucuronic acid and xylotriose reveal a "blind" pocket which selects for short decorated xylooligosaccharides substituted with the uronic acid at their nonreducing end, consistent with kinetic data . The catalytic center reveals a constellation of carboxylates; Glu292 is poised to provide protonic assistance to leaving group departure with Glu393 and Asp365 both appropriately positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water.

Mol Cell Biochem, 2002 Jan, 229(1-2), 25 - 34
The impact of insulin-like growth factor-1 on the pattern of cardiac elongation factor-2 variants in a model of overload; Jager D et al.; Because of its key role in proteosynthesis, the total content of elongation factor-2 (EF-2) and the distribution of six main EF-2 variants were investigated after Pseudomonas Exotoxin A catalyzed {37P}ADP-ribosylation using 1D-PAGE and isoelectric focusing (IEF) in a rat model of hemodynamic overload with variable degrees of cardiac hypertrophy: Chronic NO-synthase inhibition by L-NAME (N-omega-nitro-L-arginine-methyl-ester; 0.75 mg/ml drinking water) induced arterial hypertension without hypertrophy but myocardial apoptosis; additional treatment with IGF-1 (osmotic micropumps) did not modify hypertension but reduced apoptosis allowing moderate hypertrophy of the left ventricles . Total EF-2 did not significantly increase in rats with hemodynamic overload with or without IGF-1 supplementation . A positive correlation was found between an acidic EF-2 variant and apoptosis (p = 0.01) . Hypertrophy under additional IGF-1 was combined with a shift of the EF-2 variants to basic subtypes (p < 0.01) . This finding may be indicative of the trophic potency of IGF-1.

J Mol Microbiol Biotechnol, 2002 May, 4(3), 191 - 6
Control of temperature-responsive synthesis of the phytotoxin coronatine in Pseudomonas syringae by the unconventional two-component system CorRPS; Smirnova AV et al.; The phytopathogen Pseudomonas syringae produces the phytotoxin coronatine (COR) as a major virulence factor . COR and its precursor, coronafacic acid, function as molecular mimics of the plant signaling molecule jasmonate . A 32.8-kb plasmid-borne gene cluster mediates COR biosynthesis, which is optimal at 18 degrees C and non-detectable at 28 degrees C, the optimal growth temperature for P . syringae . The thermoregulation is mediated at the transcriptional level by an unconventional two-component regulatory system consisting of a histidine protein kinase, CorS, and two transcriptional activators, CorR and CorP . Dissection of this regulatory triad revealed that CorR binds to its target sequences in a thermoresponsive manner and that its DNA-binding activity is controlled by CorS . A Preliminary model for thermo-sensing by CorS is proposed based on its membrane topology and the analysis of translational fusions of CorS to reporter enzymes at different temperatures . CorP lacks a typical helix-turn-helix motif but possibly functions as a modulator of CorR or CorS activity . The thermoregulation of COR biosynthetic genes is widespread among various COR-producing P . syringae strains . Post-translational processes also contribute to the thermo-responsiveness of COR production . Additionally, COR synthesis in P . syringae is influenced by nutrient availability, rpoN encoding the alternative sigma factor sigma54, and HrpV, a negative regulator of hrp gene expression, suggesting a complex regulatory network governing phytotoxin synthesis.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 513 - 23
Pseudomonas lini sp . nov., a novel species from bulk and rhizospheric soils; Delorme S et al.; The taxonomic position of eight fluorescent Pseudomonas strains isolated from bulk and rhizospheric soils, and from water was examined . These eight strains clustered in one phenon together with Pseudomomas mandelii (CFBP 4844T), but could still be differentiated from this type strain by four phenotypic features . The eight stains exhibited internal DNA-DNA hybridization values ranging from 60 to 100%, with deltaTm below 5 degrees C (3.9 and 4.3 degrees C) for the lowest values (60 and 66%) . The percentages of hybridization with type or reference strains of other Pseudomonas species tested ranged from 12 to 60% (deltaTm = 5.5 degrees C), indicating that the eight isolates studied constituted a discrete DNA homology group . Comparison of the 16S rDNA sequence of the strain representing this group (CFBP 5737T) with the sequences of other strains belonging to the genus Pseudomonas revealed that strain CFBP 5737T was a member of this genus and that these bacteria did not cluster with any previously described species of the genus Pseudomonas . The eight isolates belonged to two siderovars different from those described so far . On the basis of the results of phenotypic, DNA-DNA and phylogenetic analyses, and of siderotyping, a new species, Pseudomonas lini sp . nov . (type strain CFBP 5737T) is proposed.

Ir Med J, 2002 Jan, 95(1), 14 - 6
Malignant otitis externa--a high index of suspicion is still needed for diagnosis; Walshe P et al.; Malignant otitis externa is a destructive inflammatory process of the petrous temporal bone which if untreated leads to osteomyelitis of the skull base and can be fatal . It is more common in immunocompromised and elderly insulin-dependant diabetic patients and is caused by infection with Pseudomonas species . Despite a range of laboratory and radiological tests it still remains difficult to diagnose, particularly in the early stages when it can be treated medically . We describe three cases which presented to this department in the past twelve months . In all cases the diagnosis was made clinically and confirmed per-operatively . Interestingly all three cases were relatively young patients who did not have an immunocompromised status and were not diabetic.

J Basic Microbiol, 2002, 42(1), 75 - 9
Regulation of pyrimidine synthesis in Pseudomonas mendocina; Santiago MF et al.; Regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was examined when its cells were grown on succinate as a carbon source . When P . mendocina was grown in the presence of orotic acid or uracil, the de novo enzyme activities were depressed with dihydroorotase activity being significantly depressed after uracil addition . Following pyrimidine limitation of a uracil auxotroph of P . mendocina deficient for orotate phosphoribosyltransferase activity, the pyrimidine biosynthetic pathway enzyme activities were affected indicating possible regulation at the level of enzyme synthesis . Of the de novo pathway enzymes assayed, dihydroorotate dehydrogenase exhibited the highest increase in its activity . The regulation of the pyrimidine biosynthetic pathway by pyrimidines in P . mendocina appeared similar to what was previously observed for the taxonomically-related species P . stutzeri.

Protein Expr Purif, 2002 Apr, 24(3), 439 - 44
Tabtoxin-resistant protein: overexpression, purification, and characterization; Liu J et al.; One of the self-protection mechanisms in Pseudomonas syringae pv . tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr) . In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels . The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE . Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum . The spectrophotometric assay demonstrated the strong detoxification activity of TTR . These results are the first report of the characterization of the purified tabtoxin-resistant protein . Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv . tabaci .

Mol Genet Genomics, 2002 Mar, 267(1), 16 - 26 Epub 2002 Feb 12.
The tobacco bZIP transcription factor BZI-1 binds to G-box elements in the promoters of phenylpropanoid pathway genes in vitro, but it is not involved in their regulation in vivo; Heinekamp T et al.; Screening of a tobacco (Nicotiana tabacum) cDNA library resulted in the isolation of a clone encoding the bZIP transcription factor BZI-1 . With respect to amino acid sequence, conservation of protein domains, genomic exon-intron structure and expression pattern, BZI-1 is closely related to CPRF2, OHP1/2, BLZ1 and REB, a group of bZIP proteins which have been described in a number of dicot and monocot species . BZI-1 exhibits the characteristics of a transcription factor . It binds to G-box and C-box cis-elements in vitro, it is localised in the nucleus, and the N-terminal region of BZI-1 functions as an activation domain in both yeast and plant cells . Since BZI-1-related transcription factors have been isolated from dicots by in vitro binding to G-box elements in the chalcone synthase ( CHS) promoter, it has been suggested that phenylpropanoid pathway genes, such as CHS and PAL (phenylalanine ammonia-lyase), are targets of these proteins in vivo . However, after infection with Pseudomonas syringae or Tobacco Mosaic Virus, no changes in pathogen-induced PAL expression were observed in transgenic plants expressing increased levels of BZI-1 or a dominant negative form of the protein, BZI-1-DeltaN . In contrast to the tissue-specific expression of CHS and PAL, BZI-1 was found to be ubiquitously expressed in tobacco plants . Furthermore, no changes in the tissue-specific expression of PAL or CHS were observed in plants that were transgenic for BZI-1-DeltaN . Expression of a VP16-BZI-1 fusion protein would be expected to result in constitutive activation of the BZI-1 target genes . However, tetracycline-dependent expression of a VP16-BZI-1 protein in tobacco plants did not result in activation of CHS or PAL . On the basis of these data, we conclude that the phenylpropanoid pathway genes analysed are not targets of BZI-1 in vivo . Thus, the pattern of in vitro DNA binding of transcription factors need not always reflect their in vivo function.

DNA Seq, 2001 Nov, 12(4), 281 - 4
Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens; Umeda F et al.; Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens . In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P . aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp . 61-3, P . oleovorans and P . aeruginosa) containing the homologous pha cluster . The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains . Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P . aureofaciens.

J Comp Physiol {B}, 2002 Feb, 172(2), 163 - 8
The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate; Duman JG; Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis . Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol . DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful . However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone . This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D . canadensis . Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.

Acta Crystallogr D Biol Crystallogr, 2002 Apr, 58(Pt 4), 697 - 9 Epub 2002 Mar 22.
Crystallization and preliminary X-ray diffraction analysis of two pH-dependent forms of a di-haem cytochrome c peroxidase from Pseudomonas nautica; Dias JM et al.; Two crystal forms of cytochrome c peroxidase from Pseudomonas nautica were obtained, one at pH 4.0 using sodium citrate as precipitant and another at pH 5.3 using ammonium phosphate and sodium citrate as precipitants . The two forms belong to different space groups P3(1)21 (pH 4.0) and P6(4)22 (pH 5.3), with unit-cell parameters a = b = 114.5, c = 90.7 A and a = b = 151.0, c = 155.9 A, respectively . Several complete data sets were collected using synchrotron radiation at ESRF and Cu K(alpha) X-ray radiation from a rotating-anode generator . These results will contribute to clarifying the haem transitions occurring during peroxidatic reaction and the required electron-transfer processes and to elucidating the catalytic mechanism of the enzyme and the role of calcium in the activation process.

J Bacteriol, 2002 Apr, 184(8), 2281 - 6
Global regulation by gidA in Pseudomonas syringae; Kinscherf TG et al.; Analysis of two virulence mutants of Pseudomonas syringae B728a revealed that the Tn 5 sites of insertion were within the gidA open reading frame (ORF) . These mutations were pleiotropic, affecting diverse phenotypic traits, such as lipodepsipeptide (syringomycin and syringopeptin) antibiotic production, swarming, presence of fluorescent pigment, and virulence . Site-specific recombination of a disrupted gidA gene into the chromosome resulted in the same phenotypic pattern as transposon insertion . Mutant phenotypes were restored by the gidA ORF on a plasmid . The salA gene, a copy number suppressor of the syringomycin-deficient phenotype in gacS and gacA mutants, was also found to suppress the antibiotic-negative phenotypes of gidA mutants, suggesting that gidA might play some role in salA regulation . Reporter studies with chromosomal salA-lacZ translational fusions confirmed that salA reporter expression decreased approximately fivefold in a gidA mutant background, with a concurrent decrease in the expression of the syringomycin biosynthetic reporter fusion syrB-lacZ . Wild-type levels of reporter expression were restored by supplying an intact gidA gene on a plasmid . Often described as being involved in cell division, more recent evidence suggests a role for gidA in moderating translational fidelity, suggesting a mechanism by which global regulation might occur . The gidA gene is essentially universal in the domains Bacteria and Eucarya but has no counterparts in Archaea, probably reflecting specific differences in the translational machinery between the former and latter domains.

Microbiol Res, 2002, 157(1), 7 - 11
The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"; Munsch P et al.; A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans" . This interaction has been considered as highly specific . However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T . Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P . tolaasii(T), produced a typical white precipitate when streaked towards P . "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P . tolaasii . We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P . tolaasii.

Biochim Biophys Acta, 2002 Feb 11, 1594(2), 207 - 18
The wild type bacterial Co(2+)/Co(2+)-phosphotriesterase shows a middle-range thermostability; Rochu D et al.; The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein {Grimsley et al., Biochemistry 36 (1997), 14366-14374} . Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co(2+)/Co(2+)-PTE . This study, carried out using capillary electrophoresis under optimised conditions in the pH range 9-10 compatible with optimal enzyme activity, provided evidence for irreversible denaturation according to the Lumry-Eyring model . A temperature-induced conformational transition (T(m) approximately equal to 58 degrees C) and an early growing of aggregates were observed . Comparison of UV spectra with heat-induced inactivation data clearly demonstrated that the PTE state populated above T(m) was neither native nor active . Differential scanning calorimetry showed only an exothermic trace due to aggregation of the denatured protein at T=76 degrees C . Accordingly, the temperature-induced denaturation process of the PTE could be described by a consecutive reaction model, including formation of an intermediate with enhanced activity at T approximately equal to 45 degrees C and an inactive unfolded state populated at T approximately equal to 58 degrees C, which leads to denatured aggregates . Thus, the wild type Co(2+)/Co(2+)-PTE displays a middle-range thermostability . Hence, for decontamination purposes under extreme Earth temperatures, wild type and engineered mutants of PTE substituted with other metal cations should be evaluated.

Res Microbiol, 2002 Mar, 153(2), 89 - 98
Occurrence and properties of glutathione S-transferases in phenol-degrading Pseudomonas strains; Santos PM et al.; Pseudomonas sp . strains, able to degrade aromatic compounds such as phenol, were chosen to investigate the occurrence and characteristics of glutathione S-transferases (GSTs) . Affinity chromatography purification showed the presence of at least one GST in each studied strain . The purified proteins exhibited a great variety in the N-terminal sequences and different enzyme activities with the standard GST substrates tested . Two Pseudomonas strains, M1 and CF600, were chosen to investigate the GST activities under different growth conditions . Therefore, cells were grown either on phenol or on different nonaromatic carbon sources in the presence and absence of increasing phenol concentrations . In strain M1 a strong correlation between the activities of the catechol 1,2-dioxygenase and GST was observed in all the tested conditions . Moreover, growth on different organic acids also affected GST activity levels, with a negative correlation with the specific growth rate determined by each substrate . These results suggest a possible function of GST as a response to specific metabolic conditions determined by phenol toxicity and/or catabolism and the metabolic status of the cells . The same experiments performed with the CF600 strain did not show induction of GST activity in any of the tested conditions, indicating that GST_CF600 probably has a different role in cell metabolism . Native gel electrophoresis gave indications that GST dimerization could be an important process in the modulation of GST activity.

Folia Microbiol (Praha), 2001, 46(5), 371 - 5
Multilocus enzyme electrophoresis analysis of Pseudomonas syringae pv . phaseolicola; Guven K; A multilocus enzyme electrophoresis technique was developed to detect variation in seven enzyme loci among isolates of Pseudomonas syringae pv . phaseolicola, representing three races from different geographical locations, the causal agent of the halo blight disease of beans . Cellulose acetate gel electrophoresis of seven enzymes revealed 19 electrotypes (ET) among 21 Pseudomonas syringae pv . phaseolicola isolates . One of the pathovar syringae and one of the pathovar tomato isolates were represented by two different ET . The population of Turkish isolates and three races of the pathovar phaseolicola appeared to be genetically diverse.

Folia Microbiol (Praha), 2001, 46(6), 515 - 8
Production, purification and characterization of intracellular alanylaminopeptidase of Pseudomonas sp; Jankiewicz U et al.; The soil bacterium Pseudomonas sp . was found to synthesize an aminopeptidase that prefers Ala-beta-naphtylamide as substrate . The enzyme was purified 660-fold by ammonium sulfate fractionation, preparative electrophoresis, ion exchange chromatography on Protein-Pak Q 8 HR and molecular sieving chromatography on Zorbax SE-250 . When purified to homogeneity, the enzyme was shown to be a monomeric protein with a molar mass of 65 kDa; it showed a maximum activity at pH 7.5 and 45 degrees C.

J Inorg Biochem, 2002 Feb, 88(3-4), 316 - 27
Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4); Andersen NH et al.; P . stutzeri cytochrome c(4) is a di-haem protein, composed of two globular domains each with His-Met coordinated haem, and a hydrogen bond network between the domains . The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and different spin states of the oxidised haem groups . We have studied unfolding of oxidised P . stutzeri cyt c(4) induced thermally and by chemical denaturants . Horse heart cyt c was a reference molecule . Isothermal unfolding induced by guanidinium chloride and acid was followed by Soret, alpha/beta, and 701-nm band absorption, and by far-UV circular dichroism spectroscopy . Multifarious patterns emerge, but the two domains clearly unfold sequentially . One phase, assigned to unfolding of the N-terminal domain, proceeds at guanidinium concentrations up to approximately 1.0 M . This is followed by two overlapping phases at higher concentrations . The intermediate state maintains Fe-Met coordination, assigned to the C-terminal domain . Interdomain interaction is reflected in decreasing values of the cooperativity parameters . Differential scanning calorimetry shows a single peak, but two peaks appear when guanidinium chloride up to 0.4 M is present . This reflects different chemical action in chemical and thermal unfolding . Acid-induced unfolding kinetics was addressed by pH jumps using diode array stopped-flow techniques . Three kinetic phases in the 701 nm Fe-Met marker band, and four phases in the Soret and alpha/beta bands, spanning 4-1000 ms could be distinguished on pH jumps from 7.5 to the range 2.5-3.5 . In this range of time and pH cyt c appears to unfold in no more than two phases . Spectral properties of the kinetic intermediates could be identified . Sequential domain unfolding, formation of high-spin states, and an intermediate state with Fe-Met coordination to a single haem group are features of the unfolding kinetics.

Bone Marrow Transplant, 2002 Feb, 29(4), 353 - 6
Radiologically guided fine needle lung biopsies in the evaluation of focal pulmonary lesions in allogeneic stem cell transplant recipients; Jantunen E et al.; Lung problems are common in allogeneic stem cell transplant (SCT) recipients . To evaluate the feasibility and diagnostic yield of radiologically guided fine needle lung biopsy (FNLB) in allogeneic SCT recipients with focal pulmonary lesions, a retrospective analysis was carried out . Between 1989 and 1998, radiologists performed a total of 30 FNLBs in 21 allogeneic SCT recipients, guided either by ultrasound (n = 17) or computed tomography (n = 13) . The median time from SCT to the first FNLB was 131 days (20-343 days) . Prophylactic platelet transfusions were given in 19 procedures (66%) . The complications of FNLB included clinically insignificant pneumothorax in four procedures (13%) and self-limiting haemoptysis in one case (3%) . The first FNLB was suggestive of invasive pulmonary aspergillosis (IPA) in five patients (24%) . Additional clinically useful findings of FNLB included Pseudomonas (two patients) and Nocardia (one patient) . The final diagnosis of pulmonary lesions was IPA in 14 patients, immunological lung problems in four patients and other in three patients . Radiologically guided FNLB is feasible in allogeneic SCT recipients and has a low complication rate . The diagnostic yield is high especially for IPA.

Plant Physiol, 2002 Mar, 128(3), 1046 - 56
Benzothiadiazole-induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis; Kohler A et al.; Systemic acquired resistance (SAR) is a plant defense state that is induced, for example, after previous pathogen infection or by chemicals that mimic natural signaling compounds . SAR is associated with the ability to induce cellular defense responses more rapidly and to a greater degree than in noninduced plants, a process called "priming." Arabidopsis plants were treated with the synthetic SAR inducer benzothiadiazole (BTH) before stimulating two prominent cellular defense responses, namely Phe AMMONIA-LYASE (PAL) gene activation and callose deposition . Although BTH itself was essentially inactive at the immediate induction of these two responses, the pretreatment with BTH greatly augmented the subsequent PAL gene expression induced by Pseudomonas syringae pv . tomato infection, wounding, or infiltrating the leaves with water . The BTH pretreatment also enhanced the production of callose, which was induced by wounding or infiltrating the leaves with water . It is interesting that the potentiation by BTH pretreatment of PAL gene activation and callose deposition was not seen in the Arabidopsis nonexpresser of PR genes 1/noninducible immunity 1 mutant, which is compromised in SAR . In a converse manner, augmented PAL gene activation and enhanced callose biosynthesis were found, without BTH pretreatment, in the Arabidopsis constitutive expresser of pathogenesis-related genes (cpr)1 and constitutive expresser of pathogenesis-related genes 5 mutants, in which SAR is constitutive . Moreover, priming for potentiated defense gene activation was also found in pathogen-induced SAR . In sum, the results suggest that priming is an important cellular mechanism in acquired disease resistance of plants that requires the nonexpresser of PR genes 1/noninducible immunity 1 gene.

Int J Med Microbiol, 2002 Feb, 291(6-7), 523 - 9
The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADP-ribosyltransferases; Han S et al.; ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division . The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release . A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins . Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing the NAD-binding pocket formed by the two perpendicular beta-sheet cores has been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases . The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues . In contrast, diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosytransferases are characterized by conserved Arg and catalytic Glu residues . Structural and mutagenic studies of the NAD-binding core of a binary toxin and a C3-like toxin identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition . Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD-binding cleft as characterized by conserved Arg and catalytic Glu residues . Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD-binding mechanisms but differing mechanisms of target protein binding via sequence variations within the ARTT motif structural framework . Thus, we propose here that the ARTT motif represents an experimentally testable general recognition motif region for many ADP-ribosyltransferases and thereby potentially provides a unified structural understanding of substrate recognition in ADP-ribosylation processes.

J Bacteriol, 2002 Apr, 184(7), 1916 - 24
Two distinct alcohol dehydrogenases participate in butane metabolism by Pseudomonas butanovora; Vangnai AS et al.; The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated . The genes coding for BOH and BDH were isolated and characterized . The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence) . The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors . BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced . When induced with butane, the gene for BOH was expressed earlier than the gene for BDH . Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P . butanovora . The P . butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol . These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol . The enzyme activity of BOH was characterized in cell extracts of the P . butanovora strain with bdh disrupted . Unlike BDH, BOH oxidized 2-butanol . The results support the involvement of two distinct NAD(+)-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P . butanovora.

Can J Microbiol, 2002 Jan, 48(1), 49 - 59
Characterization of tdt genes for the degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a; Morgan CA et al.; Resin acids are tricyclic diterpenes that are toxic to aquatic life when released in high concentrations in pulp mill effluents . These naturally formed organic acids are readily degraded by bacteria and fungi; nevertheless, many of the mechanisms involved are still unknown . We report the localization, cloning, and sequencing of genes for abietane degradation (9.18 kb; designated tdt (tricyclic diterpene) LRSABCD) from the gamma-Proteobacterium Pseudomonas diterpeniphila A19-6a . Using gene knockout mutants, we demonstrate that tdtL, encoding a putative CoA ligase, is required for growth on abietic and dehydroabietic acids . A second gene knockout in tdtD, encoding a putative cytochrome P450 monooxygenase, reduced the growth of strain A19-6a on abietic and dehydroabietic acids as sole sources of carbon and energy, but did not eliminate growth . The degree of homology between P450TdtD and P450TerpC, the closest known P450 homologue to TdtD, identifies TdtD as a new member of the P450 superfamily . Hybridization of six of the tdt genes to genomic DNA of a related resin acid degrading bacterium Pseudomonas abietaniphila BKME-9 identified tdt homologues in this strain that utilizes aromatic ring dioxygenase genes (dit) to open the ring structure of abietic and dehydroabietic acids . These results suggest the tdt and dit genes may function in concert to allow these Pseudomonas strains to degrade resin acids . Homologues of several of the tdt genes were detected in resin acid degrading Ralstonia and Comamonas species within the beta- and gamma-Proteobacteria.

FEMS Microbiol Lett, 2002 Jan 22, 207(1), 75 - 80
Natural transformation of Pseudomonas stutzeri by single-stranded DNA requires type IV pili, competence state and comA; Meier P et al.; Pseudomonas stutzeri, in addition to being transformed by duplex DNA, is also transformed by the sense or antisense strand of the genetic marker employed (hisX(+)) or by heat-denatured chromosomal DNA . Transformation was absent in non-competent cells and in mutants defective for pilus biogenesis (pilA, pilC) and function (pilT) or DNA translocation into the cytoplasm (comA) . Uptake of (3)H-thymidine-labeled single-stranded DNA was hardly detectable reflecting the 20- to 60-fold lower transformation compared to duplex DNA . The results suggest that the steps in natural transformation also accommodate single-stranded DNA and that DNA translocation from the periplasm into the cytoplasm is not necessarily coupled to the degradation of a complementary strand . Small DNA single-stranded fragments are thus not excluded from horizontal gene transfer by transformation.

Plant Cell, 2002 Feb, 14(2), 435 - 50
Large-scale structure-function analysis of the Arabidopsis RPM1 disease resistance protein; Tornero P et al.; The Arabidopsis RPM1 gene confers resistance against Pseudomonas syringae expressing either the AvrRpm1 or the AvrB type III effector protein . We present an exhaustive genetic screen for mutants that no longer recognize avrRpm1 . Using an inducible avrRpm1 expression system, we identified 110 independent mutations . These mutations represent six complementation groups . None discriminates between avrRpm1 and avrB recognition . We identified 95 rpm1 alleles and present a detailed structure--function analysis of the RPM1 protein . Several rpm1 mutants retain partial function, and we deduce that their residual activity is dependent on the level of avrRpm1 signal . In these mutants, the hypersensitive response remains activated if the signal goes above a certain threshold . Missense mutations in rpm1 are highly enriched in the nucleotide binding domain, suggesting that this region plays a key role either in the hypersensitive response associated with RPM1 activation or in RPM1 stability . Cluster analysis of rpm1 alleles defines functionally important residues that are highly conserved between nucleotide binding site leucine-rich repeat R proteins and those that are unique to RPM1 . Regions of RPM1 to which no loss-of-function alleles map may represent domains in which variation is tolerated and may contribute to the evolution of new R gene specificities.

Curr Opin Mol Ther, 2002 Feb, 4(1), 72 - 5
Technology evaluation: BL22, NCI; Barth S; BL22 (RFB4(dsFv)-PE38) is a recombinant Pseudomonas exotoxin-based immunotoxin under development by the National Cancer Institute for the treatment of B-cell malignancies . It is composed of the disulfide-stabilized Fv portion of the anti-CD22 antibody RFB4 genetically fused to a truncated form of Pseudomonas exotoxin A . It has entered phase I trials for the treatment of B-cell lymphoma.

OMICS, 2002, 6(1), 11 - 21
Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R; Wechter WP et al.; A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R . Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes . EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons . Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P . synxantha BG33R . A minimum-tile path of 47 BACs was generated and end-sequenced . The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library . Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone . Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P . synxantha.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 229 - 36
Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases Phac1 and PhaC2; Hein S et al.; This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina . The P mendocina pha gene locus, encoding two PHA synthase genes {phaC1Pm and phaC2pm flanking a PHA depolymerase gene (phaZ)}, was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2pm (1,680 bp) were determined . The amino acid sequences deduced from phaC1Pm and phaC2pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto . The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P . putida GPp104 and Ralstonia eutropha PHB-4 . In P . putida GPp 104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads . In contrast, in R . eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA . Recombinant strains of R . eutropha PHB-4 harboring either P . mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed . Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host . Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P . mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P . mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.

J Vasc Surg, 2002 Mar, 35(3), 569 - 72
Melioidosis presenting as an infected intrathoracic subclavian artery pseudoaneurysm treated with femoral vein interposition graft; Schindler N et al.; We present the first case of in situ replacement of an infected subclavian artery using superficial femoral vein and the fourth reported case of an infected arterial pseudoaneurysm caused by pseudomonas pseudomallei . Sepsis and hoarseness developed in a 58-year-old man after recent travel to Borneo, Indonesia . Indirect laryngoscopy revealed a paralyzed right vocal cord . Computed tomography and arteriography revealed a 6.5-cm pseudoaneurysm of the proximal right subclavian artery . Blood cultures grew pseudomonas pseudomallei . An abnormal cardiac stress test prompted a coronary angiography, which revealed severe coronary artery disease.The patient underwent coronary artery bypass and in situ replacement of the infected subclavian artery pseudoaneurysm with a superficial femoral vein, along with placement of a pectoralis major muscle flap to cover the vein graft . Operative cultures of the pseudoaneurysm grew pseudomonas pseudomallei . The patient was treated with a 6-week course of intravenous ceftazidime and oral doxycycline and then continued on oral amoxicillin-clavulanate . One week after discontinuing intravenous antibiotics, the patient presented to the emergency department with a rapidly expanding, pulsatile mass in the right supraclavicular space . He was taken emergently to the operating room . After hypothermic circulatory arrest was accomplished, the disrupted vein graft and aneurysm cavity were resected and the subclavian artery was oversewn proximally and distally . Parenteral ceftazidime was continued for 3 months and oral amoxicillin-clavulanate (augmentin) was continued indefinitely . There was no evidence of infection clinically or by computed tomographic scan 2 years later . Although autogenous vein replacement of infected arteries and grafts may be successful in the majority of cases, this strategy should probably be avoided when particularly virulent bacteria such as the organism in this case are present.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 138 - 46
The elusive roles of bacterial glutathione S-transferases: new lessons from genomes; Vuilleumier S et al.; Glutathione S-transferases constitute a large family of enzymes which catalyze the addition of glutathione to endogenous or xenobiotic, often toxic electrophilic chemicals . Eukaryotic glutathione S-transferases usually promote the inactivation, degradation or excretion of a wide range of compounds by formation of the corresponding glutathione conjugates . In bacteria, by contrast, the few glutathione S-transferases for which substrates are known, such as dichloromethane dehalogenase, 1,2-dichloroepoxyethane epoxidase and tetrachlorohydroquinone reductase, are catabolic enzymes with an essential role for growth on recalcitrant chemicals . Glutathione S-transferase genes have also been found in bacterial operons and gene clusters involved in the degradation of aromatic compounds . Information from bacterial genome sequencing projects now suggests that glutathione S-transferases are present in large numbers in proteobacteria . In particular, the genomes of three Pseudomonas species each include at least ten different glutathione S-transferase genes . Several of the corresponding proteins define new classes of the glutathione S-transferase family and may also have novel functions that remain to be elucidated.

Khirurgiia (Mosk), 2002, (1), 34 - 5
{Immune status in diabetic patients with pyo-necrotic lesions of lower extremities}; Zemlianoi AB et al.; Long-term and severe pyonecrotic processes in diabetic patients testify to severe disorders of immune system in this disease . High titer of antibodies to tested autostrain demonstrated its etiologic role in infectious process . The study group consisted of 29 patients (with diabetic pyonecrotic foot lesions), control group--17 patients with burns of III a, b--IV stage affecting from 20 to 60% of body surface . In diabetic patients antibodies titer to the most encountered infectious agents Staphilococcus aureus and Pseudomonas aeroginosa was lower than in burn patients with immunity deficiency . Decrease of antibodies titer in diabetic patients testifies to high insufficiency of B-immunity.

Hum Gene Ther, 2002 Mar 1, 13(4), 497 - 508
Gene therapy of murine solid tumors with T cells transduced with a retroviral vascular endothelial growth factor--immunotoxin target gene; Jin N et al.; Solid tumor growth can be inhibited by targeting its neovasculature with vascular endothelial growth factor (VEGF)-toxin fusion proteins (FPs), but these agents have been limited by their inability to localize at the tumor site . In this study, we devised a gene therapy approach intended to deliver VEGF-toxin directly to tumor . Antigen-specific cytotoxic T lymphocytes (CTLs) served as vehicles to deliver a retroviral VEGF-toxin fusion protein to its specific leukemia cell target in vivo . A retroviral vector was constructed for gene therapy with VEGF positioned downstream of its 27-amino acid leader sequence, which promoted secretion of a catalytic immunotoxin containing either truncated diphtheria toxin or Pseudomonas exotoxin A . VEGF was chosen on the basis of the expression of VEGF receptor on endothelial cells in the tumor neovasculature . The VEGF FP was first expressed and secreted by mammalian NIH 3T3 cells . Intracellular expression of both VEGF and toxin was verified by immunofluorescence . In vitro, supernatants collected from transfected cells specifically inhibited the growth of VEGF receptor-expressing human umbilical vein endothelial cells (HUVECs), but not a control cell line . In vivo findings correlated with in vitro findings . A retroviral vector containing the target gene and a nerve growth factor receptor (NGFR) reporter gene was used to transiently transduce T15, a CD8(+) CTL line that specifically recognizes C1498, a lethal C57BL/6 myeloid tumor . Transduced T15 cells injected intravenously significantly inhibited the growth of subcutaneous tumor, whereas nontransduced controls did not . Together, these data indicate that gene therapy of T cells with retrovirus containing a VEGF-immunotoxin target gene may be a valid means of inhibiting a broad range of solid tumors dependent on angiogenesis.

J Mol Microbiol Biotechnol, 2002 Mar, 4(2), 151 - 61
Analysis of regulatory elements and genes required for carbon tetrachloride degradation in Pseudomonas stutzeri strain KC; Sepulveda-Torre L et al.; Previously, we described the generation and initial characterization of four Tn5 mutants of Pseudomonas stutzeri strain KC with impaired ability to degrade carbon tetrachloride (Sepulveda-Torres et al., 1999) . In this study, we show cloning and sequencing of an 8.3 kbp region in which all four transposons were located . This fragment encodes eight potential genes and is located in the central part of the 25 kbp fragment recently identified by Lewis et al . (2000) and shown by them to be sufficient to confer carbon tetrachloride transformation capability upon other pseudomonads . The four transposon insertion mutants mapped in ORF's F and I designated by Lewis et al . (2000) . This is consistent with the results by Lewis et al . (2000) that orfFis required for carbon tetrachloride degradation . We further established that orfl is required for CCl4 degradation since the three mutants in this ORF were unable to degrade carbon tetrachloride . We present our analysis of the gene and protein sequences from the 8.3 kbp region and propose a tentative model for the role of different genes in the synthesis and activity of pyridine-2,6-bis(thiocarboxylate) (PDTC), the secreted factor responsible for carbon tetrachloride dechlorination . We also found a putative promoter that overlaps with a Fur-box-like sequence in the region upstream of mutated genes . To test this putative promoter region and Fur-box, we generated and ligated DNA fragments containing wild-type and mutant Fur-boxes to a lacZ reporter . The wild-type fragment showed promoter activity that is regulated by the concentration of iron in the medium . Finally, we screened a selection of Pseudomonas strains, including P . putida DSMZ 3601--a strain known to produce PDTC--for the presence of the genes characterized in this study . None of the strains tested positive, suggesting that Pseudomonas stutzeri strain KC may possess a distinct biosynthetic pathway for PDTC production.

Science, 2002 Mar 1, 295(5560), 1722 - 6
A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae; Guttman DS et al.; Type III secreted "effector" proteins of bacterial pathogens play central roles in virulence, yet are notoriously difficult to identify . We used an in vivo genetic screen to identify 13 effectors secreted by the type III apparatus (called Hrp, for "hypersensitive response and pathogenicity") of the plant pathogen Pseudomonas syringae . Although sharing little overall homology, the amino-terminal regions of these effectors had strikingly similar amino acid compositions . This feature facilitated the bioinformatic prediction of 38 P . syringae effectors, including 15 previously unknown proteins . The secretion of two of these putative effectors was shown to be type III--dependent . Effectors showed high interstrain variation, supporting a role for some effectors in adaptation to different hosts.

Appl Environ Microbiol, 2002 Mar, 68(3), 1403 - 7
Reduction of olive knot disease by a bacteriocin from Pseudomonas syringae pv . ciccaronei; Lavermicocca P et al.; A bacteriocin produced by Pseudomonas syringae pv . ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P . syringae subsp . savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants . Treatments with bacteriocin from P . syringae pv . ciccaronei inhibited the formation of overgrowths on olive plants caused by P . syringae subsp . savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10(5) and 10(8) CFU ml(-1), respectively . In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml(-1) at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used . Crude bacteriocin (6,000 AU ml(-1)) was also effective in controlling the multiplication of epiphytic populations of the pathogen . In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively . These results suggest that bacteriocin from P . syringae pv . ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.

J Appl Microbiol, 2002, 92(3), 517 - 25
Pyrimidine biosynthesis in Pseudomonas oleovorans; Haugaard LE et al.; AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the polyhydroxyalkanoate-producing bacterium Pseudomonas oleovorans at the level of enzyme synthesis and at the level of aspartate transcarbamoylase activity . METHODS AND RESULTS: The effect of pyrimidine supplementation on the pyrimidine biosynthetic pathway enzyme activities was analysed relative to carbon source . Two uracil auxotrophs of P . oleovorans were isolated that were deficient for aspartate transcarbamoylase or dihydroorotase activity . Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized . At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps . oleovorans enzyme . CONCLUSIONS: Pyrimidine biosynthesis is regulated in Ps . oleovorans . Taxonomically, the regulation of the pyrimidine biosynthetic pathway appeared dissimilar from previously studied Pseudomonas species . SIGNIFICANCE AND IMPACT OF THE STUDY: New insights regarding the regulation of nucleic acid metabolism are provided that could prove significant during the genetic manipulation of Ps . oleovorans to increase the synthesis of polyhydroxyalkanoates.

Br J Cancer, 2002 Jan 21, 86(2), 285 - 91
IL-4 receptors on human medulloblastoma tumours serve as a sensitive target for a circular permuted IL-4-Pseudomonas exotoxin fusion protein; Joshi BH et al.; Cytotoxins directed to interleukin-4 receptors have shown to mediate relatively selective cytotoxicity against a variety of human cancer cells in vitro and in vivo . In an ongoing Phase I clinical trial, a recombinant protein comprised of circularly permuted IL-4 fused to a mutated form of Pseudomonas exotoxin (the fusion protein termed IL-4(38-37)-PE38KDEL or cpIL4-PE) has shown antitumour activity against malignant glioma . Human medulloblastomas are neuroectodermal tumours that occur in children and have a poor prognosis . The goal of this study was to determine whether human medulloblastoma derived cell lines express interleukin-4 receptor and whether interleukin-4 receptor expression is accompanied by sensitivity to cpIL4-PE . Medulloblastoma cell lines express interleukin-4 receptor at the protein and mRNA levels as determined by binding, indirect immunofluorescence and RT--PCR studies . These cells expressed IL-4Ralpha (also known as IL-4Rbeta) and IL-13Ralpha1 (also known as IL-13Ralpha') chains, however common gamma(c), a component of the interleukin-4 receptor system in immune cells was not detected . Consistent with the expression of IL-4R, cpIL4-PE was found to be highly and specifically cytotoxic to four of five medulloblastoma cell lines . Susceptibility of medulloblastoma cell lines to cpIL4-PE seemed to correlate closely to the functional IL-4 binding sites in general as demonstrated by 125I-IL-4 binding, but did not seem to correlate with mRNA or cell surface immunoreactive receptor protein expression . The sensitivity of medulloblastoma cells to cpIL4-PE could be eliminated by concurrent incubation with IL-4 or IL-13, but not with IL-2 . None of these cell lines showed any change in proliferation upon treatment with exogenous IL-4 . These studies establish the interleukin-4 receptor as a medulloblastoma-associated target for possible tumour-directed cancer therapy . Further studies are warranted to investigate interleukin-4 receptor expression in primary medulloblastoma tumours and sensitivity to cpIL-4PE in vitro and in vivo .

Plant J, 2002 Jan, 29(2), 131 - 40
Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds; Tierens KF et al.; An Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild-type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv . tomato and Peronospora parasitica . The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens . Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild-type plants, but this induction was almost completely abolished in esa1 . This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species.

Cancer Immunol Immunother, 2002 Feb, 50(12), 691 - 700 Epub 2001 Dec 07.
Apoptotic pathways of cell death induced by an interleukin-13 receptor-targeted recombinant cytotoxin in head and neck cancer cells; Kawakami M et al.; Interleukin 13 receptor (IL-13R)-targeted cytotoxin, IL13-PE38QQR, composed of IL-13 and a mutated form of Pseudomonas exotoxin (PE), is found to be highly and specifically cytotoxic to human solid cancer cell lines . However, the mechanism of tumor cell death mediated by IL-13 toxin is still not known . To elucidate the mechanism, we utilized four head and neck cancer cell lines (SCC-25, HN12, KCCT873, and YCUM911), which express high levels of IL-13R, and IL-13 toxin is highly cytotoxic to these cells . We observed chromatin condensation and DNA fragmentation, indicating apoptotic cell death, after treatment with IL-13 toxin, as determined by bis-benzimide staining and DNA ladder assays . However, IL-13 did not induce cell death . Flow cytometric analysis suggested that these cancer cell lines increased the sub-G1/G0 phase DNA population in a dose- and time-dependent manner (ranged between 10 and 30%) after treatment with IL-13 toxin . By Western blot analysis, cleavage of caspase-3 and PARP was observed after treatment with a high concentration of IL-13 toxin, also suggesting apoptotic cell death . In addition, the results of immunofluorescence and RT-PCR assays showed that the apoptosis-regulator, Bcl-2 was downregulated after treatment with IL-13 toxin, while Bax was upregulated . Moreover, significant nitrite production was detected in the HN12 cell line after treatment with IL-13 toxin for 48--96 h . Taken together, our results suggest that IL-13 toxin-induced cytotoxicity is at least partially mediated by the apoptosis and nitric oxide pathways . This information may be useful in developing specific approaches where apoptotic bodies from tumor cells may be used to pulse antigen-presenting cells for immunotherapy of cancer.

Carbohydr Res, 2002 Mar 1, 337(5), 467 - 71
O-Specific chain structure from the lipopolysaccharide fraction of Pseudomonas reactans: a pathogen of the cultivated mushrooms; Molinaro A et al.; An O-specific polysaccharide containing 2-acetamidino-2-deoxy-beta-D-glucopyranose (Glcp2Am), 2,4-diacetamido-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAc4NAc, bacillosamine) and 2,4-di-(N-acetyl-L-alanylamino)-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAlaAc4NAlaAc) was isolated from the phenol-soluble lipopolysaccharide fraction of the mushroom-associated bacterium Pseudomonas reactans . The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a linear trisaccharide-repeating unit, as shown below:-->3)-beta-D-QuipNAlaAc4NAlaAc-(1-->3)-alpha-D-Glcp2Am-(1-->3)-alpha-D-QuipNAc4NAc(1-->To our knowledge, this is the first complete O-chain structure reported for the lipopolysaccharide of a mushroom-associated bacterium.

Diagn Microbiol Infect Dis, 2002 Feb, 42(2), 141 - 3
Leg ulcer due to Pseudomanas luteola in a patient with sickle cell disease; Tsakris A et al.; Pseudomonas luteola is rarely implicated in clinical infections, usually in association with indwelling catheters or prostheses . This report describes the first case of deteriorated leg ulcer caused by a multiply antibiotic resistant P . luteola strain in a patient with homozygous sickle cell disease.

JOP, 2000 Nov, 1(4), 208 - 10
Microvascular complications in cystic fibrosis-related diabetes mellitus: a case report; Scott AI et al.; CONTEXT, The prevalence of cystic fibrosis-related diabetes mellitus is increasing and is associated with increased survival from cystic fibrosis . CASE REPORT, This study describes a case of the premature onset of disabling and widespread microvascular complications resulting from cystic fibrosis-related diabetes mellitus . Previously asymptomatic retinopathy was diagnosed on recognition of diabetic nephropathy . CONCLUSIONS, The treatment of pulmonary exacerbations has become more complex due to the nephrotoxic potential of intravenous aminoglycoside drugs which are frequently used to control chronic Pseudomonas infection in cystic fibrosis.

Plant Mol Biol, 2002 Feb 1, 48(3), 267 - 76
Infection of Arabidopsis with a necrotrophic pathogen, Botrytis cinerea, elicits various defense responses but does not induce systemic acquired resistance (SAR); Govrin EM et al.; Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species . In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells . Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms . We analyzed the role of SAR in B . cinerea infection of Arabidopsis . We show that although B . cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of Pseudomonas syringae, or against subsequent B . cinerea infections . Induction of SAR with avirulent P . syringae or by chemical treatment with salicylic acid (SA) or benzothiadiazole also failed to inhibit B . cinerea growth, although removal of basal SA accumulation by expression of a bacterial salicylate hydroxylase (NahG) gene or by infiltration of 2-aminoindan-2-phosphonic acid, an inhibitor of phenylpropanoid pathway, increased B . cinerea disease symptoms . In addition, we show that B . cinerea induced expression of genes associated with SAR, general stress and ethylene/jasmonate-mediated defense pathways . Thus, B . cinerea does not induce SAR nor is it affected by SAR, making it a rare example of a necrogenic pathogen that does not cause SAR.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2275 - 80
Genomewide identification of Pseudomonas syringae pv . tomato DC3000 promoters controlled by the HrpL alternative sigma factor; Fouts DE et al.; The ability of Pseudomonas syringae pv . tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor . To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters . Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000 . This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes . We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N(epsilon)-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production . Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.

Lett Appl Microbiol, 2002, 34(1), 7 - 12
Exposure to non-acid fresh meat decontamination washing fluids sensitizes Escherichia coli O157:H7 to organic acids; Samelis J et al.; AIMS: To investigate whether Escherichia coli O157:H7 maintains acid tolerance in water meat decontamination washing fluids . METHODS AND RESULTS: A rifampicin-resistant derivative of E . coli O157:H7 strain ATCC 43895 was inoculated (10(5) cfu ml(-1)) in spray-washings from meat sprayed with cold (10 degrees C) or hot (85 degrees C) water, stored at 10 degrees C for up to 14 days, and its acid tolerance was assessed at 2 and 8 days by exposure to broth or new washings adjusted to pH 3.5 or 3.7 with lactic or acetic acid . The pathogen survived in the water washings, but it was outgrown by the natural, Pseudomonas-like flora, and it was sensitized to acid . CONCLUSIONS: The acid tolerance of E . coli O157:H7 decreases following exposure to non-acid, but otherwise stressful, conditions prevailing in water meat washings at 10 degrees C . SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the more intense use of water-based technologies should be included in meat decontamination strategies because they may contribute to enhanced meat safety by inducing acid sensitization in E . coli O157:H7.

Environ Microbiol, 2001 Nov, 3(11), 691 - 8
Characterization of nickel-resistant bacteria isolated from serpentine soil; Mengoni A et al.; In the present study, heterotrophic nickel-resistant bacteria were isolated and characterized from three different serpentine outcrops in central Italy populated by the nickel-hyperaccumulating plant Alyssum bertolonii . Bacteria were isolated from the rhizosphere of the plant and from soil portions at various distances from the plant . The proportion of nickel-resistant cfu was higher in proximity to the plant than in free soil . A total of 138 isolates was collected and grouped into 47 different operational taxonomic units (OTUs) by means of amplified ribosomal DNA restriction analysis (ARDRA) and into 25 heavy-metal resistant phenotypes . The phylogenetic position of strains belonging to 20 OTUs, representing more than the 70% of the total isolates, was determined by 16S rDNA sequencing . These analyses showed that the most represented genera in all three different outcrops were Pseudomonas and Streptomyces . Pseudomonas strains were found to be predominant in the plant rhizosphere, whereas Streptomyces strains were mainly present in the soil.

Biochem Biophys Res Commun, 2002 Feb 22, 291(2), 361 - 6
Direct atomic force microscopy visualization of integration host factor-induced DNA bending structure of the promoter regulatory region on the Pseudomonas TOL plasmid; Seong GH et al.; Atomic force microscopy (AFM) was used to analyze DNA bending induced by integration host factor (IHF) . The direct AFM visualization of IHF-DNA complexes on the OP1 promoter regulatory regions on the Pseudomonas TOL plasmid showed that there was no intrinsic DNA bend in the OP1 promoter region, but a sharp DNA bend was induced by binding of IHF to the region between the upstream regulatory sequence and the promoter sequence . The DNA bending angles were distributed with a mean bend angle of 123 degrees . The IHF-DNA complexes were shown to bend at the IHF binding site giving rise to an asymmetric structure . These results provide direct evidence that IHF is required functionally for activation of OP1 transcription and support the DNA-loop model that the sharp DNA bend induced by binding of IHF facilitates the contact between RNA polymerase bound by the promoter sequence and XylR protein attached to the upstream sequence in the OP1 promoter . (c)2002 Elsevier Science (USA).

J Bacteriol, 2002 Mar, 184(5), 1481 - 7
Novel group I intron in the tRNA(Leu)(UAA) gene of a gamma-proteobacterium isolated from a deep subsurface environment; Vepritskiy AA et al.; A group I intron has been found to interrupt the anticodon loop of the tRNA(Leu)(UAA) gene in a bacterium belonging to the gamma-subdivision of Proteobacteria and isolated from a deep subsurface environment . The subsurface isolate SMCC D0715 was identified as belonging to the genus Pseudomonas . The group I intron from this isolate is the first to be reported for gamma-proteobacteria, and the first instance of a tRNA(Leu)(UAA) group I intron to be found in a group of bacteria other than cyanobacteria . The 231-nucleotide (nt) intron's sequence has group I conserved elements and folds into a bona fide group I secondary structure with canonical base-paired segments P1 to P9 and a paired region, P10 . The D0715 intron possesses the 11-nt motif CCUACG . UAUGG in its P8 region, a feature not common in bacterial introns . To date, phylogenetic analysis has shown that bacterial introns form two distinct families, and their complex distribution suggests that both lateral transfer and common ancestry have taken part in the evolutionary history of these elements.

Mol Plant Microbe Interact, 2002 Jan, 15(1), 43 - 53
Characterization of the salA, syrF, and syrG regulatory genes located at the right border of the syringomycin gene cluster of Pseudomonas syringae pv . syringae; Lu SE et al.; Sequence analysis of the right border of the syr gene cluster of Pseudomonas syringae pv . syringae strain B301D revealed the presence of the salA gene 8,113 bp downstream of syrE . The predicted SalA protein of strain B301D differs by one amino acid from that of strain B728a . Two homologs of salA, designated syrF and syrG, were identified between syrE and salA . All three proteins contain helix-turn-helix DNA-binding motifs at their C termini and exhibit homology to regulatory proteins of the LuxR family . A salA mutant failed to produce syringomycin, whereas syrF and syrG mutants produced 12 and 50%, respectively, of syringomycin relative to the wild-type strain . The salA, syrF, and syrG mutants were significantly reduced in virulence, forming small, nonspreading lesions in immature cherry fruits . Translational fusions to the uidA gene were constructed to evaluate expression of syrB1 in regulatory mutant backgrounds and to determine the relationship among the three regulatory loci . Expression of a syrB1::uidA fusion required functional salA and syrF genes and, in series, the expression of a syrF::uidA fusion required a functional salA gene . These results demonstrate that salA is located upstream of syrF in the regulatory hierarchy controlling syringomycin production and virulence in P . syringae pv . syringae.

Am J Pathol, 2002 Feb, 160(2), 481 - 90
Stat6-deficient mice develop airway hyperresponsiveness and peribronchial fibrosis during chronic fungal asthma; Blease K et al.; Signal transducer and activator of transcription 6 (Stat6) is critical for Th2-mediated responses during allergic airway disease . To investigate the role of Stat6 in fungus-induced airway hyperresponsiveness and remodeling, Stat6-deficient (Stat6-/-) and Stat6-wildtype (Stat6+/+) mice were sensitized to Aspergillus fumigatus and airway disease was subsequently assessed in both groups at days 21, 30, 38, and 44 after an intratracheal challenge with live A . fumigatus conidia . At all times after conidia, histological analysis revealed an absence of goblet cell hyperplasia and markedly diminished peribronchial inflammation in Stat6-/- mice in contrast to Stat6+/+ mice . Airway hyperresponsiveness and peribronchial fibrosis in Stat6-/- mice were significantly reduced at day 21 after conidia compared with Stat6+/+ mice, but both groups exhibited significant, similar increases in these parameters at all subsequent times after conidia . In separate experiments, IL-13-responsive cells in Stat6-/- mice were targeted via the daily intranasal administration of 200 ng of IL-13-PE38QQR (IL13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, from days 38 to 44 after the conidia challenge . IL13-PE treatment abolished airway hyperresponsiveness, but not peribronchial fibrosis in Stat6-/- mice . Taken together, these data demonstrate that the chronic development of airway hyperresponsiveness during fungal asthma is IL-13-dependent but Stat6-independent.

J Assoc Physicians India, 2001 Aug, 49, 850 - 1
Pulmonary nocardial infection and pseudomonas infection of the tongue in a patient with dermatomyositis; Shah NM et al.; Opportunistic infections in immunocompromised patients are common . We report the case of a 63 year old female patient with dermatomyositis who while on oral steroids developed nocardial infection of the lung and pseudomonas infection of the tongue simultaneously . Nocardial infections are not very commonly seen in patients with dermatomyositis . Pseudomonas infection of the tongue is a rarity . We report this case for its rarity as regards the type and site of infections and review the relevant literature.

J Soc Biol, 2001, 195(3), 235 - 42
{Endosomes and toxin translocation}; Beaumelle B et al.; In this review we discuss data obtained by our group regarding the entry of toxins, especially ricin, diphtheria toxin (DT) and Pseudomonas exotoxin A (PE) into animal cells . We studied the translocation process of these toxins using endosomes purified from lymphocytes . This process is rate-limiting for toxicity and enables these toxins to reach the cytosol where they will inactivate the protein synthesis system and kill the cell . We could show that each of these toxins uses a different strategy to cross the endosome membrane . Whereas ricin transmembrane transport only relies on cytosolic ATP hydrolysis, PE first requires exposure to the low endosomal pH (pH-6), presumably to insert into the endosome membrane, before being translocated via a process which also requires cytosolic ATP hydrolysis . DT translocation is directly triggered and energized by the endosome-cytosol pH gradient . Using conjugates with dihydrofolate reductase we could indirectly show that ricin and PE require unfolding for translocation . A deletion approach enabled to produce a more cytotoxic PE mutant by increasing its translocation activity.

J Agric Food Chem, 2002 Feb 13, 50(4), 706 - 9
Evaluation of peracid formation as the basis for resistance to infection in plants transformed with haloperoxidase; Jacks TJ et al.; Nonheme haloperoxidase (HPO-P) isolated from Pseudomonas pyrrocinia catalyzed the peroxidation of alkyl acids to peracids . Among acids tested as substrates, acetic acid was most readily peroxidized . The reaction product peracetate possessed potent antifungal activity: 50% death (LD(50)) of Aspergillus flavus occurred at 25 microM peracetate . Viability of A . flavus was inhibited by up to 80% by leaf extracts of tobacco plants transformed with the HPO-P gene from P . pyrrocinia compared to viability of fungi exposed to extracts from controls . To elucidate if peracid formation by HPO-P was the basis for antifungal activity in transgenic leaf tissues, lethalities of hydrogen peroxide-acetate-HPO-P combinations against A . flavus were examined in vitro . LD(50) of A . flavus exposed to the combinations occurred at 30 mM acetate when concentrations of hydrogen peroxide and HPO-P were held constant . This value was identical to the LD(50) produced by 30 mM acetate in the absence of hydrogen peroxide-HPO-P and therefore did not account for enhanced antifungal activity in transgenic plants . For clarification, kinetics of the enzymic reaction were examined . According to the concentration of acetate needed for enzyme saturation (K(m) = 250 mM), acetate was lethal prior to its oxidation to peracetate . Results indicate that peracid generation by HPO-P was not the basis for enhanced antifungal activity in transgenic plants expressing the HPO-P gene.

J Surg Res, 2002 Feb, 102(2), 169 - 77
Expression of HER2 in human gastric cancer cells directly correlates with antitumor activity of a recombinant disulfide-stabilized anti-HER2 immunotoxin; Shinohara H et al.; BACKGROUND: Amplification of the human epidermal growth factor receptor 2 (HER2) gene and overexpression of the HER2 protein have been associated with an unfavorable prognosis . We determined the efficacy of an anti-HER2 immunotoxin, erb-38 {e23(dsFv)PE38}, against human gastric cancer cells . METHODS: Immunotoxin was made by fusing the disulfide-stabilized Fv fragments (dsFv) of a monoclonal antibody e23 to a truncated mutant of M(r) 38 Pseudomonas exotoxin (PE38) that lacks its cell-binding domain . RESULTS: The immunotoxin-mediated cytotoxicity directly correlated with the expression levels of the HER2 gene and protein in human gastric cancer cells . Interestingly, MKN-45P cells, a variant line of MKN-45 producing peritoneal dissemination and ascites in vivo, expressed a higher level of HER2 and were more sensitive to erb-38 than MKN-45 cells . RFB-4, a control anti-CD22 immunotoxin, was cytotoxic against none of the tested human gastric cancer cells, also suggesting that the lysis mediated by erb-38 was specific for HER2 expression . Three consecutive iv injections of erb-38 at doses of 0.5 or 5 microg/body eradicated experimental liver metastases and peritoneal disseminations produced by MKN-45P in a dose-dependent manner . CONCLUSIONS: We conclude that an erb-38 anti-HER2 immunotoxin has specific antitumor activities against human gastric cancer cells overexpressing HER2 . (c)2001 Elsevier Science.

Biosci Biotechnol Biochem, 2001 Dec, 65(12), 2701 - 9
Cloning, sequencing, and expression of the gene encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae NGJ1; Maruyama K et al.; A DNA fragment that carried the gene (proA) encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase was cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1, and the coding region was assigned to the nucleotide sequence based on the N-terminal amino acid sequence of the enzyme purified from the organism . The proA gene was 684 bp long, corresponding to a protein of 227 amino acid residues with a calculated molecular mass of 24,067 Da . The genes encoding a putative transporter and a 4-oxalomesaconate hydratase were upstream, and a 3'-truncated gene encoding 2-pyrone-4,6-dicarboxylate lactonase was downstream from the proA gene in the same orientation on the DNA fragment . The proA gene product was overproduced in Escherichia coli and briefly purified to homogeneity from the crude extract by a two-step purification . The molecular and catalytic properties of the gene product were similar to those of the P . ochraceae enzyme.

Plant Cell, 2002 Jan, 14(1), 275 - 86
EDS5, an essential component of salicylic acid-dependent signaling for disease resistance in Arabidopsis, is a member of the MATE transporter family; Nawrath C et al.; The eds5 mutant of Arabidopsis (earlier named sid1) was shown previously to accumulate very little salicylic acid and PR-1 transcript after pathogen inoculation and to be hypersusceptible to pathogens . We have isolated EDS5 by positional cloning and show that it encodes a protein with a predicted series of nine to 11 membrane-spanning domains and a coil domain at the N terminus . EDS5 is homologous with members of the MATE (multidrug and toxin extrusion) transporter family . EDS5 expression is very low in unstressed plants and strongly induced by pathogens and UV-C light . The transcript starts to accumulate 2 hr after inoculation of Arabidopsis with an avirulent strain of Pseudomonas syringae or UV-C light exposure, and it stays induced for approximately 2 days . EDS5 also is expressed after treatments with salicylic acid, indicating a possible positive feedback regulation . EDS5 expression after infection by certain pathogens as well as after UV-C light exposure depends on the pathogen response proteins EDS1, PAD4, and NDR1, indicating that the signal transduction pathways after UV-C light exposure and pathogen inoculation share common elements.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 136 - 49
Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre; Galkin AG et al.; Sequence alignment shows that residue Arg 284 (according to the numbering of the residues in formate dehydrogenase, FDH, from the methylotrophic bacterium Pseudomonas sp . 101) is conserved in NAD-dependent FDHs and D-specific 2-hydroxyacid dehydrogenases . Mutation of Arg 284 to glutamine and alanine results in a change of the catalytic, thermodynamic and spectral properties of FDH . In comparison to wild-type, the affinity of the mutants for the substrate (K(formate)m) or the transition state analogue (K(azide)i) decreases and correlates with the ability of the side chain of residue 284 to form H-bonds . In contrast, the affinity for the coenzyme (K(NAD)d or K(NAD)m) is either not affected or increases and correlates inversely with the partial positive charge of the side chain . The temperature dependence of circular dichroism (CD) spectra of the wild-type FDH and its Ala mutant has been studied over the 5-90 degrees C temperature range . Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55 degrees C) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70 degrees C which is followed by the loss of enzyme activity . CD spectra of the wild-type and mutant proteins were deconvoluted and contributions from various types of secondary structure estimated . It is shown that the co-operative transition at 55-70 degrees C in the FDH protein globule is triggered by a loss of alpha-helical secondary structure . The results confirm the conclusion, from the crystal structures, that Arg 284 is directly involved in substrate binding . In addition this residue seems to exert a major structural role by supporting the catalytic conformation of the enzyme active centre.

Zhonghua Wai Ke Za Zhi, 1998 Mar, 36(3), 138 - 40
{Primary epididymal tumors}; Pan B et al.; OBJECTIVE: To improve the diagnosis of primary epididymal tumors . METHOD: In 23 primary epididymal tumors (22 benign and 1 malignant), 14 were adenomatoid tumors, 6 were leiomyomas, 1 was multiple fibrous pseudomonas and 1 was sclerosing hemangioma . Epididymal adenocarcinoma was seen in a 57-year-old man . RESULT: Epididymal tumors might be easily misdiagnosed as epididymal tuberculosis, chronic epididymis and spermaocele . Apart from certain benign clinical characteristics, benign epididymal tumors could be diagnosed by B-ultrasonography and aspiration biopsy . Most malignant tumors of epididymis presented as rapidly growing scrotal masses and cured by the removal of tumors or the whole epididymis of the same side . 19 of 22 cases were free from recurrence . CONCLUSION: Malignant epididymal tumors should be treated as malignant tumors of testis and their prognosis is extremely poor.

Appl Environ Microbiol, 2002 Feb, 68(2), 957 - 62
Coexistence of two distinct copies of naphthalene degradation genes in Pseudomonas strains isolated from the western Mediterranean region; Ferrero M et al.; We analyzed the occurrence of the naphthalene degradation upper-pathway (nah) genes in the western Mediterranean region . The amplification, restriction, and sequence analysis of internal fragments for several nah genes (nahAc, nahB, nahC, and nahE) from naphthalene-degrading strains isolated from this geographical area proved the coexistence of two distinct sets of nah genes.

Appl Environ Microbiol, 2002 Feb, 68(2), 865 - 73
Highly different levels of natural transformation are associated with genomic subgroups within a local population of Pseudomonas stutzeri from soil; Sikorski J et al.; A highly sensitive and specific PCR-based method of monitoring 16S rRNA genes of Pseudomonas stutzeri was developed for searching P . stutzeri DNA in environmental samples . This monitoring was combined with a reliable and sensitive method for isolating P . stutzeri colony formers from soil and sediment, depending on their utilization of ethylene glycol, starch, and maltose . With these techniques, P . stutzeri populations (n = 2 to 170) were obtained from five of six sites giving positive PCR signals (including three marine sediment and two soil samples) . The phylogenetic positions of isolates from the five sites, based on their 16S ribosomal DNA sequences, indicated that the environmental isolates were affiliated with different genomovars of P . stutzeri . Using the broad-host-range plasmid pNS1 with kanamycin and gentamicin resistance determinants as the transforming DNA, naturally transformable strains were identified among the isolates from all sites . For one population from soil, the genetic relationship of the 120 members was determined by randomly amplified polymorphic DNA-PCR with three PCR primers . Among the population members which are taxonomically closely related as determined by 16S sequence comparisons of group representatives, a rather high genetic diversity and a characteristic clustering into subgroups were found . Remarkably, within the population, nontransformability and different levels of transformability (a frequency between about 10(-9) and 10(-4) per cell) were often associated with distinct genetic subgroups . It is concluded that transformability is widespread among environmental P . stutzeri strains and that its specific level is a heritable trait that may vary strongly within a local population.

Appl Environ Microbiol, 2002 Feb, 68(2), 634 - 41
Molecular characterization and substrate specificity of nitrobenzene dioxygenase from Comamonas sp . strain JS765; Lessner DJ et al.; Comamonas sp . strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy . We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765 . The components of NBDO were designated Reductase(NBZ), Ferredoxin(NBZ), Oxygenase(NBZalpha), and Oxygenase(NBZbeta), with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively . Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp . strain JS42 . The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase(NBZ) revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765 . NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.

Appl Environ Microbiol, 2002 Feb, 68(2), 582 - 7
L-glucitol catabolism in Stenotrophomonas maltophilia Ac; Brechtel E et al.; The carbohydrate catabolism of the bacterium Stenotrophomonas maltophilia Ac (previously named Pseudomonas sp . strain Ac), which is known to convert the unnatural polyol L-glucitol to D-sorbose during growth on the former as the sole source of carbon and energy, was studied in detail . All enzymes operating in a pathway that channels L-glucitol via D-sorbose into compounds of the intermediary metabolism were demonstrated, and for some prominent reactions the products of conversion were identified . D-Sorbose was converted by C-3 epimerization to D-tagatose, which, in turn, was isomerized to D-galactose . D-Galactose was the initial substrate of the De Ley-Doudoroff pathway, involving reactions of NAD-dependent oxidation of D-galactose to D-galactonate, its dehydration to 2-keto-3-deoxy-D-galactonate, and its phosphorylation to 2-keto-3-deoxy-D-galactonate 6-phosphate . Finally, aldol cleavage yielded pyruvate and D-glycerate 3-phosphate as the central metabolic intermediates.

Curr Microbiol, 2002 Mar, 44(3), 189 - 95
Physiological characterization and genetic engineering of Pseudomonas corrugata for medium-chain-length polyhydroxyalkanoates synthesis from triacylglycerols; Solaiman DK et al.; Pseudomonas belonging to the rRNA-DNA homology group I produce medium-chain-length (mcl)-polyhydroxyalkanoates (PHA) . We show that P . corrugata, a member of this group, accumulates 0.5-1.0 g of mcl-PHA/L of culture when grown on glucose (Gl) or oleic acid (Ol) . The predominant monomers of Gl-PHA and Ol-PHA are beta-hydroxydecanoate and beta-hydroxyoctanoate, respectively . The molecular masses and polydispersity of P . corrugata PHAs are higher than those typically found with other Pseudomonas . We electrotransformed P . corrugata with a plasmid pCN51lip-1 carrying Pseudomonas lipase genes to generate strain III111-1 . The recombinant strain grew on intact triacylglycerols (TAGs) to 1.9-2.7 g of cell-dry-weight/L of culture . The yields and the predominant repeat-units of PHAs obtained from the lard- and tallow-grown III111-1 were similar to those of Ol-PHA from wild-type cells . In contrast to other Pseudomonas species, P . corrugata III111-1 grown on TAGs at temperatures up to 36 degrees C was not significantly affected with regard to cell yields, amounts of PHA produced, and the repeat unit compositions of the polymer.

Curr Microbiol, 2002 Feb, 44(2), 132 - 5
Cloning and molecular analysis of poly(3-hydroxyalkanoate) biosynthesis genes in Pseudomonas aureofaciens; Nishikawa T et al.; Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs) . To clone the PHA synthase gene(s) (phaC), the genomic library of P . aureofaciens was constructed using a cosmid vector . The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P . putida GPp104 . The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert . Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD) . The heterologous expression of pha genes from P . aureofaciens was confirmed . P . putida GPp104 regained the ability to accumulate PHA on introduction of pVK6 . Wild-type strains P . oleovorans and P . fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6.

J Agric Food Chem, 2002 Jan 30, 50(3), 477 - 83
Structured lipids via lipase-catalyzed incorporation of eicosapentaenoic acid into borage (Borago officinalis L.) and evening primrose (Oenothera biennis L.) oils; Senanayake SP et al.; Enzymatic acidolysis of borage oil (BO) or evening primrose oil (EPO) with eicosapentaenoic acid (20:5n-3; EPA) was studied . Of the six lipases that were tested in the initial screening, nonspecific lipase PS-30 from Pseudomonas sp . resulted in the highest incorporation of EPA into both oils . This enzyme was further studied for the influence of enzyme load, temperature, time, type of organic solvent, and mole ratio of substrates . The products from the acidolysis reaction were analyzed by gas chromatography (GC) . The highest incorporation of EPA in both oils occurred at 45-55 degrees C and at 150-250 enzyme activity units . One unit of lipase activity was defined as nanomoles of fatty acids (oleic acid equivalents) produced per minute per gram of enzyme . Time course studies indicated that EPA incorporation was increased up to 26.8 and 25.2% (after 24 h) in BO and EPO, respectively . Among the solvents examined, n-hexane served best for the acidolysis of EPA with both oils . The effect of the mole ratio of oil to EPA was studied from 1:1 to 1:3 . As the mole ratio of EPA increased, the incorporation increased from 25.2-26.8 to 37.4-39.9% (after 24 h) . The highest EPA incorporations of 39.9 and 37.4% in BO and EPO, respectively, occurred at the stoichiometric mole ratio of 1:3 for oil to EPA.

Mol Cells, 2001 Dec 31, 12(3), 398 - 402
A divalent recombinant immunotoxin formed by a disulfide bond between the extension peptide chains; Park JH et al.; Recombinant immunotoxin for the treatment of cancer was made by connecting toxins to 'carcinoma-specific' antibodies that selectively bind to cancer cells, then kills them without harming the normal cells . The divalent recombinant immunotoxin, {B3(Fab)-ext-PE38}2, is a derivative of B3(Fab)-PE38 . B3(Fab)-PE38 was made by fusing the Fab domain of the monoclonal antibody (MAb) B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE) . In this study, B3(Fab)-ext-PE38 was constructed, which has the hinge region of the B3(Fab)-PE38 extended with the peptide extension, G4C(G4S)2, and connected to the C3 connector . The Cys residue of the extension peptide chain makes the disulfide bond between the two Fab domains . The extension sequence (ext) makes the dimerization of B3(Fab)-ext-PE38 easier to form the divalent immunotoxin, because it decreases the steric hindrance between the two PE38s . The constructed genes were expressed in E . coli as inclusion bodies . Polypeptides that were obtained from the inclusion body were refolded, and the active forms were purified . The ID50 values of the divalent molecule, {B3(Fab)-ext-PE38}2, were about 4 ng/ml on A431 cell lines, about 1 ng/ml on CRL1739 cell lines, and 5 ng/ml on MCF-7 cell lines . The {B3(Fab)-ext-PE38}2 showed about a 12-fold higher cytotoxicity on CRL1739 cell lines than B3(scFv)-PE40 did.

J Basic Microbiol, 2001, 41(6), 363 - 6
Properties of a thermostable and solvent stable extracellular lipase from a Pseudomonas sp . AG-8; Sharma AK et al.; An extracellular lipase isolated from Pseudomonas sp . AG-8, had an optimal activity at 45 degrees C and pH 8.0-8.5 . It retained more than 80% of its initial activity after keeping for 1 h at 65 degrees C . The enzyme was stable in 5 M NaCl and 6 M urea . Triton X-100 increased the lipase activity by 2.4 fold . Ca2+ ions activated the enzyme, while Zn2+, Fe2+, Fe3+ strongly inhibited its activity . Ethanol, methanol and acetone at 20% (v/v) enhanced the lipase activity by 2.9, 3.6 and 4.5 fold respectively . Dimethylsulphoxide at 90% (v/v) enhanced the enzyme activity up to 5.7 fold.

Clin Cancer Res, 2002 Jan, 8(1), 281 - 6
Interleukin 4 receptor-directed cytotoxin therapy for human head and neck squamous cell carcinoma in animal models; Strome SE et al.; Receptors for interleukin 4 (IL-4R) are overexpressed on the surface of various human solid tumors including renal cell carcinoma, glioblastoma, Kaposi's sarcoma, and head and neck squamous cell carcinoma (SCCHN) . On the basis of this preferential receptor overexpression, a novel IL-4R-targeted cytotoxin, IL-4 (38-37)-PE38KDEL, was developed in which circularly permuted IL-4 {IL-4 (38-37)} was fused to mutated form of Pseudomonas exotoxin (PE38KDEL) . Despite the recognized expression of the IL-4R on SSCHN, the utility of a receptor-specific fusion protein for the treatment of this disease remains unknown . The purpose of this study was to establish the utility of IL-4 (38-37)-PE38KDEL for the treatment of established SSCHN in animal models of human disease . Expression of IL-4R in SCCHN was confirmed by immunohistochemistry with eight of eight tissue sections expressing IL-4R . Protein synthesis inhibition assays demonstrated growth inhibition of two cell lines in IL-4 (38-37)-PE38KDEL in a dose-dependent fashion . In two SCCHN s.c . xenografted nude mouse models, i.p . and intratumoral injection of IL-4 (38-37)-PE38KDEL mediated tumor regression with no visual toxicity observed in any of the animals . Subcultured tumor cells after intratumoral treatment with IL-4 toxin did not develop resistance to the drug . These data demonstrate that IL-4 (38-37)-PE38KDEL is effective in mediating significant antitumor effects in SCCHN and may represent an attractive therapeutic option for patients with advanced cancers of the upper aerodigestive tract.

Clin Cancer Res, 2002 Jan, 8(1), 258 - 66
Internalization property of interleukin-4 receptor alpha chain increases cytotoxic effect of interleukin-4 receptor-targeted cytotoxin in cancer cells; Kawakami K et al.; Although the receptor for interleukin-4 (IL-4R) is highly expressed on solid human cancer cells, its significance and internalization function is still unclear . To address these issues, we reconstituted Chinese hamster ovarian (CHO-K1) cells with various components of the IL-4R by transient transfection and performed internalization assays using radiolabeled IL-4 . Radiolabeled IL-4 internalized through the IL-4Ralpha chain in a time-dependent manner . When the IL-4Ralpha chain was cotransfected with the IL-13Ralpha1 or -gamma(c) chain, the IL-4 internalization level was identical to IL-4Ralpha transfectants, suggesting that the IL-4Ralpha chain plays a major role in IL-4 internalization . These results were confirmed by determining the cytotoxicity of a chimeric protein composed of IL-4 and a mutated form of Pseudomonas exotoxin {IL4(38-37)-PE38KDEL} in CHO-K1 cells transfected with increasing concentrations of IL-4Ralpha cDNA . To use the internalization property of the IL-4Ralpha chain in the context of IL-4R-targeted cytotoxin therapy, we transiently transfected pancreatic and brain tumor cells with IL-4Ralpha chain . Surprisingly, these tumor cells acquired 4-75-fold higher binding activity to IL-4 compared with control cells . Consequently, the cytotoxic activity of IL-4 toxin to these cancer cells was enhanced 5-13-fold compared with control cells as assessed by protein synthesis inhibition and clonogenic assays . Taken together, a combination approach that involves transfer of the IL-4Ralpha gene and IL-4R-targeted cytotoxin therapy may serve as a novel approach for cancer therapy.

Chest, 2002 Jan, 121(1), 73 - 80
Association between genetically determined pancreatic status and lung disease in adult cystic fibrosis patients; Loubieres Y et al.; STUDY OBJECTIVES: The association between genotype and phenotype in cystic fibrosis (CF) has been clearly established for pancreatic status, but not for lung disease . DESIGN: Retrospective study . SETTING: A respiratory unit of a teaching hospital . PATIENTS: We studied 51 adult CF patients for whom current data and genotype were available . Thirty-seven patients carried two severe mutations associated with pancreatic insufficiency phenotype (group S) . Fourteen patients carried at least one mild (and dominant) mutation associated with pancreatic sufficiency phenotype (group M) . MEASUREMENTS: We compared the course of the disease between the two groups, looking for a genotype/phenotype association for lung disease . RESULTS: The mean age of the population was 30 years . Patients with two severe mutations presented more severe disease with earlier onset (1.7 years vs 7.9 years, p = 0.0001) . They presented with a more severe respiratory impairment, with a lower mean FEV(1) (29% of predictive value vs 58% of predictive value, p < 0.001); a higher Pseudomonas colonization rate (97% vs 57%, p < 0.01); a more frequent end-stage respiratory insufficiency, defined by a FEV(1) < 30% (73% vs 29%, p < 0.05); and a more marked yearly decline of FEV(1) (3% vs 1.4%, p < 0.001) . By multivariate logistic regression analysis, carrying two severe mutations was the only independent predictor of a terminal respiratory insufficiency (relative risk, 6.75; 95% confidence interval, 1.79 to 26.50; p = 0.003) . CONCLUSION: This study suggests that pulmonary disease appears to be associated with the severity of CF transmembrane regulator mutations.

Pediatr Infect Dis J, 2002 Jan, 21(1), 83 - 5
Is noma neonatorum a presentation of ecthyma gangrenosum in the newborn?
Freeman AF, Mancini AJ, Yogev R.
Noma neonatorum was suggested as a distinct entity characterized by a gangrenous process of the nose, oral cavity, eyelids and perineum that was almost universally fatal in premature infants with Pseudomonas sepsis . We report the first case of noma neonatorum in a 26-week-gestation twin born in the United States . Our case is consistent with previous descriptions of noma neonatorum; however, we question the distinction between noma neonatorum and a neonatal presentation of ecthyma gangrenosum.

Comp Biochem Physiol C Toxicol Pharmacol, 2000 Jan, 125(1), 65 - 83
Cyclohexadienyl dehydrogenase from Pseudomonas stutzeri exemplifies a widespread type of tyrosine-pathway dehydrogenase in the TyrA protein family; Xie G et al.; The uni-domain cyclohexadienyl dehydrogenases are able to use the alternative intermediates of tyrosine biosynthesis, prephenate or L-arogenate, as substrates . Members of this TyrA protein family have been generally considered to fall into two classes: sensitive or insensitive to feedback inhibition by L-tyrosine . A gene (tyrA(c)) encoding a cyclohexadienyl dehydrogenase from Pseudomonas stutzeri JM300 was cloned, sequenced, and expressed at a high level in Escherichia coli . This is the first molecular-genetic and biochemical characterization of a purified protein representing the feedback-sensitive type of cyclohexadienyl dehydrogenase . The catalytic-efficiency constant k(cat)/K(m) for prephenate (7.0x10(7) M/s) was much better than that of L-arogenate (5.7x10(6) M/s) . TyrA(c) was sensitive to feedback inhibition by either L-tyrosine or 4-hydroxyphenylpyruvate, competitively with respect to either prephenate or L-arogenate and non-competitively with respect to NAD(+) . A variety of related compounds were tested as inhibitors, and the minimal inhibitor structure was found to require only the aromatic ring and a hydroxyl substituent . Analysis by multiple alignment was used to compare 17 protein sequences representing TyrA family members having catalytic domains that are independent or fused to other catalytic domains, that exhibit broad substrate specificity or narrow substrate specificity, and that possess or lack sensitivity to endproduct inhibitors . We propose that the entire TyrA protein family lacks a discrete allosteric domain and that inhibitors act competitively at the catalytic site of different family members which exhibit individuality in the range and extent of molecules recognized as substrate or inhibitor.

Plant Physiol, 2002 Jan, 128(1), 30 - 7
Functional analysis of tomato Pti4 in Arabidopsis; Wu K et al.; Pti4 is a tomato (Lycopersicon esculentum) transcription factor that belongs to the ERF (ethylene-responsive element binding factor) family of proteins . It interacts with the Pto kinase in tomato, which confers resistance to the Pseudomonas syringae pv tomato pathogen that causes bacterial speck disease . To study the function of Pti4, transgenic Arabidopsis plants were generated that expressed tomato Pti4 driven by the strong constitutive promoters, cauliflower mosaic virus 35S and tCUP . Global gene expression analysis by Affimetric GeneChip indicated that expression of Pti4 in transgenic Arabidopsis plants induced the expression of GCC box-containing PR genes . We also demonstrated that Pti4 enhanced GCC box-mediated transcription of a reporter gene . The data suggests that tomato Pti4 could act as a transcriptional activator to regulate expression of GCC box-containing genes . Furthermore, we show that the expression of tomato Pti4 in transgenic Arabidopsis plants produced a phenotype similar to that seen in plants treated with ethylene, thus providing evidence that the Pti4 gene is involved in the regulation of a subset of ethylene-responsive genes containing the GCC box.

Biochem Biophys Res Commun, 2002 Jan 18, 290(2), 770 - 4
Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp . from the chemostat; Thakur IS et al.; Pseudomonas sp . strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase . The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography . The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP . The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha . The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy . The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan . Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.

Avian Dis, 2001 Oct-Dec, 45(4), 972 - 7
Efficacy comparisons of disinfectants used by the commercial poultry industry; Ruano M et al.; Several commercially available disinfectants used by the poultry industry were evaluated for their effectiveness against selected bacteria and viruses . When tested in the absence of organic matter, most disinfectant products were effective at the manufacturer's recommended level within 10 min of contact time . However, when organic matter was present, longer contact times and/or higher disinfectant dosages were needed to maintain effectiveness . Pseudomona aeruginosa and infectious laryngotracheitis virus were very resistant organisms in the presence of organic matter . Evaluation of disinfectant efficacy against several microbials in the absence or presence of organic matter was highly practical, flexible, and reproducible.

J Biol Chem, 2002 Mar 22, 277(12), 10256 - 64 Epub 2002 Jan 08.
Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp . 130; Huang X et al.; Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp . 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA) . The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry . Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA . By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond . Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity . None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA . Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y . Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself . The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure . Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis.

Int J Cancer, 2002 Jan 20, 97(3), 349 - 56
Replacement of N-terminal portions of TGF-alpha with corresponding heregulin sequences affects ligand-induced receptor signaling and intoxication of tumor cells by chimeric growth-factor toxins; Schmidt M et al.; ErbB receptor tyrosine kinases play an important role in developmental processes and tumor formation . Their activity is regulated by a family of structurally related ligands that bind to distinct ErbB receptor subsets, with transforming growth factor (TGF)-alpha preferentially interacting with epidermal growth-factor receptor (EGFR) and heregulin (HRG)-beta1 recognizing ErbB3 and ErbB4 . To investigate the contribution of N-terminal ligand sequences to binding specificity, we have constructed 2 chimeric growth factors termed H181T8 and H194T20, which contain N-terminal HRG-beta1 sequences linked to complementary fragments of TGF-alpha . For bacterial expression and analysis of cell binding, the chimeric ligands were genetically fused to truncated Pseudomonas exotoxin A (ETA) . H181T8-ETA and H194T20-ETA toxins both were cytotoxic for human tumor cell lines overexpressing EGFR but did not significantly affect the growth of cells that express ErbB receptors other than EGFR . Binding of H181T8, which contains HRG-beta1 residues 177-181, induced rapid autophosphorylation of EGFR, but in contrast to a previously described chimeric ligand based on EGF was unable to activate other ErbB receptors . H194T20, which contains HRG-beta1 residues 177-194, despite specific binding to EGFR was unable to induce autophosphorylation of any of the ErbB family members . However, H194T20 enhanced and modified the activity of parental TGF-alpha and HRG-beta1 when these ligands were simultaneously present . Our results show that modification of the N-terminal TGF-alpha sequence can have a significant effect on the signaling properties of the ligand and suggest that different EGF-like ligands can synergize in the activation of ErbB receptors .

Cancer Gene Ther, 2001 Nov, 8(11), 861 - 8
Gene transfer of interleukin 13 receptor alpha2 chain dramatically enhances the antitumor effect of IL-13 receptor-targeted cytotoxin in human prostate cancer xenografts; Kawakami K et al.; IL-13Ralpha2 chain, the primary interleukin-13 (IL-13) binding protein, plays an important role in IL-13 binding and internalization . Based on these findings, in our previous study we transiently transfected four cancer cell lines that do not express IL-13Ralpha2 chain and demonstrated that these cells acquired increased sensitivity to IL-13 receptor-targeted recombinant cytotoxin, IL13-PE38QQR, which is composed of IL-13 and a mutated form of a Pseudomonas exotoxin . Although some prostate cancer cell lines express functional IL-13R, they are not highly sensitive to IL-13 cytotoxin . Here we investigated whether human prostate cancer and normal prostate epithelial cell lines express IL-13Ralpha2 chain and whether they can be sensitized to the cytotoxic effect of IL-13 cytotoxin after transient or stable gene transfer of IL-13Ralpha2 chain . Gene transfer of IL-13Ralpha2 chain improved binding activity of IL-13 and sensitivity to IL-13 cytotoxin in vitro . In vivo experiments demonstrated that gene transfer of IL-13Ralpha2 chain dramatically enhanced the antitumor activity of IL-13 cytotoxin in human prostate cancer xenograft models . These results suggest that IL-13R-targeted cytotoxin therapy of prostate cancer may be dramatically enhanced by gene transfer of IL-13Ralpha2 chain and this strategy, the combination of gene therapy and cytotoxin therapy, may be utilized in the treatment of localized prostate cancer.

Appl Environ Microbiol, 2002 Jan, 68(1), 280 - 8
Homology of Escherichia coli R773 arsA, arsB, and arsC genes in arsenic-resistant bacteria isolated from raw sewage and arsenic-enriched creek waters; Saltikov CW et al.; The occurrence and diversity of the Escherichia coli R773 ars operon were investigated among arsenic-resistant enteric and nonenteric bacteria isolated from raw sewage and arsenic-enriched creek waters . Selected isolates from each creek location were screened for ars genes by colony hybridization and PCR . The occurrence of arsA, arsB, and arsC determined by low-stringency colony hybridization (31 to 53% estimated mismatch) was 81, 87, and 86%, respectively, for 84 bacteria isolated on arsenate- and arsenite-amended media from three locations . At moderate stringency (21 to 36% estimated mismatch), the occurrence decreased to 42, 56, and 63% for arsA, arsB, and arsC, respectively . PCR results showed that the ars operon is conserved in some enteric bacteria isolated from creek waters and raw sewage . The occurrence of the arsBC genotype was about 50% in raw sewage enteric bacteria, while arsA was detected in only 9.4% of the isolates (n = 32) . The arsABC and arsBC genotypes occurred more frequently in enteric bacteria isolated from creek samples: 71.4 and 85.7% (n = 7), respectively . Average sequence divergence within arsB for six creek enteric bacteria was 20% compared to that of the E . coli R773 ars operon . Only 1 of 11 pseudomonads screened by PCR was positive for arsB . The results from this study suggest that significant divergence has occurred in the ars operon among As-resistant E . coli strains and in Pseudomonas spp.

Appl Environ Microbiol, 2002 Jan, 68(1), 1 - 10
Cloning, sequencing, and expression of the cold-inducible hutU gene from the antarctic psychrotrophic bacterium Pseudomonas syringae; Janiyani KL et al.; A promoter-fusion study with a Tn 5-based promoter probe vector had earlier found that the hutU gene which encodes the enzyme urocanase for the histidine utilization pathway is upregulated at a lower temperature (4 degrees C) in the Antarctic psychrotrophic bacterium Pseudomonas syringae . To examine the characteristics of the urocanase gene and its promoter elements from the psychrotroph, the complete hutU and its upstream region from P . syringae were cloned, sequenced, and analyzed in the present study . Northern blot and primer extension analyses suggested that the hutU gene is inducible upon a downshift of temperature (22 to 4 degrees C) and that there is more than one transcription initiation site . One of the initiation sites was specific to the cells grown at 4 degrees C, which was different from the common initiation sites observed at both 4 and 22 degrees C . Although no typical promoter consensus sequences were observed in the flanking region of the transcription initiation sites, there was a characteristic CAAAA sequence at the -10 position of the promoters . Additionally, the location of the transcription and translation initiation sites suggested that the hutU mRNA contains a long 5'-untranslated region, a characteristic feature of many cold-inducible genes of mesophilic bacteria . A comparison of deduced amino acid sequences of urocanase from various bacteria, including the mesophilic and psychrotrophic Pseudomonas spp., suggests that there is a high degree of similarity between the enzymes . The enzyme sequence contains a signature motif (GXGX(2)GX(10)G) of the Rossmann fold for dinucleotide (NAD(+)) binding and two conserved cysteine residues in and around the active site . The psychrotrophic enzyme, however, has an extended N-terminal end.

Acta Pharm Hung, 2001, 71(1), 119 - 26
{Lipase-catalyzed kinetic resolution of 2-substituted cycloalkanols}; Forro E; Racemates of cis- and trans-2-cyanocyclopentanol and -cyclohexanol, cis- and trans-2-dialkylaminomethylcyclopentanol, -cyclohexanol and -cycloheptanol and Boc-protected cis- and trans-2-methylhydrazinocyclopentanol and -cyclohexanol were resolved through lipase PS (from Pseudomonas cepacia) or Novozym 435 (from Candida antarctica B)-catalysed asymmetric acylation . High enantioselectivity (E > 200) was observed when vinyl acetate was used as acylating agent, with diethyl ether or with diisopropyl ether as solvent . Reaction rates were markedly affected by the solvent and by the quantity of the enzyme . The size of the cycloalkane ring had a clear effect on the rate of enantioselective acylation: the acetylations of the five-membered cycloalcanols proceeded more rapidly than those of the six-membered ones and much more rapidly than those of the seven-membered systems . It can also be concluded that the trans isomers react more rapidly than the cis counterparts, the only exception being found in the case of 2-cyanocyclohexanols . In good correlation with the "Kazlauskas rule", in all cases, the (R) enantiomer is acylated faster than the (S) enantiomer, yielding an (R) ester and an (S) alcohol, which products from large-scale experiments were separated by column chromatography . During these studies, a total of 18 racemates of cis- and trans-2-substituted cycloalkanols were resolved by using lipases as catalysts, and 52 enantiomers (50 of them new) were characterized by NMR, elemental analysis and ocasionally MS.

Mol Plant Microbe Interact, 2001 Dec, 14(12), 1453 - 7
Overexpression of Pti5 in tomato potentiates pathogen-induced defense gene expression and enhances disease resistance to Pseudomonas syringae pv . tomato; He P et al.; The tomato Pti5 gene encodes a pathogen-inducible ethylene response element-binding protein-like transcription factor that interacts with the disease resistance gene product Pto . Overexpression of Pti5 or Pti5-VP16, a translational fusion with a constitutive transcriptional activation domain, in tomato enhanced resistance to Pseudomonas syringae pv . tomato . Constitutive expression of Pti5 or Pti5-VP16 did not affect the basal level of pathogenesis-related gene expression, but it accelerated pathogen-induced expression of GluB and Catalase . The results demonstrate a positive role of Pti5 in defense gene regulation and disease resistance and suggest that a pathogen-activated posttranscriptional regulatory step is necessary for the pathogen induction of the defense gene expression.

Mol Plant Microbe Interact, 2001 Dec, 14(12), 1426 - 35
A physical map of the syringomycin and syringopeptin gene clusters localized to an approximately 145-kb DNA region of Pseudomonas syringae pv . syringae strain B301D; Scholz-Schroeder BK et al.; Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv . syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment . The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively . Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases . Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment . In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment . In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P . syringae pv . syringae strain B728a, was localized to the syr gene cluster . A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins . Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters . Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P . syringae pv . syringae that contributes to virulence in plant hosts.

Ying Yong Sheng Tai Xue Bao, 2000 Jun, 11(3), 425 - 7
{Effect of plumular axis-cutted cotton on growth and development of cotton bollworm}; Li P et al.; The method of plumular axis cutting was used to induce the resistance of cotton plants to cotton bollworms(Heliothis armigera) . The bollworm was cultured in the laboratory, and the effect of induced cotton on the duration of larval development and weights of larvae and pupae was studied . In the treatments of infection and control, the lengths of larval period were delayed by 3 days and 0.5 days, the weights of larvae were decreased by 19.60% and 11.45%, and the pupae weights were decreased by 10.81% and 6.54%, respectively . After the 8 days old larvae were reared with induced cotton leaves for 3 days, the infected and uninfected cutted plants resulted in a decrease of the relative growth rates of larvae by 22.9% and 17.2% respectively, and in a decrease of the relative feeding rates by 26.1% and 21.4% respectively . It is suggested that plumular axis cutting could induce the resistance of cotton plants to bollworm, and influence the growth and development speed of bollworms through retarding their feeding and digestion . Combining with the treatment of Pseudomona gladioli D-2251 strain could obviously increase the insect-resistance of cotton plants.

Kansenshogaku Zasshi, 2001 Nov, 75(11), 970 - 6
{A case of diffuse panbronchiolitis with SLE complicated by fungal infection}; Wada Y et al.; We report a patient who developed SLE during the course of diffuse panbronchiolitis (DPB) and had candidiasis later . The patient fulfilled the criteria for diagnosis of SLE after appearance of fever and general peripheral arthritis . Regarding serum virus antibody values at the time of SLE diagnosis, IgG and IgM of human Parvovirus B19 (B19) were positive by the EIA test and also by the serum PCR test . For continuously Pseudomonas aeruginozae in sputum cultures because of existing DPB, immunosuppressasnt therapy with prednisolone and mizoribine was given while suppressing proliferation of bacterial infections with antibiotics . As a result, the intensity of SLE decreased smoothly . About 1 month after beginning of the treatment, the chest X-ray revealed infiltrative densities in the lingual area of the left lobule and in S3 of the right lobule . Judging from the clinical course and various examination findings, concurrence of candidiasis was suspected . Fungal infection in this patient was progressive, so various antifungal agents were used concurrently . Furthermore, immunoglobulin therapy was supplemented while determining serum immunoglobulin levels, and doses of prednisolone and mizoribine were reduced rapidly . Afterward the patient followed a satisfactory clinical course . About 2 years later SLE recurred, aspergillosis developed concurrently and the disease progressed rapidly to its termination . DPB itself is difficult to control and often complicated with various diseases . Therefore, immunosuppresant therapy for complications is sometimes used in addition to the treatment of DPB . More careful observations on the clinical course are necessary in dealing with this disease.

Appl Microbiol Biotechnol, 2001 Nov, 57(4), 548 - 54
Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp . strain DCA1; Hage JC et al.; Pseudomonas sp . strain DCA1, which is capable of utilizing 1,2-dichloroethane (DCA) as sole carbon and energy source, was used to oxidize chlorinated methanes, ethanes, propanes, and ethenes . Chloroacetic acid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCAI and did not compete for the monooxygenase . All of the tested compounds except tetrachloroethylene (PER) were oxidized by cells expressing DCA monooxygenase . Strain DCAI could not utilize any of these compounds as a growth substrate . Co-metabolic oxidation during growth on DCA was studied with 1,2-dichloropropane . Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCAI . This inhibition was not caused by competition for the monooxygenase . It was shown that the oxidation of 1,2dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol.

Curr Pharm Biotechnol, 2001 Dec, 2(4), 313 - 25
Toxin-labeled monoclonal antibodies; Kreitman RJ; To arm monoclonal antibodies (MAbs) with the power to kill malignant cells, they have been connected to toxins to create chimeric proteins called immunotoxins . Conventional immunotoxins contain a MAb chemically conjugated to a toxin which is mutated or chemically modified to minimize binding to normal cells . Examples include anti-B4-blocked ricin, targeting CD5, and RFB4-deglycosylated ricin A chain, targeting CD22 . Conventional immunotoxins are capable of inducing responses in patients with hematologic malignancies, with dose-limiting toxicities being vascular leak syndrome, thrombocytopenia, and hepatic damage . Newer immunotoxins contain a recombinant ligand, either the variable domains (Fv) of a MAb, or a growth factor, fused to a truncated bacterial toxin . Bacterial toxins commonly used for this purpose include diphtheria toxin and Pseudomonas exotoxin . DAB389lL2 (Ontak) is a recently approved growth factor fusion toxin containing human interleukin-2 and diphtheria toxin and is effective in chemotherapy-resistant cutaneous T-cell lymphoma . Anti-Tac(Fv)-PE38 (LMB-2) and RFB4(dsFv)-PE38 (BL22) are two recombinant immunotoxins, targeting CD25 and CD22, respectively, in which Fvs of MAbs targeting these antigens are fused to truncated Pseudomonas exotoxin . Both LMB-2 and BL22 have exhibited clinical activity in patients with hematologic malignancies, with less vascular leak syndrome and probably less immunogenicity than the larger conventional immunotoxin conjugates . New recombinant immunotoxins are currently being engineered and developed to target other hematologic and solid tumor antigens.

DNA Seq, 2001, 12(2), 125 - 9
Genetic divergence in the algT-muc operon controlling alginate biosynthesis and response to environmental stress in Pseudomonas syringae; Keith LM et al.; The algT-muc gene cluster (rpoE operon) is important for alginate production and survival during environmental stress in Pseudomonas syringae . The algT-muc operon was cloned and sequenced from P . syringae to determine whether the organization of this gene cluster was conserved in this plant pathogen . Interestingly, analysis of the algT-muc region in P . syringae revealed a unique arrangement when compared to other bacteria and lacked a mucC homologue . The relative importance of the mucC gene in the algT (rpoE) operon of various bacterial species is discussed.

Ying Yong Sheng Tai Xue Bao, 2001 Jun, 12(3), 359 - 62
{Ecological factors affecting prevalence of kiwifruit bacterial canker and bacteriostatic action of bacteriocides on Pseudomonas syringae pv . actinidae}; Li Y et al.; According to the systematic study for 5 years, this paper dealt with the ecological factors affecting prevalence of kiwifruit bacterial canker . The disease was more serious in the orchards with above 750 m in elevation, and more serious at mountain slope facing south than facing north . Incidence differed significantly among cultivars, with easy disease infection for Jinfeng, and strong disease resistance for Jinkui . The older the trees, the more the diseased plants . The one year old twigs had highest death twig incidence of diseased twigs and death twig incidence than other twigs . Among 6 tested bacteriocides, Jiaruinong and streptomycin showed best bacteriostatic effect.

Ying Yong Sheng Tai Xue Bao, 2001 Jun, 12(3), 355 - 8
{Prevalent forecast of kiwifruit bacterial canker caused by Pseudomonas syringae pv . actinidiae}; Li Y et al.; The prevalent analysis of kiwifruit bacterial canker for several years showed that the effective ecological factors of severe degree were the precipitation (x1) in the second and last ten-days of March, and the average temperature (x2) of January . The model was y = 2.1359 + 0.0107x1 - 0.6061x2 . The main factor of the prevalence was the relative variation of ten-days average temperature and precipitation in Winter and in early Spring, and the regression equation was y = -8.127 + 22.739x - 13.254x2 . The forecast effect of the equation was obviously significant after testing.

Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 517 - 22 Epub 2001 Dec 26.
Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response; Torres MA et al.; Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses . The origin of these ROI has been controversial . Arabidopsis respiratory burst oxidase homologues (rboh genes) have been proposed to play a role in ROI generation . We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes . Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv . tomato DC3000(avrRpm1) and the oomycete parasite Peronospora parasitica . We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000(avrRpm1) inoculation . However, enhanced cell death is observed after infection of mutant lines with P . parasitica . Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.

Cell Mol Biol Lett, 2001, 6(4), 913 - 23
Protein fingerprinting as a complementary tool for the classification of Pseudomonas bacteria; Zarnowski R et al.; A collection of total 42 bacterial strains belonging to the genus Pseudomonas were characterised based on protein fingerprinting using sodium dodecyl sulphate polyacrylamide gel electrophoregrams of cell-free extracts . Densitometrical analysis revealed unique and distinct profiles characteristic of the studied species . This comparison differentiated the isolates into four main clusters and twelve subclusters . The obtained protein patterns have proved to be an effective and reliable method both for the classification of bacteria and for showing similarities and variability among them.

Science, 2001 Dec 21, 294(5551), 2556 - 8
Role of the Hrp pilus in type III protein secretion in Pseudomonas syringae; Jin Q et al.; Bacterial surface appendages called pili and needle-like filaments are associated with protein and/or DNA transfer to recipient plant, human, or bacterial cells during pathogenesis or conjugation . Although it has long been suspected that pili function as a conduit for protein or DNA transfer, direct evidence has been lacking . The Hrp pilus of Pseudomonas syringae is assembled by the type III secretion system . We used an in situ immunogold labeling procedure to visualize the extrusion of an effector protein, AvrPto, from the tip of the Hrp pilus, providing direct evidence that a bacterial pilus can function as a conduit for protein delivery.

Biomacromolecules, 2001 Spring, 2(1), 313 - 21
Crystallization, melting, and enzymatic degradation of biodegradable poly(butylene succinate-co-14 mol % ethylene succinate) copolyester; Gan Z et al.; The crystal structure and growth kinetics of melt-crystallized poly(butylene succinate-co-14 mol % ethylene succinate) {P(BS-co-14 mol % ES)} copolyester have been investigated at a wide crystallization temperature range of 30 to 90 degrees C . By means of wide-angle X-ray diffraction (WAXD), the copolyester composed of BS and ES units has been identified to have the same crystal structure with that of poly(butylene succinate) (PBS) homopolymer, suggesting that only PBS sequences crystallize while that the ES units are in an amorphous form . On the basis of secondary nucleation theory, two regimes of II and III have been observed and their transition temperature is around 80 degrees C . The spherulitic morphologies of P(BS-co-14 mol % ES) copolyester developed from banded spherulites to the normal ones without banding extinction patterns as the crystallization temperature increased . The melting behavior of P(BS-co-14 mol % ES) copolyester under different conditions has been studied by differential scanning calorimetry (DSC) . Four melting peaks and one exothermal peak on the melting curves were observed during heating process, and their origination is discussed . The enzymatic degradation was carried out on the melt-crystallized P(BS-co-14 mol % ES) thin film by an extracellar PHB depolymerase from Pseudomonas stutzeri and the morphologies of lamellar crystals before and after degradation have been examined by atomic force microscopy (AFM) . The results have indicated that enzymatically degradable ES units exist on the surface of lamellar crystals and are hydrolyzed by the enzyme, while that the crystalline cores composed of PBS chains are not degraded.

Biomacromolecules, 2001 Spring, 2(1), 211 - 6
Glucose/lipid mixed substrates as a means of controlling the properties of medium chain length poly(hydroxyalkanoates); Ashby RD et al.; Glucose-triacylglycerol (TAG) mixed substrates were used to modulate the physical and mechanical properties of medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) . Pseudomonas resinovorans NRRL B-2649 grew and produced mcl-PHAs on glucose and TAGs (coconut oil, C; soybean oil, S) after 24 h in a shake flask culture . However, with the exception of coconut oil, maximum cell productivity was not reached in any of the cultures until 72 h post-inoculation . Here, 50:50 mixtures of glucose and coconut oil (glc/C) or glucose and soybean oil (glc/S) resulted in intermediate cell productivities with a maximum of 57% and 48% of the CDW at 72 h, respectively . In addition, mixed substrates resulted in mcl-PHAs with compositions that varied slightly over time . PHA-glc/C and PHA-glc/S were composed of 7 mol % and 8 mol % 3-hydroxydodecenoic acid (C(12:1)), respectively at 72 h . These concentrations were intermediate to the C(12:1) concentration of PHA-glc and respective PHA-TAG . Also, significant amounts of 3-hydroxytetradecanoic acid (C(14:0)), 3-hydroxytetradecenoic acid (C(14:1)), and 3-hydroxytetradecadienoic acid (C(14:2)) were present in PHA-glc/C and PHA-glc/S, which were derived from the respective TAG, as glucose resulted in almost no C(14:)(X) monomers . The molar masses of each of the polymers remained relatively constant between 24 and 96 h . At 72 h, the number-average molar masses (M(n)) of PHA-glc/C and PHA-glc/S were 178,000 and 163,000 g/mol, respectively, which were also intermediate to the M(n) of PHA-glc (225,000 g/mol) and the respective PHA-TAG (PHA-C = 153,000 g/mol; PHA-S = 75,000 g/mol) . These physical differences caused variations in the mechanical properties of mcl-PHA films, thus providing a new and effective method of modifying their properties.

Biomacromolecules, 2001 Spring, 2(1), 142 - 7
Cloning and characterization of the Pseudomonas sp . 61-3 phaG gene involved in polyhydroxyalkanoate biosynthesis; Matsumoto K et al.; Pseudomonas sp . 61-3 produces a blend of poly(3-hydroxybutyrate) {P(3HB)} homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) {P(3HB-co-3HA)} random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars . The phaG(Ps) gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated . Additional copies of the phaG(Ps) gene in Pseudomonas sp . 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA . Disruption of the chromosomal phaG(Ps) gene resulted in an increase in the fraction of the 3HB unit in PHA . The site-directed mutagenesis of the phaG(Ps) gene was carried out to investigate the role of a HX(4)D motif which has been proposed to be related to PhaG activity.

J Biol Chem, 2002 Feb 15, 277(7), 5575 - 82 Epub 2001 Dec 03.
Changing the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution; Meyer A et al.; The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC, HbpA) was changed by directed evolution using error-prone PCR . In situ screening of mutant libraries resulted in the identification of proteins with increased activity towards 2-tert-butylphenol and guaiacol (2-methoxyphenol) . One enzyme variant contained amino acid substitutions V368A/L417F, which were inserted by two rounds of mutagenesis . The double replacement improved the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH . With guaiacol as substrate, the two substitutions increased V(max) from 0.22 to 0.43 units mg(-1) protein and decreased the K'(m) from 588 to 143 microm, improving k'(cat)/K'(m) by a factor of 8.2 . With 2-tert-butylphenol as the substrate, k'(cat) was increased more than 5-fold . Another selected enzyme variant contained amino acid substitution I244V and had a 30% higher specific activity with 2-sec-butylphenol, guaiacol, and the "natural" substrate 2-hydroxybiphenyl . The K'(m) for guaiacol decreased with this mutant, but the K'(m) for 2-hydroxybiphenyl increased . The primary structure of HbpA shares 20.1% sequence identity with phenol 2-monooxygenase from Trichosporon cutaneum . Structure homology modeling with this three-domain enzyme suggests that Ile(244) of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol . In contrast, Val(368) and Leu(417) are not close to the active site and would not have been obvious candidates for modification by rational design.

J Biol Chem, 2002 Mar 22, 277(12), 10555 - 61 Epub 2001 Dec 17.
Monitoring the switch from housekeeping to pathogen defense metabolism in Arabidopsis thaliana using cDNA arrays; Scheideler M et al.; Plants respond to pathogen attack by deploying several defense reactions . Some rely on the activation of preformed components, whereas others depend on changes in transcriptional activity . Using cDNA arrays comprising 13,000 unique expressed sequence tags, changes in the transcriptome of Arabidopsis thaliana were monitored after attempted infection with the bacterial plant pathogen Pseudomonas syringae pv . tomato carrying the avirulence gene avrRpt2 . Sampling at four time points during the first 24 h after infiltration revealed significant changes in the steady state transcript levels of approximately 650 genes within 10 min and a massive shift in gene expression patterns by 7 h involving approximately 2,000 genes representing many cellular processes . This shift from housekeeping to defense metabolism results from changes in regulatory and signaling circuits and from an increased demand for energy and biosynthetic capacity in plants fighting off a pathogenic attack . Concentrating our detailed analysis on the genes encoding enzymes in glycolysis, the Krebs cycle, the pentose phosphate pathway, the biosynthesis of aromatic amino acids, phenylpropanoids, and ethylene, we observed interesting differential regulation patterns . Furthermore, our data showed potentially important changes in areas of metabolism, such as the glyoxylate metabolism, hitherto not suspected to be components of plant defense.

Biochemistry, 2001 Dec 25, 40(51), 15602 - 11
Inhibitor complexes of the Pseudomonas serine-carboxyl proteinase; Wlodawer A et al.; Crystal structures of the serine-carboxyl proteinase from Pseudomonas sp . 101 (PSCP), complexed with a number of inhibitors, have been solved and refined at high- to atomic-level resolution . All of these inhibitors (tyrostatin, pseudo-tyrostatin, AcIPF, AcIAF, and chymostatin, as well as previously studied iodotyrostatin and pseudo-iodotyrostatin) make covalent bonds to the active site Ser287 through their aldehyde moieties, while their side chains occupy subsites S1-S4 of the enzyme . The mode of binding of the inhibitors is almost identical for their P1 and P2 side chains, while significant differences are observed for P3 and P4 (if present) . Kinetic parameters for the binding of these nanomolar inhibitors to PSCP have been established and correlated with the observed mode of binding . The preferences of this enzyme for a larger side chain in P2 as well as Tyr or Phe in P1 are explained by the size, shape, and characteristics of the S2 and S1 regions of the protein structure, respectively . Networks of hydrogen bonds involving glutamic and aspartic acids have been analyzed for the atomic-resolution structure of the native enzyme . PSCP contains a calcium-binding site that consists of Asp328, Asp348, three amide carbonyl groups, and a water molecule, in almost perfect octahedral coordination . The presence of Ca(2+) cation is necessary for the activity of the enzyme.

J Bacteriol, 2002 Jan, 184(1), 216 - 23
Degradation of aromatics and chloroaromatics by Pseudomonas sp . strain B13: cloning, characterization, and analysis of sequences encoding 3-oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase; Gobel M et al.; 3-oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway . This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp . strain B13 that encode these enzymes . DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one . ORF1, of unknown function, has a length of 414 bp . ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813 . ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661 . CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a . ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity . The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF . Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF . ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase . An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found . The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.

Eur J Biochem, 2001 Dec, 268(24), 6486 - 91
The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity; Forte E et al.; The cytochrome cbb3 is an isoenzyme in the family of cytochrome c oxidases . This protein purified from Pseudomonas stutzeri displays a cyanide-sensitive nitric oxide reductase activity (Vmax=100+/-9 mol NO x mol cbb3(-1) x min(-1) and Km=12+/-2.5 microm), which is lost upon denaturation . This enzyme is only partially reduced by ascorbate, and readily re-oxidized by NO under anaerobic conditions at a rate consistent with the turnover number for NO consumption . As shown by transient spectroscopy experiments and singular value decomposition (SVD) analysis, these results suggest that the cbb3-type cytochromes, sharing structural features with bacterial nitric oxide reductases, are the enzymes retaining the highest NO reductase activity within the heme-copper oxidase superfamily.

J Org Chem, 2001 Dec 14, 66(25), 8395 - 401
Improved preparation and use of room-temperature ionic liquids in lipase-catalyzed enantio- and regioselective acylations; Park S et al.; Polar organic solvents such as methanol or N-methylformamide inactivate lipases . Although ionic liquids such as 3-alkyl-1-methylimidazolium tetrafluoroborates have polarities similar to these polar organic solvents, they do not inactivate lipases . To get reliable lipase-catalyzed reactions in ionic liquids, we modified their preparation by adding a wash with aqueous sodium carbonate . Lipase-catalyzed reactions that previously did not occur in untreated ionic liquids now occur at rates comparable to those in nonpolar organic solvents such as toluene . Acetylation of 1-phenylethanol catalyzed by lipase from Pseudomonas cepacia (PCL) was as fast and as enantioselective in ionic liquids as in toluene . Ionic liquids permit reactions in a more polar solvent than previously possible . Acetylation of glucose catalyzed by lipase B from Candida antarctica (CAL-B) was more regioselective in ionic liquids because glucose is up to one hundred times more soluble in ionic liquids . Acetylation of insoluble glucose in organic solvents yielded the more soluble 6-O-acetyl glucose, which underwent further acetylation to give 3,6-O-diacetyl glucose (2-3:1 mixture) . However, acetylation of glucose in ionic liquids yielded only 6-O-acetyl glucose (>13:1 and up to >50:1).

FEBS Lett, 2001 Nov 30, 509(1), 17 - 21
Ca(2+)-induced folding of a family I.3 lipase with repetitive Ca(2+) binding motifs at the C-terminus; Amada K et al.; In order to understand a role of the Ca(2+) ion on the structure and function of a Ca(2+)-dependent family I.3 lipase from Pseudomonas sp . MIS38, apo-PML, holo-PML, holo-PML*, and the N-terminal domain alone (N-fragment) were prepared and biochemically characterized . Apo-PML and holo-PML represent refolded proteins in the absence and presence of the Ca(2+) ion, respectively . Holo-PML* represents a holo-PML dialyzed against 20 mM Tris-HCl (pH 7.5) . The results suggest that the C-terminal domain of PML is almost fully unfolded in the apo-form and its folding is induced by Ca(2+) binding . The folding of this C-terminal domain may be required to make a conformation of the N-terminal catalytic domain functional.

J Microbiol Methods, 2002 Jan, 48(1), 69 - 86
A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere; van Overbeek LS et al.; Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade . Knowledge on the ecological behaviour of R . solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields.A polyphasic approach was used to study the invasion of plants by a selected R . solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P . fluorescens UA5-40 . Thus, this study combined plating (spread and drop plate methods), reporter gene technology (gfp mutants) and serological (imunofluorescence colony staining {IFC}) and molecular techniques (fluorescent in situ hybridization {FISH}, PCR with R . solanacearum specific primers and PCR-DGGE on plant DNA extracts) . The behaviour of R . solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants.The results showed that an interaction between the pathogen and the control strains at the root surface was likely . In particular, R . solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P . corrugata IDV1(chr:gfp1) cells as compared to those on untreated roots . Concomitant with the presence of P . corrugata IDV1(chr:gfp1), plant invasion by the pathogen was hampered, but not abolished.PCR-DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R . solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen . Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e . P . corrugata IDV1(chr:gfp1); R . solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent.R . solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection . In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists.The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural practice.

Ann Clin Biochem, 2001 Nov, 38(Pt 6), 701 - 7
Determination of D-sorbitol in human erythrocytes by an enzymatic fluorometric method with an improved deproteinization procedure; Umeda M et al.; We developed an improved enzymatic assay of D-sorbitol in human erythrocytes by employing highly specific D-sorbitol dehydrogenase from Pseudomonas sp . (EC 1.1.1.14) and replacing perchloric acid (HClO4) and potassium carbonate (K2CO3), generally used for deproteinization, with sodium hydroxide (NaOH) and zinc sulphate (ZnSO4) . In this assay, erythrocytes were separated from plasma by centrifugation and washed once with physiological saline . Subsequently, the erythrocytes were lysed with distilled water and proteins precipitated with NaOH and ZnSO4 . After centrifugation, the resulting colourless supernatant was mixed with a glycine buffer (pH 9.0) containing NAD+ and D-sorbitol dehydrogenase . After incubation for 30 min at 37 degrees C, the NADH produced was measured fluorimetrically . The fluorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normalized for haemoglobin content . The method had an analytical range of 1-180 micromol/L . The intra- and inter-assay precisions were < 3.3% and < 5.8%, respectively . The detection limit was 0.65 micromol/L . In terms of the linearity, precision and sensitivity, the improved method using NaOH and ZnSO4 was superior to the conventional method using HClO4 and K2CO3.

Biochim Biophys Acta, 2001 Nov 7, 1568(1), 83 - 9
Dioxygenative cleavage of C-methylated hydroquinones and 2,6-dichlorohydroquinone by Pseudomonas sp . HH35; Schmidt S et al.; The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp . strain HH35 was elucidated . This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and (1)H NMR and UV spectroscopy . The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack . With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group . In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.

Acta Pharmacol Sin, 2001 Jan, 22(1), 15 - 20
Inhibitory effect of recombinant TGFalpha-PE40 on neointimal proliferation after arterial balloon injury; Wang B et al.; AIM: To investigate inhibitory effects of recombinant transforming growth factor alpha-Pseudomonas exotoxin 40 fusion protein (TGFalpha-PE40; TP40) on neointimal proliferation after arterial balloon injury . METHODS: Forty male rabbits fed a cholesterol rich diet were randomly divided into TP40 15 microg, 30 microg, 60 microg, physiologic saline control, and normal artery groups (n=8) . Rabbits in the treatment groups were treated by local administration of TP40 (15 microg, 30 microg, and 60 microg per rabbit) 24 h postinjury, and those in the control group were treated by physiologic saline 24 h postinjury . Remained 8 rabbits in normal artery group were treated by TP40 (60 microg) . Optical microscope, electron microscope, and computer image analysis were used to study arterial segments 2 weeks after treatment . RESULTS: Irregular thickening of the arterial intima, large amounts of smooth muscle cells (SMC) within the neointima, and stenosis of the arterial cavity were observed in the physiologic saline control group . Great inhibition of intimal proliferation and prevention of stenosis of the arterial cavity were observed in the TP40 treated groups that were examined 2 weeks postinjury by optical microscope . The uninjured carotids were histologically normal . Lots of destructural and necrotic SMC were observed in media in TP40 60 microg group by electron microscope . Computer image analysis showed that the neointimal area of the TP40-treated groups was markedly smaller than that of the saline control group (P < 0.01) . CONCLUSION: Recombinant TP40 greatly inhibited neointimal proliferation after arterial balloon injury.

FEMS Microbiol Lett, 2001 Nov 27, 205(1), 9 - 16
The coenzyme A-dependent, non-beta-oxidation pathway and not direct deacetylation is the major route for ferulic acid degradation in Delftia acidovorans; Plaggenborg R et al.; The gene loci fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, are involved in the ferulic acid catabolism in Delftia acidovorans . The amino acid sequence deduced from ech exhibited 51% identity to the enoyl-CoA hydratase/aldolase from Pseudomonas sp . strain HR199, indicating that the enzyme from D . acidovorans represents a new lineage of this protein . The genes fcs and ech were expressed in Escherichia coli enabling the recombinant strain to transform ferulic acid to vanillin as revealed by photometric and HPLC analysis . An fcs deficient mutant of D . acidovorans was unable to grow on ferulic acid . The obtained data suggest that in contrast to a previous publication the biotechnologically interesting direct non-oxidative deacetylation mechanism of ferulic acid cleavage is not realized in D . acidovorans . Instead, ferulic acid degradation in D . acidovorans proceeds via a coenzyme A-dependent non-beta-oxidative pathway.

Carbohydr Res, 2001 Dec 7, 336(4), 329 - 36
Structural heterogeneity in the lipopolysaccharides of Pseudomonas syringae with O-polysaccharide chains having different repeating units; Zdorovenko EL et al.; Studies by sugar and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy revealed a structural heterogeneity in the O-polysaccharides of Pseudomonas syringae pvs . coronafaciens IMV 9030 and atrofaciens IMV 8281 owing to the presence of different types of repeating units . In strain IMV 9030, the major repeating units are a linear alpha-L-rhamnose trisaccharide and a tetrasaccharide (A, n=0 or 1) . A minor repeating unit is a branched pentasaccharide with an alpha-L-rhamnose main chain and a lateral 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) residue (B, X=2, n=1) . In strain IMV 8281, all repeating units are branched and differ in size and position of substitution of one of the alpha-L-rhamnose residues (tetrasaccharide, B, X=3, n=0; pentasaccharides, B, X=2 or 3, n=1) . {structure--see text} Reinvestigation of the structure of the branched O-polysaccharide of P . syringae pv . tomato IPGR 140 showed that, together with the major tetrasaccharide repeating unit (B, X=3, n=0) {Knirel, Y . A., et al . Carbohydr . Res . 1993, 243, 199-204}, it has a minor pentasaccharide repeating unit (B, X=3, n=1).

J Biochem (Tokyo), 2001 Dec, 130(6), 789 - 97
Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system; Takeuchi K et al.; The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone . The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin . We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K . Such shifts were not observed for the low-spin EPR signals . Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K . These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system . The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.

Z Naturforsch {C}, 2001 Sep-Oct, 56(9-10), 687 - 94
The structure of the pyoverdin isolated from various Pseudomonas syringae pathovars; Julich M et al.; From seven different pathovars of Pseudomonas syringae representing various genetic subgroups, and one strain of Pseudomonas viridiflava the same pyoverdin siderophore (1) was isolated, probably identical with the pyoverdin whose amino acid composition (but not their sequence) had been reported before . 1 is the first pyoverdin where two of the ligands for Fe3+ are beta-hydroxy Asp units . Its remarkably high complexing constant for Fe3+ at pH 5 as compared with other pyoverdins offers a definite advantage in plant infection . The structure elucidation of 1 will be described and the taxonomical implications regarding pyoverdins with different structures ascribed previously to P . syringae strains will be discussed.

Z Naturforsch {C}, 2001 Sep-Oct, 56(9-10), 680 - 6
The pyoverdins of Pseudomonas sp . 96-312 and 96-318; Schlegel K et al.; The structures of the pyoverdins isolated from the Pseudomonas spp . 96-312 and 96-318 were elucidated by spectroscopic and degradation techniques . As observed before for Pseudomonas spp . producing pyoverdins with a C-terminal cyclopeptidic substructure, the two strains can recognize to some extent structurally different pyoverdins as long as they have also a similar cyclopeptidic C-terminus.

Cancer Res, 2001 Nov 15, 61(22), 8058 - 61
In situ expression of interleukin-4 (IL-4) receptors in human brain tumors and cytotoxicity of a recombinant IL-4 cytotoxin in primary glioblastoma cell cultures; Joshi BH et al.; We have reported that human malignant glioma cell lines express high levels of plasma membrane interleukin-4 receptors (IL-4R) . We have also reported that biopsy/surgical samples or primary explant cell cultures from brain tumors express mRNA and protein for the IL-4Ralpha chain, a primary IL-4-binding protein . However, whether IL-4R are expressed in brain tumors in situ has not been resolved . In addition, expression of IL-4R on the cell surface of various normal brain tissues is not known . We examined the expression of IL-4R by using a monoclonal antibody to the IL-4Ralpha chain (also known as IL-4R beta) in surgical/biopsy samples of brain tumor tissues by immunohistochemistry . Our data indicate that 15 of 18 glioblastoma multiforme (GBMs) tumors obtained from two different institutions and 12 other brain tumor samples are moderately to intensely positive for IL-4Ralpha . In contrast, although IL-4Ralpha mRNA was expressed, no IL-4R protein was detectable in two adult and one pediatric brain tissue specimens . In addition, a commercially available human neural tissue grid containing fixed tissues from various areas of brain showed no positive staining for the IL-4Ralpha chain . IL-4Ralpha expression was also demonstrated on astrocytoma grades I, II, and III . Because IL-4 cytotoxin comprised of a circularly permutated IL-4 and a mutated form of Pseudomonas exotoxin {IL4(38-37)-PE38KDEL} is cytotoxic to IL-4R-expressing cells, we tested whether primary GBM explant cell cultures are sensitive to IL-4 cytotoxin . Our data indicate that 13 of 15 GBM cell cultures were 25-74 times more sensitive to IL-4 cytotoxin compared with normal human astrocytes or the NT2 neuronal cell line . These observations indicate that human brain tumors in situ overexpress IL-4R compared with normal brain tissues, thus confirming our previous conclusions that IL-4R in brain tumors may serve as an attractive target for anticancer therapy.

Int J Biol Macromol, 2001 Dec 10, 29(4-5), 295 - 301
Direct biosynthesis of poly(3-hydroxyalkanoates) bearing epoxide groups; Imamura T et al.; The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas cichorii YN2 cultured with C6-C12 1-alkenes was studied . PHAs containing repeating units with terminal epoxide groups were obtained when C7-C12 1-alkenes were fed separately as the only carbon source, but no epoxidized unit was detected when 1-hexene was fed . The content of epoxidized units in the polymers was in the range of 4-20 mol%, which was not dependent on the C atom length of the 1-alkene used as a substrate . The polymers produced undergo a glass transition at around -40 degrees C, and number average molecular weights were in the range of 150000-200000 as determined by GPC relative to polystyrene, with M(w)/M(n) ratios of 1.9-2.5 . As an intermediate, the corresponding 1,2-epoxyalkane was found in the culture medium . According to this result, the epoxidation of the 1-alkene is the initial step in the synthetic pathway of the epoxy unit in the polymer.

Curr Opin Investig Drugs, 2001 Sep, 2(9), 1309 - 13
hIL-13-PE38QQR . NeoPharm; Barth S; NeoPharm, under license from the NIH and the FDA, is developing a chimeric human IL-13 fused in frame to a genetically engineered truncated Pseudomonas exotoxin (PE38QQR) molecule, for its potential as an antitumor agent {266296}, {281418}, {290480} . NeoPharm filed an IND in 1999 for renal cell carcinoma (RCC) and glioma {319690}, {325001}; an additional IND was filed in March 2000 for the treatment of glioblastoma . In December 2000, NeoPharm initiated phase I/II trials of IL-13-PE38QQR involving patients with refractory glioblastoma multiforme . This trial was being conducted by the New Approaches to Brain Tumor Therapy, a research consortium sponsored by the NCI . At that time, the first patient with brain cancer had completed treatment with IL-13-PE38QQR {393197} . In October 1999, NeoPharm initiated phase I trials of hIL-13-PE38QQR for the treatment of patients with RCC {343878} . In February 2000, Dirks & Co estimated the potential US market for hIL-13-PE38QQR to be $5.8 billion {414515}.

Curr Opin Investig Drugs, 2001 Sep, 2(9), 1282 - 93
Chimeric fusion proteins--Pseudomonas exotoxin-based; Kreitman RJ; Recombinant fusion toxins have several potential advantages over conventional immunotoxin chemical conjugates, including (i) a defined toxin-ligand junction; (ii) efficient and relatively inexpensive production in large scale from bacteria; (iii) shorter plasma half-lives which might avoid diffuse endothelial damage which leads to vascular leak syndrome (VLS); and (iv) ability to genetically engineer mutations in the recombinant toxin to increase its potency or lower its non-specific toxicity . Two major varieties of recombinant fusion toxins include growth factor fusion toxins, containing a growth factor fused to truncated toxin, and recombinant immunotoxins, containing the variable fragments of an antibody fused to truncated toxin . In either case the ligand is used to bind selectively to tumor cells while the toxin kills the target cell following internalization . The bacterial toxins Pseudomonas exotoxin and diphtheria toxin are most often used for making recombinant fusion toxins . This review will focus on several agents containing truncated Pseudomonas exotoxin, which are undergoing preclinical and clinical development.

Arch Biochem Biophys, 2001 Dec 1, 396(1), 106 - 10
Receptor-associated protein binding blocks ubiquitinylation of the low density lipoprotein receptor-related protein; Misra UK et al.; The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M(r) approximately 515,000 and 85,000 . LRP is a receptor for activated alpha2-macrogobulin (alpha2M*), Pseudomonas exotoxin A, and many other proteins . We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or alpha2M* bound to LRP on macrophages . Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor . Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either alpha2M* or Pseudomonas exotoxin A . By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur . While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone . The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands . (c)2001 Elsevier Science.

J Immunol, 2001 Dec 1, 167(11), 6583 - 92
IL-13 fusion cytotoxin ameliorates chronic fungal-induced allergic airway disease in mice; Blease K et al.; IL-13 has emerged as a major contributor to allergic and asthmatic responses, and as such it represents an attractive target in these diseases . In this study, IL-13-responsive cells in the lung were targeted via the intranasal administration of IL-13-PE38QQR (IL-13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, to Aspergillus fumigatus-sensitized mice challenged with A . fumigatus spores, or conidia . Mice received 50, 100, or 200 ng of IL-13-PE or diluent alone (i.e., control group) on alternate days from day 14 to day 28 after the conidia challenge . The control group of mice exhibited significant airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis at day 28 after conidia . Although the two lower doses of IL-13-PE had limited therapeutic effects in mice with fungal-induced allergic airway disease, the highest dose of IL-13-PE tested significantly reduced all features of airway disease compared with the control group . Whole lung mRNA expression of IL-4Ralpha and IL-13Ralpha1 was markedly reduced, whereas bronchoalveolar lavage and whole lung levels of IFN-gamma were significantly elevated in mice treated with 200 ng of IL-13-PE compared with the control group . This study demonstrates that a therapy designed to target IL-13-responsive cells in the lung ameliorates established fungal-induced allergic airway disease in mice.

Phytochemistry, 1998 Nov 20, 49(6), 1579 - 1583
Chlorosis-inducing products from Pseudomonas syringae pathovars: new N-coronafacoyl compounds; Mitchell RE et al.; Liquid cultures of Pseudomonas syringae pv . tomato strains that also produced the phytotoxin coronatine, were found to have a new chlorosis-inducing activity, not previously described . Bioassay-guided fractionation and HPLC analysis revealed two new peaks that were chlorosis-inducing on leaves of bean plants . Mass spectrometry and NMR analyses of the compounds led to the derivation of their structures as coronafacoyl-L-serine and coronafacoyl-L-threonine, respectively . The amino acid C-2 configurations were determined by GC analysis following hydrolysis with 6M HCl . Both compounds have more polar and more acidic properties than coronatine, norcoronatine, and other known coronafacoyl conjugates with non-polar amino acids . Investigation of Pseudomonas syringae pv . glycinea strains that are known to be prolific producers of the coronatine family of compounds revealed the production of coronafacoylserine and coronafacoylthreonine also.

Biomacromolecules, 2000 Winter, 1(4), 713 - 20
Biodegradable poly(ethylene succinate) (PES) . 2 . Crystal morphology of melt-crystallized ultrathin film and its change after enzymatic degradation; Gan Z et al.; Poly(ethylene succinate) (PES) ultrathin films with an initial thickness of approximately 100 nm were prepared by the solution cast method on either cover glass or freshly cleaved mica as the substrate . The ultrathin films were then melt-crystallized at a given temperature for a certain period of time . The surface morphologies of these films on the substrates were observed by an atomic force microscope (AFM) and an optical microscope (OM) under ambient conditions . Two different crystal morphologies having fibril-like structure and flat-on lamellar crystals with a certain width were formed, and their growth mechanisms were discussed in association with previous kinetic data . It has been shown that at a higher crystallization temperature such as 85 degrees C (smaller degree of undercooling) the crystal aggregates tend to form lozenge-shaped hedrites which evolved from a single crystal . The enzymatic degradation of PES crystals on the ultrathin films was carried out by using a PHB depolymerase from Pseudomonas stutzeri at room temperature . The crystal morphologies before and after enzymatic degradation were examined by AFM . The lamellar crystals were hydrolyzed into many small fragments, and these fragments had the same thickness as that of the lamellar crystals before enzymatic degradation . The analysis of morphological results for PES lamellar crystals has revealed the existence of many defects on the surface of melt-crystallized lamellar crystals . These defects were preferentially attacked by the enzyme molecules . Hydrolysis starts from the chains folding in crystal defect area and proceeds along the lateral edges, i.e., along the direction perpendicular to the folding chain.

Biomacromolecules, 2000 Fall, 1(3), 350 - 9
Selective enzymatic degradations of poly(L-lactide) and poly(epsilon-caprolactone) blend films; Liu L et al.; Solution cast films were prepared from poly(L-lactide) (PLLA) and poly(epsilon-caprolactone) (PCL) as well as from three blends, namely B75, B50, and B25 with PLLA/PCL proportions of 75/25, 50/50, and 25/75, respectively . The enzymatic degradation of square samples (10 x 10 x 0.2 mm) cut from the films was investigated at 37 degrees C in a pH = 8.6 Tris buffer containing proteinase K or in a pH = 7.0 phosphate buffer containing Pseudomonas lipase . It was confirmed that proteinase K can degrade amorphous domains of PLLA, but cannot degrade crystalline PLLA or PCL . In contrast, Pseudomonas lipase can degrade both amorphous and crystalline PCL but cannot degrade PLLA . The two faces of solution cast films showed different morphologies due to the solvent evaporation process . The lower face appeared more crystalline than the upper face because of the plasticizing effect of solvent entrapped inside which allowed crystallization to proceed . Therefore, the lower face was more resistant to enzymatic attack by proteinase K in the cases of PLLA and the blends . The two polymers in the blends exhibited well separated crystalline domains . PCL seemed to constitute the continuous phase of the blends with formation of large size spherulites when the PCL content was over 50% . The selective degradation of PCL or PLLA components revealed the inner morphology of the blends where microspherelike or islandlike patterns were observed.

Biomacromolecules, 2000 Fall, 1(3), 335 - 8
Enzymatic synthesis of polyesters from lactones, dicarboxylic acid divinyl esters, and glycols through combination of ring-opening polymerization and polycondensation; Namekawa S et al.; Enzymatic copolymerization of lactones, divinyl esters, and glycols has been performed using lipase as catalyst to produce ester copolymers . The monomers used in this study were 12-, 13-, and 16-membered lactones, divinyl esters of adipic and sebasic acids, and alpha,omega-glycols . Candida antarctica and Pseudomonas cepacia lipases showed relatively high catalytic activity for the present copolymerization, yielding the copolymer having relatively high molecular weight in moderate yields . From 13C NMR analysis, the resulting product was not a mixture of homopolymers, but a copolymer derived from the monomers . NMR data and reaction monitoring results indicate that two different modes of polymerization, ring-opening polymerization and polycondensation, simultaneously take place through enzyme catalysis in one-pot to produce ester copolymers.

Biomacromolecules, 2000 Fall, 1(3), 320 - 4
Function of the catalytic domain of poly(3-hydroxybutyrate) depolymerase from Pseudomonas stutzeri; Hiraishi T et al.; The mechanism of enzymatic hydrolysis for (R)-3-hydroxybutyrate (3HB) oligomers with poly{(R)-3-hydroxybutyrate} {P(3HB)} depolymerase (PhaZpst) from Pseudomonas stutzeri was investigated by two deletion mutants lacking the substrate-binding domain and linker region, PhaZpst delta sbd and PhaZpstcore . The two deletion mutants had no ability for hydrolysis of water-insoluble P(3HB), while the hydrolysis activities of two deletion mutants for water-soluble 3HB oligomer and its derivatives (dimer, trimer, and tetramer) were identical with those of the wild type, indicating that the function of catalytic domain is independent of its substrate-binding domain and linker region . The hydrolyzed products analysis of 3HB oligomers by HPLC showed that the active site of catalytic domain recognizes at least two 3HB units for hydrolysis . The initial rates of hydrolysis of dimer derivative were lower by 2 orders of magnitude than those of trimer and tetramer derivatives, suggesting that 3HB oligomer derivatives larger than trimer are favorite substrates for PhaZpst.

Biomacromolecules, 2001 Fall, 2(3), 934 - 9
Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) copolymer from sugars by recombinant Ralstonia eutropha harboring the phaC1Ps and the phaGPs genes of Pseudomonas sp . 61-3; Matsumoto K et al.; Heterologous expression of the phaGPs and the phaClPs genes encoding 3-hydroxyacyl acyl carrier protein-coenzyme A transacylase and polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp . 61-3, respectively, was performed in the phbCRe negative mutant, Ralstonia eutropha PHB-4 . The recombinant strain of the R . eutropha PHB-4 produced PHA copolymers consisting of 3-hydroxybutyrate (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units of 6-12 carbon atoms from sugars . The 3HB fraction in copolymers was very high (95-97 mol%) . The PHA content in the recombinant strain could further be increased by the additional introduction of the phbABRe genes from R . eutropha encoding beta-ketothiolase and NADPH-depedent acetoacetyl-coenzyme A reductase . Differential scanning calorimetry analysis of the PHA copolymers produced by the recombinant R . eutropha PHB-4 have indicated that the PHA is a random copolymer of 3HB and mcl-3HA units.

Biomacromolecules, 2000 Spring, 1(1), 17 - 22
Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant strains of Pseudomonas sp . 61-3; Matsusaki H et al.; Pseudomonas sp . 61-3 (phbC::tet) strain, which is a phbCPs-disrupted mutant, accumulated a random copolymer consisting of (R)-3-hydroxybutyrate (3HB) and (R)-medium-chain-length 3-hydroxyalkanoate (3HA) units of 6-12 carbon atoms, but the 3HB fraction in the copolymer was less than 50 mol %, resulting in the formation of an amorphus polymer . Therefore, the genes encoding beta-ketothiolase (PhbARe) and NADPH-dependent acetoacetyl-CoA reductase (PhbBRe) from Ralstonia eutropha were expressed under the control of promoters for Pseudomonas sp . 61-3 pha locus or R . eutropha phb operon together with the phaC1Ps gene (PHA synthase 1 gene) from Pseudomonas sp . 61-3 in the phbCPs-disrupted mutant . The recombinant strains synthesized P(3HB-co-3HA) copolymers with very high 3HB compositions (up to 94 mol %) from glucose . The number-average molecular weights of P(3HB-co-3HA) were in the range of 349 x 10(3) to 605 x 10(3) . The structure and physical properties of P(3HB-co-3HA) copolymers were characterized by 1H- and 13C-NMR spectrometry, differential scanning calorimetry, and mechanical tensile measurement . P(94% 3HB-co-3HA) copolymer was demonstrated to have good physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene.

FEBS Lett, 2001 Nov 9, 508(1), 29 - 35
Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb(3)) purified from Pseudomonas stutzeri; Urbani A et al.; Cytochrome cbb(3) is a cytochrome c-oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases . We have developed a purification method yielding large amounts of pure cbb(3) complex from the soil bacterium Pseudomonas stutzeri . This cytochrome cbb(3) complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b-type and three c-type haems . The protein complex behaves as a monomer with an overall molecular weight of 114.0+/-8.9 kDa and a s(0)(20,w) value of 8.9+/-0.3 S as determined by analytical ultracentrifugation . Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1 x 0.1 x 0.3 mm . This is the first crystallisation report of a (cbb(3))-type oxidase.

Plant Physiol, 2001 Nov, 127(3), 1089 - 101
The role of NDR1 in avirulence gene-directed signaling and control of programmed cell death in Arabidopsis; Shapiro AD et al.; Arabidopsis plants containing the ndr1-1 mutation are incapable of mounting a hypersensitive response to bacteria carrying avrRpt2, but show an exaggerated cell death response to bacteria carrying avrB (Century et al., 1995) . We show here that ndr1-1 plants are severely impaired in induction of systemic acquired resistance and PR1-driven transcription of a reporter gene in response to Pseudomonas syringae strains carrying avrRpt2 but not in response to P . syringae carrying avrB . The ndr1-1 mutation also impaired salicylic acid (SA) accumulation in response to treatments that produced reactive oxygen species (ROS) and impaired induction of systemic acquired resistance in response to in situ production of ROS . Hydrogen peroxide accumulated in wild-type Arabidopsis leaves beginning 4 to 7 h postinoculation with P . syringae carrying either avrRpt2 or avrB . In ndr1-1 plants, P . syringae carrying avrRpt2 elicited no detectable hydrogen peroxide production . Hydrogen peroxide production in response to bacteria carrying avrB was similar to that of Columbia in kinetics but of lesser intensity at early time points . These data are interpreted to indicate that NDR1 links ROS generation to SA production and that the phenotypic consequences of the ndr1-1 mutation are caused by a reduced ability to accumulate SA upon pathogen infection.

J Biol Chem, 2002 Jan 25, 277(4), 2823 - 9 Epub 2001 Nov 08.
Precursor structure of cephalosporin acylase . Insights into autoproteolytic activation in a new N-terminal hydrolase family; Kim Y et al.; Autocatalytic proteolytic cleavage is a frequently observed post-translational modification in proteins . Cephalosporin acylase (CA) is a recently identified member of the N-terminal hydrolase family that is activated from an inactive precursor by autoproteolytic processing, generating a new N-terminal residue, which is either a Ser or a Thr . The N-terminal Ser or Thr becomes a nucleophilic catalytic center for intramolecular and intermolecular amide cleavages . The gene structure of the open reading frame of CAs generally consists of a signal peptide followed by the alpha-subunit, a spacer sequence, and the beta-subunit, which are all translated into a single polypeptide chain, the CA precursor . The precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and second by intermolecular cleavage . We solved the first CA precursor structure (code 1KEH) from a class I CA from Pseudomonas diminuta at a 2.5-A resolution that provides insight into the mechanism of intramolecular cleavage . A conserved water molecule, stabilized by four hydrogen bonds in unusual pseudotetrahedral geometry, plays a key role to assist the OG atom of Ser(1beta) to generate a strong nucleophile . In addition, the site of the secondary intermolecular cleavage of CA is proposed to be the carbonyl carbon of Gly(158alpha) (Kim, S., and Kim, Y., (2001) J . Biol . Chem., 276, 48376-48381), which is different from the situation in two other class I CAs.

Annu Rev Phytopathol, 1999, 37, 175 - 196
POLYKETIDE PRODUCTION BY PLANT-ASSOCIATED PSEUDOMONADS; Bender C et al.; Polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics . Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes; however, plant-associated pseudomonads also produce a variety of compounds via the polyketide pathway including the phytotoxin coronatine, the antibiotic mupirocin, and the antifungal compounds pyoluteorin and 2,4-diacetylphloroglucinol . This review focuses on the mode of action, regulation, biosynthesis, and genetics of these four compounds and the potential use of Pseudomonas-derived polyketide synthases in the generation of novel compounds with unique activities.

Chemosphere, 2001 Nov, 45(6-7), 927 - 34
Mineralization of aged atrazine and mecoprop in soil and aquifer chalk; Kristensen GB et al.; The effect of ageing on the bioavailability and sorption of the herbicides atrazine and mecoprop was studied in soil and aquifer chalk sampled at an agricultural field near Aalborg, Denmark . The herbicides were incubated in sterile soil or chalk up to 3 months prior to inoculation with 5 x 10(7) cells g(-1) (dry weight) of a mecoprop degrading highly enriched culture (PM) or 1 x 10(9) cells g(-1) (dry weight) of the atrazine degrading Pseudomonas sp . strain ADP . As a measure of the bioavailable residues accumulated 14CO2 was measured for 2 months . In both soil and chalk ageing limited the rate of atrazine mineralization, and in chalk the extent of mineralization was reduced as well . The fraction of sorbed atrazine in the soil ranged between 50% and 62%, whereas a maximum of 12% was sorbed in chalk . No impact on the mineralization of aged mecoprop was seen as no sorption of this herbicide on either soil or chalk was measured.

Curr Microbiol, 2001 Nov, 43(5), 374 - 7
The rpoS gene in Pseudomonas syringae is important in surviving exposure to the near-UV in sunlight; Miller CD et al.; The near-UV component of sunlight decreased culturability of the leaf epiphyte and plant pathogen Pseudomonas syringae . Exposure of the wild-type cells for 4 h to UV-A and UV-B in sunlight was ten fold more detrimental than exposure to sunlight with just UV-A . Sensitivity to UV-A especially increased in a mutant of P . syringae lacking the global regulatory sigma factor, RpoS . No RpoS-mutant cells were culturable after 4 h of exposure to near-UV sunlight . These findings suggest that both UV-A and UV-B wavelengths cause damage to the bacterial cell and that the RpoS protein regulates protective measures for the leaf-associated pseudomonad.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2001, 36(9), 1589 - 95
Enhancement of biodegradability of polychlorinated dibenzo-p-dioxins; Du X et al.; Enhancement of biodegradability of polychlorinated dibenzo-p-dioxins (PCDDs) was studied with new isolated bacterial strains from soil and oxic-sediments contaminated by PCDDs . The results indicated that mono- and di-chlorinated dibenzo-p-dioxins could be utilized as a sole carbon source and degraded by isolated bacterial strains, but tri-chlorinated dibenzo-p-dioxin (TrCDD) was hardly degraded . The biodegradability of TrCDD and tetra-chlorinated dibenzo-p-dioxin (TCDD) by the strain Pseudomonas sp . EE41, a new isolated one, could be enhanced through primary nutrient of co-metabolism of o-dichlorobenzene (o-DCB) . In this case, TrCDD (1.2mg/l for 3 weeks) was degraded by 33.1% and the degradation rate enhanced more than 2 fold; also TCDD (0.1 mg/l for 3 weeks) biodegraded by 37.8% . Most highly chlorinated, Penta-, Hexa-, Hepta-, and Octa-chlorinated, dibenzo-p-dioxins (P-CDD, H6, H7-CDD and OCDD) tested in this study could not be degraded while accumulated in bacterial cells.

Environ Monit Assess, 2001 Oct, 71(3), 243 - 54
Lead solubilization and accumulation by two strains of Pseudomonas obtained from a contaminated alfisol's effluent in southwestern Nigeria; Ekundayo EO et al.; Two strains of Pseudomonas species (B2 and D5) selected from an array of lead solubilizing and accumulating bacteria obtained from the effluent contaminated soil samples of a battery manufacturing factory were studied . Increase in pH between 4.0 and 6.0 favoured the growth of isolates: Peak log10 cfu mL(-1) values of 7.1, 7.5 and 8.5 were obtained at pH 4, 5 and 6, respectively . Cell bound lead concentrations for B2 (0.34 mg mL(-1)) and D5 (0.30 mg mL(-1)) obtained by direct contact with Pbs were greater than lead concentrations of 0.89 and 0.25 mg mL(-1) for B2 and D5, respectively, obtained in dialyzed cultures . These cell bound lead concentration in undialyzed cultures were also greater than lead concentrations of 0.03 and 0.07 mg mL(-1) for B2 and D5 in culture supernatants . Glucose addition did nor improve lead accumulation in the isolates . Exploitation of such isolates for the biotreatment of lead laden effluent was conducted.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1729 - 31 Epub 2001 Oct 25.
Crystallization and preliminary X-ray crystallographic analysis of native and selenomethionyl recombinant tabtoxin-resistance protein complexed with acetyl-coenzyme A; He H et al.; Tabtoxin-resistance protein (TTR), an acetyltransferase from Pseudomonas syringae pv . tabaci, was overexpressed in Eschericha coli M15 and the TTR fusion protein complexed with acetyl-coenzyme A (AcCoA) was purified and crystallized . Diffraction data were collected to 3.0 A resolution in-house and the crystal was found to belong to space group P2(1), with unit-cell parameters a = 47.6, b = 66.6, c = 53.5 A, beta = 104.3 degrees . Furthermore, a selenomethionine (SeMet) TTR fusion protein derivative was overexpressed in the same expression system and its complex with AcCoA was purified in a reductive environment . The SeMet TTR derivative crystallized in two forms: the first was identical to that observed for native crystals and the second belonged to space group C2, with unit-cell parameters a = 101.7, b = 45.6, c = 84.2 A, beta = 105.8 degrees . Data from the P2(1) crystal form were collected in-house to 2.3 A resolution . Subsequently, three different wavelength data sets of the C2 crystal form to 1.55 A resolution were collected at the Advanced Photon Source at Argonne National Laboratory.

Appl Environ Microbiol, 2001 Nov, 67(11), 5233 - 9
An altered Pseudomonas diversity is recovered from soil by using nutrient-poor Pseudomonas-selective soil extract media; Aagot N et al.; We designed five Pseudomonas-selective soil extract NAA media containing the selective properties of trimethoprim and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino Acids used in the classical Pseudomonas-selective Gould's S1 medium . All of the isolates were confirmed to be Pseudomonas by a Pseudomonas-specific OprF antibody and a Pseudomonas-specific PCR targeting 16S ribosomal DNA . The Pseudomonas isolates were characterized by classical physiological tests, repetitive extragenic palindromic-PCR, Fourier transform infrared spectroscopy, and carbon source utilization patterns . Several of these analyses showed that the amount of Casamino Acids significantly influenced the diversity of the recovered Pseudomonas isolates . Furthermore, the data suggested that specific Pseudomonas subpopulations were represented on the nutrient-poor media . The NAA 1:100 medium, containing ca . 15 mg of organic carbon per liter, consistently gave significantly higher Pseudomonas CFU counts than Gould's S1 when tested on four Danish soils . NAA 1:100 may, therefore, be a better medium than Gould's S1 for enumeration and isolation of Pseudomonas from the low-nutrient soil environment.

Appl Environ Microbiol, 2001 Nov, 67(11), 4992 - 8
Two distinct monooxygenases for alkane oxidation in Nocardioides sp . strain CF8; Hamamura N et al.; Alkane monooxygenases in Nocardioides sp . strain CF8 were examined at the physiological and genetic levels . Strain CF8 can utilize alkanes ranging in chain length from C(2) to C(16) . Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea . Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane . A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne . These results suggest the presence of two monooxygenases in strain CF8 . Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase from Pseudomonas oleovorans were used to clone a related gene from strain CF8 . Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C(6) . These results indicate the presence of two distinct monooxygenases for alkane oxidation in Nocardioides sp . strain CF8.

J Org Chem, 1999 Apr 16, 64(8), 2638 - 2647
Molecular Basis for Enantioselectivity of Lipase from Pseudomonas cepacia toward Primary Alcohols . Modeling, Kinetics, and Chemical Modification of Tyr29 to Increase or Decrease Enantioselectivity; Tuomi WV et al.; Lipase from Pseudomonas cepacia (PCL) shows good enantioselectivity toward primary alcohols . An empirical rule can predict which enantiomer of a primary alcohol reacts faster, but there is no reliable strategy to increase the enantioselectivity . We used a combination of molecular modeling of lipase-transition state analogue complexes and kinetic measurements to identify the molecular basis of the enantioselectivity toward two primary alcohols: 2-methyl-3-phenyl-1-propanol, 1, and 2-phenoxy-1-propanol, 2 . In hydrolysis of the acetate esters, PCL favors the (S)-enantiomer of both substrates (E = 16 and 17, respectively), but, due to changes in priorities of the substituents, the (S)-enantiomers of 1 and 2 have opposite shapes . Computer modeling of transition state analogues bound to PCL show that primary alcohols bind to PCL differently than secondary alcohols . Modeling and kinetics suggest that the enantioselectivity of PCL toward 1 comes from the binding of the methyl group at the stereocenter within a hydrophobic pocket for the fast-reacting enantiomer, but not for the slow-reacting enantiomer . On the other hand, the enantioselectivity toward 2 comes from an extra hydrogen bond between the phenoxy oxygen of the substrate to the phenolic OH of Tyr29 . This hydrogen bond may slow release of the (R)-alcohol and thus account for the reversal of enantioselectvity . To decrease the enantioselectivity of PCL toward 2-acetate by a factor of 2 to E = 8, we eliminated the hydrogen bond by acetylation of the tyrosyl residues with N-acetylimidazole . To increase the enantioselectivity of PCL toward 2-acetate by a factor of 2 to E = 36, we increased the strength of the hydrogen bond by nitration of the tyrosyl residues with tetranitromethane . This is one of the first examples of a rationally designed modification of a lipase to increase enantioselectivity.

J Org Chem, 1999 Jan 8, 64(1), 153 - 155
Oximidines I and II: Novel Antitumor Macrolides from Pseudomonas sp .
Kim JW, Shin-Ya K, Furihata K, Hayakawa Y, Seto H.
Our screening for cell-cycle inhibitors in transformed cells resulted in the isolation of novel active compounds, oximidines I (1) and II (2), from Pseudomonas sp . Q52002 . The molecular formulas of 1 and 2 were established as C(23)H(24)N(2)O(7) and C(23)H(24)N(2)O(6), respectively, by high-resolution FABMS . The structures of the oximidines were elucidated by NMR spectral analysis, including a variety of two-dimensional techniques . The absolute stereochemistry of 1 was determined by using the modified Mosher method . The oximidines are novel 12-membered macrolides containing an O-methyloxime moiety . Oximidines I and II selectively inhibited the growth of rat 3Y1 cells transformed with E1A, ras, or src oncogenes.

J Bacteriol, 2001 Nov, 183(22), 6694 - 8
The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide; Sugimoto M et al.; Several Sphingomonas spp . utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain . Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound . The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli . The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent . It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn . PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates . Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E . coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%).

J Org Chem, 1998 Sep 4, 63(18), 6128 - 6131
An Efficient Derivation of the Versatile Chiron Antipode 1-tert-Butyldimethylsilylpenta-1,4-diyn-3-ol: Application to the Synthesis of (15E,R,R)-Duryne; Sharma A et al.; The title chiron, 1-tert-butyldimethylsilylpenta-1,4-diyn-3-ol (5), an essential segment of various bioactive polyacetylenic alcohols, has been efficiently resolved via a lipase-catalyzed acylation strategy . Lipases from different Pseudomonas species and Candida rugosa (CRL) furnished its (S)-antipode (as esters) with 86-96% ee . However, the (R)-alcohol 5 could be prepared with acceptable enantiomeric purity (93% ee) only using CRL-vinyl acetate-diisopropyl ether as the reagent protocol and under double filtration conditions . The chiron alcohol was then extended to the target compound (15E,R,R)-duryne (I) by its pyranylation, alkylation with the required achiral C(20)-alpha,omega-dibromide 15, and suitable functionalization.

J Org Chem, 1997 Jan 10, 62(1), 103 - 108
Pholipeptin, a Novel Cyclic Lipoundecapeptide fromPseudomonas fluorescens; Ui H et al.; An inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC), pholipeptin (1), was purified from the culture broth of Pseudomonas sp . by solvent extraction and column chromatography . Acid hydrolysis of 1 gave Leu, Ile, Ser, Thr, and Asp moieties . Although 1 was a peptide compound, fragmentation by mild hydrolysis was not accomplished under any conditions . So, we performed the structure elucidation using various 2D NMR techniques . In the NMR studies, the addition of a small amount of trifluoroacetic acid gave relatively sharp and resolved signals, such that the structure of this novel cyclic lipodepsipeptide consisting of 11 amino acids and a 3-hydroxydecanoic acid moiety could be determined . Chirality of the constituent amino acids was analyzed by chiral HPLC, but two Asp residues could not be distinguished because they were contained as a racemic mixture . Finally, their chiralities were determined by NMR analysis of (13)C-labeled 1 into which {L-(13)C}Asp had been biosynthetically incorporated.

J Org Chem, 1996 Sep 6, 61(18), 6282 - 6288
Synthesis of Enantiomerically Pure Bis(hydroxymethyl)-Branched Cyclohexenyl and Cyclohexyl Purines as Potential Inhibitors of HIV; Rosenquist A A et al.; The synthesis of the enantiomerically pure bis(hydroxymethyl)-branched cyclohexenyl and cyclohexyl purines is described . Racemic trans-4,5-bis(methoxycarbonyl)cyclohexene {(+/-)-6} was reduced with lithium aluminum hydride to give the racemic diol (+/-)-7 . Resolution of (+/-)-7 via a transesterification process using lipase from Pseudomonas sp . (SAM-II) gave both diols in enantiomerically pure form . The enantiomerically pure diol (S,S)-7was benzoylated and epoxidized to give the epoxide 9 . Treatment of the epoxide 9 with trimethylsilyl trifluoromethanesulfonate and 1,5-diazabicyclo{5.4.0}undec-5-ene followed by dilute hydrochloric acid gave (1R,4S,5R)-4,5-bis{(benzoyloxy)methyl}-1-hydroxycyclohex-2-ene (10) . Acetylation of 10 gave (1R,4S,5R)-1-acetoxy-4,5-bis{(benzoyloxy)methyl}cyclohex-2-ene (11) . (1R,4S,5R)-1-Acetoxy-4,5-bis{(benzoyloxy)methyl}cyclohex-2-ene (11) was converted to the adenine derivative 12 and guanine derivative 13 via palladium(0)-catalyzed coupling with adenine and 2-amino-6-chloropurine, respectively . Hydrogenation of 12 and 13 gave the correspondning saturated adenine derivative 14 and guanine derivative 15 . (1R,4S,5R)-4,5-Bis{(benzoyloxy)methyl}-1-hydroxycyclohex-2-ene (10) was converted to the adenine derivative 16 and guanine derivative 17 via coupling with 6-chloropurine and 2-amino-6-chloropurine, respectively, using a modified Mitsunobu procedure . Hydrogenation of 16 and 17 gave the corresponding saturated adenine derivative 18 and guanine derivative 19 . Compounds 12-19 were evaluated for activity against human immunodeficiency virus (HIV), but were found to be inactive . Further biological testings are underway.

Crit Rev Immunol, 2001, 21(1-3), 299 - 310
Overexpressed cell surface interleukin-4 receptor molecules can be successfully targeted for antitumor cytotoxin therapy; Kawakami K et al.; A variety of human solid cancer cell lines and primary cell cultures has been reported to overexpress high-affinity receptors (R) for interleukin-4 (IL-4), a pleiotropic immunoregulatory cytokine . The significance of IL-4R expression is not known; however, IL-4 is able to upregulate adhesion molecules, inhibit cell proliferation, and mediate signal transduction in tumor cell lines . To target IL-4R, we produced a chimeric protein composed of a circular permuted IL-4 and a mutated form of Pseudomonas exotoxin {termed IL4(38-37)-PE38KDEL or cpIL4-PE} . The recombinant cpIL4-PE was highly cytotoxic to cancer cells, but not toxic to normal B cells, T cells, monocytes, and CD34+, even though these cells express detectable numbers of IL-4R . The cytotoxicity was specific because excess of recombinant IL-4 neutralized the cpIL4-PE effect . To further develop this molecule, in vivo antitumor activity was tested in animal models of human cancer . This agent showed remarkable antitumor activity in AIDS-Kaposi's sarcoma, glioblastoma multiforme, and breast cancer models in immunodeficient animals . cpIL4-PE caused partial or complete regression of established human tumors . Preclinical efficacy and toxicity studies provided a therapeutic window in which this cancer-targeted agent could be used . On the basis of these studies, we initiated a Phase I clinical trial for the treatment of recurrent glioblastoma multiforme . Our preliminary clinical results suggest that cpIL4-PE has antitumor activity against the deadliest form of brain tumors, without detectable toxicity to normal brain tissues . Thus, IL-4 receptors represent novel targets for cancer cytotoxin therapy.

Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6597 - 601
NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen; Century KS et al.; We have employed Arabidopsis thaliana as a model host plant to genetically dissect the molecular pathways leading to disease resistance . A . thaliana accession Col-0 is susceptible to the bacterial pathogen Pseudomonas syringae pv . tomato strain DC3000 but resistant in a race-specific manner to DC3000 carrying any one of the cloned avirulence genes avrB, avrRpm1, avrRpt2, and avrPph3 . Fast-neutron-mutagenized Col-0 M2 seed was screened to identify mutants susceptible to DC3000(avrB) . Disease assays and analysis of in planta bacterial growth identified one mutant, ndr1-1 (nonrace-specific disease resistance), that was susceptible to DC3000 expressing any one of the four avirulence genes tested . Interestingly, a hypersensitive-like response was still induced by several of the strains . The ndr1-1 mutation also rendered the plant susceptible to several avirulent isolates of the fungal pathogen Peronospora parasitica . Genetic analysis of ndr1-1 demonstrated that the mutation segregated as a single recessive locus, located on chromosome III . Characterization of the ndr1-1 mutation suggests that a common step exists in pathways of resistance to two unrelated pathogens.

Mol Plant Microbe Interact, 2001 Oct, 14(10), 1131 - 9
Activation of an EDS1-mediated R-gene pathway in the snc1 mutant leads to constitutive, NPR1-independent pathogen resistance; Li X et al.; The Arabidopsis NPR1 protein is an essential regulatory component of systemic acquired resistance (SAR) . Mutations in the NPR1 gene completely block the induction of SAR by signals such as salicylic acid (SA) . An Arabidopsis mutant, snc1 (suppressor of npr1-1, constitutive 1), was isolated in a screen for suppressors of npr1-1 . In the npr1-1 background, the snc1 mutation resulted in constitutive resistance to Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2 . High levels of SA were detected in the mutant and shown to be required for manifestation of the snc1 phenotype . The snc1 mutation was mapped to the RPP5 resistance (R) gene cluster and the eds1 mutation that blocks RPP5-mediated resistance suppressed snc1 . These data suggest that a RPP5-related resistance pathway is activated constitutively in snc1 . This pathway does not employ NPR1 but requires the signal molecule SA and the function of EDS1 . Moreover, in snc1, constitutive resistance is conferred in the absence of cell death, which is often associated with R-gene mediated resistance.

Int J Mol Med, 2001 Nov, 8(5), 579 - 84
An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines; Tur MK et al.; Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma . A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5 . By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA') . Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry . The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml . This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

Curr Opin Biotechnol, 2001 Oct, 12(5), 439 - 45
Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads; Schweizer HP; Improved tools for Pseudomonas research include small, broad-host-range vectors that allow regulated expression from the lac operon and T7 promoters whose biology is well understood and adaptable to many bacteria . To facilitate studies on gene regulation, tracking and monitoring of bacteria in diverse environments, and the construction of biosensors, various reporter genes with versatile assay formats have been developed that can be delivered on plasmid, transposon and integration-proficient vectors.

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 724 - 30
Characterization of the eugenol hydroxylase genes (ehyA/ehyB) from the new eugenol-degrading Pseudomonas sp . strain OPS1; Brandt K et al.; During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis . When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant . The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp . strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp . strain HR199 . EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp . strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P . putida . Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for an eugenol dehydrogenase of P fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB . Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase . The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.

Eur J Med Chem, 2001 Jul-Aug, 36(7-8), 691 - 5
Diesters of glycosylglycerols active in cancer chemoprevention; Colombo D et al.; Enzymatic transesterification, mediated by Pseudomonas cepacia lipase (lipase PS), led to the pure 1,6'-diacylderivatives of 2-O-beta-D-glucosyl-sn-glycerol and 2-O-beta-D-galactosyl-sn-glycerol, the acyl chains being derived from short-medium length fatty acids . A study of the in vitro inhibitory effects of these diacylderivatives on Epstein-Barr virus early antigen activation induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate revealed that maximum activity was reached for the hexanoyl chain and that the introduction of a second acyl chain did not significantly modify the inhibitory potential referring to the corresponding 1- or 6'-monoesters.

Plant Cell, 2001 Oct, 13(10), 2225 - 40
A humidity-sensitive Arabidopsis copine mutant exhibits precocious cell death and increased disease resistance; Jambunathan N et al.; The copines are a newly identified class of calcium-dependent, phospholipid binding proteins that are present in a wide range of organisms, including Paramecium, plants, Caenorhabditis elegans, mouse, and human . However, the biological functions of the copines are unknown . Here, we describe a humidity-sensitive copine mutant in Arabidopsis . Under nonpermissive, low-humidity conditions, the cpn1-1 mutant displayed aberrant regulation of cell death that included a lesion mimic phenotype and an accelerated hypersensitive response (HR) . However, the HR in cpn1-1 showed no increase in sensitivity to low pathogen titers . Low-humidity-grown cpn1-1 mutants also exhibited morphological abnormalities, increased resistance to virulent strains of Pseudomonas syringae and Peronospora parasitica, and constitutive expression of pathogenesis-related (PR) genes . Growth of cpn1-1 under permissive, high-humidity conditions abolished the increased disease resistance, lesion mimic, and morphological mutant phenotypes but only partially alleviated the accelerated HR and constitutive PR gene expression phenotypes . The disease resistance phenotype of cpn1-1 suggests that the CPN1 gene regulates defense responses . Alternatively, the primary function of CPN1 may be the regulation of plant responses to low humidity, and the effect of the cpn1-1 mutation on disease resistance may be indirect.

Biotechnol Appl Biochem, 2001 Oct, 34(Pt 2), 81 - 4
Enhanced thermal stability of an alkaline protease, AprP, isolated from a Pseudomonas sp . by mutation at an autoproteolysis site, Ser-331; Jang JW et al.; The thermal stability of the alkaline protease extracellular subtilisin-type serine protease (AprP) from Pseudomonas sp . KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site . N-terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser-307 and Ser-331 . To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate . The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not . The half-lives of the S331D mutant at 55 degrees C and 60 degrees C were 1.5 and 2.4 times longer than that of the wild-type enzyme, respectively . In addition, the catalytic efficiency was also enhanced.

Int J Biol Macromol, 2001 Oct 22, 29(3), 145 - 50
Biosynthesis of polyhydroxyalkanoate copolyester containing cyclohexyl groups by Pseudomonas oleovorans; Kim DY et al.; Production of polyhydroxyalkanoates (PHAs) substituted with cyclohexyl groups by Pseudomonas oleovorans grown with 4-cyclohexylbutyric acid (4-CHB) and its mixtures with nonanoic acid (NA) was investigated . Addition of NA to medium gave rise to an increase in the total concentration of 3-hydroxy-4-cyclohexylbutyrate repeating unit in the PHAs, indicating that the bioconversion rate of 4-CHB to polyester was significantly improved by the cometabolic effect . Increasing the proportion of NA from 1.0 to 7.5 mM at a concentration of 10 mM total carbon substrate also accelerated the uptake speed of 4-CHB by the organism and resulted in an increase of the ratio of 3-hydroxynonanoate to 3-hydroxyheptanoate from 1.28 to 2.05 . Differential scanning calorimetric analysis of the PHAs bearing the corresponding functional groups showed one melting transition and one glass transition temperature varying according to the composition . These results indicated that random copolyesters were obtained from the carbon substrates used in this study.

Cell Immunol, 2001 Jul 10, 211(1), 37 - 42
Suppression of an IL-13 autocrine growth loop in a human Hodgkin/Reed-Sternberg tumor cell line by a novel IL-13 antagonist; Oshima Y et al.; IL-13 has been proposed to be an autocrine growth factor for Hodgkin/Reed-Sternberg tumor cells (H/RS cells) . Since we have recently identified and produced a novel IL-13 antagonist (IL-13E13K) that can suppress the biological activity of IL-13, here we examined whether IL-13E13K can inhibit growth of Hodgkin lymphoma (HL)-derived cell lines . IL-13E13K not only inhibited the growth of an unstimulated H/RS cell line (L1236) but also cells that were stimulated by exogenous IL-13 in a dose-dependent manner . Several HL-derived cell lines expressed IL-13 message and protein and message for various chains of IL-13R . H/RS cell lines expressed mRNA for the IL-13R alpha 1, IL-4R alpha, and IL-2R gamma chains . However, none of these cell lines expressed the IL-13R alpha 2 chain . An H/RS cell line (L1236) internalized the ligand-receptor complex after binding to a fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin A (IL-13-PE38QQR, or IL-13 cytotoxin), as IL-13 cytotoxin was specifically cytotoxic to H/RS cells in vitro . These results indicate that IL-13E13K and IL-13 cytotoxin can effectively suppress growth of a L1236 H/RS cell line . Therefore, additional studies should be performed to determine the expression of IL-13 and IL-13R in primary clinical samples of Hodgkin's lymphoma and both agents should be further tested in vitro and in vivo as possible therapeutic agents for HL.

Environ Microbiol, 2001 Aug, 3(8), 512 - 24
Measuring mass transfer processes of octane with the help of an alkSalkB::gfp-tagged Escherichia coli; Jaspers MC et al.; Diffusion of octane from oily droplets in different microscale settings was measured using Escherichia coli expressing the stable green fluorescent protein (GFP) from the alkB promoter of Pseudomonas oleovorans . GFP fluorescence intensities were determined quantitatively at the single-cell level after 1.0 or 2.5 h incubation and compared with different calibration series using known concentrations of octane . By immobilizing the E . coli sensor cells on the bottom glass plate of a microscope flow chamber, it was possible to monitor the diffusion process for octane in aqueous solution as a function of time and distance from non-aqueous phase droplets of octane alone or oily octane mixtures . When a gas phase was included in the flow chambers, octane transport could be demonstrated from the oily mixtures to the cells through both gas and liquid phase . Assays of non-immobilized sensor cells in microdroplets in the presence or absence of soil particles incubated with octane through the vapour phase revealed a slight reduction in the total amount of induced E . coli cells in the presence of soil . Our results indicate the power of using GFP-marked single-cell biosensors in determining microscale bioavailability of organic pollutants.

Microbiol Res, 2001, 156(2), 191 - 4
Suppression of damping-off in maize seedlings by Pseudomonas corrugata; Pandey A et al.; Two strains of Pseudomonas corrugata, (1 and 7), isolated from subtropical and temperate soils in Sikkim Himalaya, respectively, were subjected to Petri-dish as well as plant-based bioassay to examine their potential for disease suppression against three major pathogens of maize . A mixture of Pythium ultimum, P . arrhenomanes and Fusarium graminearum was introduced in the soil; maize seed inoculated with one of the two strains of Pseudomonas corrugata (1 or 7) were sown in pots containing such soil . The bacterial inoculation resulted in significant disease suppression as well as growth promotion of seedlings . The bacterial strains were also evaluated for their intrinsic antibiotic resistance against a range of concentrations of ten antibiotics . While the bacteria were found to be sensitive to gentamycin and rifampicin, they exhibited resistance against ampicillin, carbenicillin and penicillin, even at high concentrations.

Microbiol Res, 2001, 156(2), 151 - 8
Isolation of a benzoate-utilizing Pseudomonas strain from soil and production of catechol from benzoate by transpositional mutants; Wang CL et al.; Pseudomonas sp . Ba-0511 was isolated from soil by enrichment cultivation on a medium containing 6 mg/ml of sodium benzoate . The bacterium could grow on a medium containing 20 mg/ml of sodium benzoate by a successive enrichment culture . One hundred and twelve transpositional mutants of the bacterium produced catechol from benzoate and accumulated it outside of the cells . Among the mutants, strain BA+63 produced a maximal amount of catechol (2.3 mg/ml) from 6 mg/ml of sodium benzoate after growing for 10.5 h . The conversion rate of benzoate to catechol was 50% on a molar basis . The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 6 mg/ml of sodium benzoate reached 3.3 mg/ml at the conversion rate of 72% after 5 h of incubation . The resting cells converted m-methylbenzoic acid to 3- and 4-methylcatechol and m-chlorobenzoic acid to 3- and 4-chlorocatechol.

J Bacteriol, 2001 Oct, 183(20), 5937 - 41
Lipase and its modulator from Pseudomonas sp . strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator; Kim EK et al.; A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp . strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli . The limK gene is located immediately downstream of the lipK gene . Enzymatically active lipase was produced only in the presence of the limK gene . The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs) . The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp . strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species . Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK . When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK . These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding . We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Mol Cells, 2001 Aug 31, 12(1), 25 - 31
Lignification induced by pseudomonads harboring avirulent genes on Arabidopsis; Lee S et al.; The responses of Arabidopsis thaliana ecotypes to the bacterial pathogen Pseudomonas syringae pv . maculicola 4326 (Psm4326) harboring cloned avirulence genes avrB and avrRpt2 from P . syringae pv . glycinea were examined . Psm4326 containing avirulent genes, avrB and avrRpt2 induced lignification and peroxidase activities in the bacteria infiltrated leaves of Col-O only and not in Mt-O, Bla-2 and Po-1 . However, Arabidopsis ecotypes infiltrated with Psm4326 harboring with and without avirulent genes all showed differential induction of mRNA for peroxidase gene and lignin accumulation up to 24 h after infiltration . Only avrB gene in Col-O showed strong corelationship between peroxidase mRNA expression as well as lignification gradually up to 36 h after infiltration . These results extend previous observations that avirulence genes from pathogens of one host plant can be recognized by non-host plants and provide the genetic framework for analysis of the plant-specific response to the bacterial avirulent gene products in A . thaliana.

Lett Appl Microbiol, 2001 Oct, 33(4), 280 - 4
Biosurfactant-rhamnolipid effects on yeast cells; Vasileva-Tonkova E et al.; AIM: The aim of this work was to study the effect of the novel surfactant PS from Pseudomonas sp . S-17 on Saccharomyces cerevisiae 83-20 yeast cells and to compare it with the effect of the well known surfactant Triton X-100 . METHODS AND RESULTS: The effect of surfactants was investigated on the cells during growth, and on the separated cells . The cell-permeabilizing effect of surfactants was studied by following the release of protein and some enzyme activities . The biosurfactant did not affect the culture growth kinetics, and altered the polypeptide profiles of cells and membrane proteins in the same way as Triton X-100 . CONCLUSION: Results of this study demonstrate that biosurfactant PS and Triton X-100 have a similar type of action, mainly surface located, and that they do not affect the intracellular structures of yeast cells . SIGNIFICANCE AND IMPACT OF THE STUDY: A novel surfactant PS was isolated from Pseudomonas sp . S-17 . A mild effect of PS on yeast cells was demonstrated . The results indicate the ecological safety of the biosurfactant and its potential use in the development of environmentally-benign and efficient cleaning technologies.

J Appl Microbiol, 2001 Sep, 91(3), 412 - 20
Genetic characterization of Pseudomonas 'NZI7'--a novel pathogen that results in a brown blotch disease of Agaricus bisporus; Godfrey SA et al.; AIMS: To characterize a novel pseudomonad isolate capable of causing brown blotch disease of Agaricus bisporus . METHODS AND RESULTS: Using the white-line-in-agar (WLA) assay, fluorescent pseudomonads isolated from a New Zealand mushroom farm were screened for the lipodepsipeptide tolaasin, a characteristic marker of Pseudomonas tolaasii . One isolate, NZI7, produced a positive WLA assay and caused brown lesions of A . bisporus comparable with those produced by Ps . tolaasii . However, genetic analysis suggested that Ps . tolaasii and NZI7 were genetically dissimilar, and that NZI7 is closely related to Pseudomonas syringae . Nucleotide sequence analyses of a gene involved in tolaasin production indicated that similar genes are present in both NZI7 and Ps . tolaasii . CONCLUSION: NZI7 represents a novel Pseudomonas species capable of causing brown blotch disease of A . bisporus . SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic identification of Ps . tolaasii based on A . bisporus browning and positive WLA may have limited specificity.

Acta Med Port, 2001 May-Jun, 14(3), 285 - 91
{Transient neutropenia in previously healthy children}; Martins P et al.; Transient neutropenia in otherwise healthy children is not unusual, the main causes being infections and drugs . The risk of developing an infectious complication in previously healthy children with transient neutropenia is low . These infections are usually superficial and caused by common bacteria . We present two cases of previously healthy children with Pseudomonas sepsis who presented with severe neutropenia, not submitted to recent hospitalization or invasive therapeutic or monitoring proceedings . We discuss the etiology of transitory neutropenia and the reasons for this severe community-acquired infection, caused by an unusual agent . The authors also mention their therapeutic attitudes for previously healthy children with transient neutropenia . They should be guided by clinical presentation and neutropenia severity.

Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 545 - 9
Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-xylene monooxygenase activity in Pseudomonas stutzeri OX1; Ryoo D et al.; Pseudomonas stutzeri OX1 naphthalene-oxidation activity is induced 3.0-fold by tetrachloroethylene (PCE) and 3.1-fold by trichloroethylene (TCE) at 100 microM . With the mutant P . stutzeri M1, which does not express toluene-o-xylene monooxygenase (ToMO, product of the tou operon), no naphthalene-oxidation activity induction by PCE and TCE was found; hence, PCE and TCE induce ToMO of P . stutzeri OX1 . The chlorinated phenols 2-, 3-, and 4-chlorophenol induced ToMO expression 0.58-, 0.23- and 0.37-fold, respectively, compared to the direct inducer of the pathway, o-cresol . Using P . putida PaW340 (pPP4062, pFP3028), which has the tou promoter fused to the reporter catechol-2,3-dioxygenase, and the regulator gene touR, it was determined that the tou promoter was induced directly 5.7-, 7.1-, and 5.1-fold for 2-, 3-, and 4-chlorophenol, respectively (compared to an 8.8-fold induction with o-cresol) . In addition, it was found that TCE and PCE do not directly induce the tou pathway and that components other than the tou structural and regulatory genes are necessary for induction . Gas chromatography results also showed that 100 microM TCE induced its own degradation (8-9%) in 16 h in P . stutzeri OX1, and all of the stoichiometric chloride from the degraded TCE was detected in solution.

J Bacteriol, 2001 Oct, 183(19), 5589 - 98
Enhancer-binding proteins HrpR and HrpS interact to regulate hrp-encoded type III protein secretion in Pseudomonas syringae strains; Hutcheson SW et al.; In Pseudomonas syringae strains, the hrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis . HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established . Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity . hrpR and hrpS were shown to be expressed as an operon: a promoter was identified 5' to hrpR, and reverse transcriptase PCR detected the presence of an hrpRS transcript . The hrpR promoter and coding sequence were conserved among P . syringae strains . The coding sequences for hrpR and hrpS were cloned into compatible expression vectors, and their activities were monitored in Escherichia coli transformants carrying an hrpL'-lacZ fusion . HrpS could function as a weak activator of the hrpL promoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain . This finding is consistent with a requirement for both HrpR and HrpS in the activation of the hrpL promoter . By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex . Physical interaction of HrpR and HrpS was confirmed by column-binding experiments . The results show that HrpR and HrpS physically interact to regulate the sigma(54)-dependent hrpL promoter in P . syringae strains.

Biol Sci Space, 1997 Dec, 11(4), 339 - 45
Protein crystallization in microgravity; Aibara S et al.; A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992 . The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization . Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp . F-126, were obtained as single crystals of good diffraction quality . Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR . Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

HortScience, 1997 Feb, 32(1), 96 - 100
Spectral quality affects disease development of three pathogens on hydroponically grown plants; Schuerger AC et al.; Plants were grown under light-emitting diode (LED) arrays with various spectra to determine the effects of light quality on the development of diseases caused by tomato mosaic virus (ToMV) on pepper (Capsicum annuum L.), powdery mildew {Sphaerotheca fuliginea (Schlectend:Fr.) Pollaci} on cucumber (Cucumis sativus L.), and bacterial wilt (Pseudomonas solanacearum Smith) on tomato (Lycopersicon esculentum Mill.) . One LED (660) array supplied 99% red light at 660 nm (25 nm bandwidth at half-peak height) and 1% far-red light between 700 to 800 nm . A second LED (660/735) array supplied 83% red light at 660 nm and 17% far-red light at 735 nm (25 nm bandwidth at half-peak height) . A third LED (660/BF) array supplied 98% red light at 660 nm, 1% blue light (BF) between 350 to 550 nm, and 1% far-red light between 700 to 800 nm . Control plants were grown under broad-spectrum metal halide (MH) lamps . Plants were grown at a mean photon flux (300 to 800 nm) of 330 micromoles m-2 s-1 under a 12-h day/night photoperiod . Spectral quality affected each pathosystem differently . In the ToMV/pepper pathosystem, disease symptoms developed slower and were less severe in plants grown under light sources that contained blue and UV-A wavelengths (MH and 660/BF treatments) compared to plants grown under light sources that lacked blue and UV-A wavelengths (660 and 660/735 LED arrays) . In contrast, the number of colonies per leaf was highest and the mean colony diameters of S . fuliginea on cucumber plants were largest on leaves grown under the MH lamp (highest amount of blue and UV-A light) and least on leaves grown under the 660 LED array (no blue or UV-A light) . The addition of far-red irradiation to the primary light source in the 660/735 LED array increased the colony counts per leaf in the S . fuliginea/cucumber pathosystem compared to the red-only (660) LED array . In the P . solanacearum/tomato pathosystem, disease symptoms were less severe in plants grown under the 660 LED array, but the effects of spectral quality on disease development when other wavelengths were included in the light source (MH-, 660/BF-, and 660/735-grown plants) were equivocal . These results demonstrate that spectral quality may be useful as a component of an integrated pest management program for future space-based controlled ecological life support systems.

Biochem J, 2001 Sep 15, 358(Pt 3), 607 - 14
The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity; Beylot MH et al.; To investigate the mechanism by which Pseudomonas cellulosa releases arabinose from polysaccharides and oligosaccharides, a gene library of P . cellulosa genomic DNA was screened for 4-methylumbelliferyl-alpha-L-arabinofuranosidase (MUAase) activity . A single MUAase gene (abf51A) was isolated, which encoded a non-modular glycoside hydrolase family (GH) 51 arabinofuranosidase (Abf51A) of 57000 Da . The substrate specificity of the Abf51A showed that it preferentially removed alpha1,2- and alpha1,3-linked arabinofuranose side chains from either arabinan or arabinoxylan, and hydrolysed alpha1,5-linked arabino-oligosaccharides, although at a much lower rate . The activity of Abf51A against arabinoxylan was similar to a GH62 arabinofuranosidase encoded by a P . cellulosa gene . Glu-194 and Glu-321 of Abf51A are conserved in GH51 enzymes, and it has been suggested that these amino acids comprise the key catalytic acid/base and nucleophile residues, respectively . To evaluate this hypothesis the biochemical properties of E194A and E321A mutants of Abf51A were evaluated . The data were consistent with the view that Glu-194 and Glu-321 comprise the key catalytic residues of Abf51A . These data, in conjunction with the results presented in the accompanying paper {Beylot, Emami, McKie, Gilbert and Pell (2001) Biochem . J . 358, 599-605}, indicate that P . cellulosa expresses a membrane-bound GH51 arabinofuranosidase that plays a pivotal role in releasing arabinose from a range of polysaccharides and oligosaccharides.

Biochem J, 2001 Sep 15, 358(Pt 3), 599 - 605
Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase; Beylot MH et al.; In the accompanying paper {Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem . J . 358, 607-614} the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively . In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides . Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides . Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P . cellulosa . To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P . cellulosa (Deltaabf51A) . The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium . Arabinofuranosidase activity in Deltaabf51A against aryl-alpha-arabinofuranosides, arabinan and alpha1,5-linked arabino-oligosaccharides was approx . 1% of the wild-type bacterium . The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P . cellulosa . These data indicate that P . cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.

Plant J, 2001 Aug, 27(3), 203 - 11
The Arabidopsis aberrant growth and death2 mutant shows resistance to Pseudomonas syringae and reveals a role for NPR1 in suppressing hypersensitive cell death; Rate DN et al.; A novel Arabidopsis mutant has been identified with constitutive expression of GST1-GUS using plants with a pathogen-responsive reporter transgene containing the beta-glucuronidase (GUS) coding region driven by the GST1 promoter . The recessive mutant, called agd2 (aberrant growth and death2), has salicylic acid (SA)-dependent increased resistance to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae, elevated SA levels, a low level of spontaneous cell death, callose deposition, and enlarged cells in leaves . The enhanced resistance of agd2 to virulent P . syringae requires the SA signaling component NONEXPRESSOR OF PR1 (NPR1) . However, agd2 renders the resistance response to P . syringae carrying avrRpt2 NPR1-independent . Thus agd2 affects both an SA- and NPR1-dependent general defense pathway and an SA-dependent, NPR1-independent pathway that is active during the recognition of avirulent P . syringae . agd2 plants also fail to show a hypersensitive cell death response (HR) unless NPR1 is removed . This novel function for NPR1 is also apparent in otherwise wild-type plants: npr1 mutants show a stronger HR, while NPR1-overproducing plants show a weaker HR when infected with P . syringae carrying the avrRpm1 gene . Spontaneous cell death in agd2 is partially suppressed by npr1, indicating that NPR1 can suppress or enhance cell death depending on the cellular context . agd2 plants depleted of SA show a dramatic exacerbation of the cell-growth phenotype and increased callose deposition, suggesting a role for SA in regulating growth and this cell-wall modification . AGD2 may function in cell death and/or growth control as well as the defense response, similarly to what has been described in animals for the functions of NFkappaB.

Ned Tijdschr Geneeskd . 2001 Apr 7;145(14):685.
{Diagnostic image (32) . Gout tophus}; Blaauwgeers JL et al.; A 65-year old man had gout and a secondary infection of a tophus with Pseudomonas at the metatarsophalangeal joint of the right first toe.

Hepatology, 2001 Sep, 34(3), 535 - 47
Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice; Wolf D et al.; Liver resident NK1.1+ T cells are supposed to play a pivotal role in the onset of inflammatory liver injury in experimental mouse models such as concanavalin A (Con A)-induced hepatitis . These cells, expressing the adhesion receptor, CD44, are largely depleted from the liver by a single intravenous injection of low-molecular-weight fragments of hyaluronic acid (LMW-HA) . Here, we report that LMW-HA pretreatment protected mice from liver injury in several models of T-cell- and macrophage-dependent, tumor necrosis factor alpha (TNF-alpha)-mediated inflammatory liver injury, i.e., from liver injury induced by either Con A or Pseudomonas exotoxin A (PEA) or PEA/lipopolysaccharide (LPS) . Interestingly, apart from inhibition of cellular adhesion, pretreatment of mice with LMW-HA was also capable of preventing hepatocellular apoptosis and activation of caspase-3 induced by direct administration of recombinant murine (rmu) TNF-alpha to D-galactosamine (GalN)-sensitized mice . LMW-HA-induced hepatoprotection could be neutralized by pretreatment with the nuclear factor-kappaB (NF-kappaB) inhibitor, pyrrolidine dithiocarbamate (PDTC), demonstrating the involvement of NF-kappaB in the observed protective mechanism . Indeed, injection of LMW-HA rapidly induced the production of TNF-alpha by Kupffer cells and the translocation of NF-kappaB into hepatocellular nuclei . Both LMW-HA-induced TNF-alpha production and NF-kappaB translocation were blocked by pretreatment with PDTC . Our findings provide evidence for an unknown mechanism of LMW-HA-dependent protection from inflammatory liver disease, i.e., induction of TNF-alpha- and NF-kappaB-dependent cytoprotective proteins within the target parenchymal liver cells.

Antonie Van Leeuwenhoek, 2001 Jun, 79(2), 149 - 61
Bacterial diversity in soil samples from two uranium waste piles as determined by rep-APD, RISA and 16S rDNA retrieval; Selenska-Pobell S et al.; The bacterial diversity in two uranium waste piles was studied . Total DNA was recovered from a large number of soil samples collected from different sites and depths in the piles using two procedures for direct lysis . Significant differences in the bacterial composition of the samples were revealed by the use of rep-APD, RISA and 16S ARDREA . The 16S rDNA analyses showed that the uranium wastes were dominated by Acidithiobacillusferrooxidans and by several Pseudomonas species classified in the gamma-subdivision of the Proteobacteria . The three kinds of A . ferrooxidans 16S and IGS rDNA specific fragments that were found corresponded to the three phylogenetic groups recognised in this species . This microdiversity probably reflects the genetic adaptation of the uranium waste strains to different concentrations of heavy metals.

Antonie Van Leeuwenhoek, 2001 Jun, 79(2), 135 - 40
Evolution of catabolic pathways and metabolic versatility in Pseudomonas stutzeri OX1; Barbieri P et al.; Pseudomonas stutzeri OX1 is able to degrade toluene and ortho-xylene via the direct oxygenation of the aromatic ring . The genetic studies carried out suggest that the genes coding for the monooxygenase involved in the early steps of this catabolic route have been acquired by gene transfer . P stutzeri OX1 is also potentially able to utilize meta- and para-xylene as growth substrates . These two isomers are metabolized through a different pathway (TOL pathway) . Both catabolic routes can be activated or inactivated by means of genomic rearrangements . The relevance of such recombination mechanisms in the evolution and the adaptability of P . stutzeri is discussed.

Syst Appl Microbiol, 2001 Jul, 24(2), 157 - 65
Flagellin gene sequence variation in the genus Pseudomonas; Bellingham NF et al.; Flagellin gene (fliC) sequences from 18 strains of Pseudomonas sensu stricto representing 8 different species, and 9 representative fliC sequences from other members of the gamma sub-division of proteobacteria, were compared . Analysis was performed on N-terminal, C-terminal and whole fliC sequences . The fliC analyses confirmed the inferred relationship between P . mendocina, P . oleovorans and P . aeruginosa based on 16S rRNA sequence comparisons . In addition, the analyses indicated that P . putida PRS2000 was closely related to P . fluorescens SBW25 and P . fluorescens NCIMB 9046T, but suggested that P . putida PaW8 and P . putida PRS2000 were more closely related to other Pseudomonas spp . than they were to each other . There were a number of inconsistencies in inferred evolutionary relationships between strains, depending on the analysis performed . In particular, whole flagellin gene comparisons often differed from those obtained using N- and C-terminal sequences . However, there were also inconsistencies between the terminal region analyses, suggesting that phylogenetic relationships inferred on the basis of fliC sequence should be treated with caution . Although the central domain of fliC is highly variable between Pseudomonas strains, there was evidence of sequence similarities between the central domains of different Pseudomonas fliC sequences . This indicates the possibility of recombination in the central domain of fliC genes within Pseudomonas species, and between these genes and those from other bacteria.

J Org Chem, 2001 Aug 24, 66(17), 5649 - 54
Biosynthesis of acaterin: coupling of C(5) unit with octanoate; Sekiyama Y et al.; Acaterin (1), produced by Pseudomonas sp . A 92, is a secondary metabolite having a 2-penten-4-olide structure . Feeding experiments with (2)H- and (13)C-labeled decanoic acid, their 3-oxygenated congeners, and octanoic acid have suggested that 1 is biosynthesized via coupling of a C(5) unit with octanoate, rather than via introduction of a C(3) unit at the alpha position of a decanoate derivative . Further feeding study of {2,3-(13)C(2)}decanoic acid concluded that the former route is operating in the biosynthesis of 1.

Indian J Exp Biol, 2001 May, 39(5), 464 - 8
Siderophore production by fluorescent pseudomonads colonizing roots of certain crop plants; Yeole RD et al.; Twelve fluorescent Pseudomonas isolates colonizing roots of four crop plants, chilli, cotton, groundnut and soybean, were examined for extracellular siderophore production in different media under iron deficient conditions . While all the organisms produced siderophores, they varied in the quantity of siderophores produced and in their preference to the medium . The siderophores were invariably hydroxamates (pyoverdine) of trihydroxamate type which formed bidentate ligands with Fe III ions.

Ann Anat, 2001 Jul, 183(4), 309 - 17
Postinflammatory changes of the diaphragmatic stomata; Michailova KN; Numerous investigations concerning the fine morphology of diaphragmatic stomata have been performed, but its ultrastructural changes in experimental conditions remain unclear . The present study demonstrates the peritoneal side of the diaphragm in adult Wistar rats by transmission electron microscopy . Ten experimental animals were observed 5 and 8 days after Pseudomonas aeuriginosa instillation (PI) into the peritoneal cavity . A control group of 6 rats showed flat mesothelial covering on basal lamina (BL) and connective tissue layer, as well as cubic mesothelial cells, single stomata over underlying lymphatic lacunae (LL) . Five days after PI the mesothelial cells had more numerous microvilli, microvesicles, vacuoles, lysosomes and a lesser number of specialized contacts . The multiplication of the extravasal cells and larger intercellular spaces lead to thickenings of the connective tissue around LL . LL were larger and located in close proximity of the mesothelium . Intercellular spaces in the mesothelial layer and different types of contacts between mesothelial cells and endothelial protrusions of LL (with common BL or without BL) were encountered . Eight days after PI the mesothelium, endothelium of LL, their BL and surrounding connective tissue were interrupted and structurally modified to form typical new channels--stomata . The larger portion of the channels were formed of mesothelial cells, while the endothelial cells participated in the submesothelial part . LL were more numerous than in the previous period, and were arranged in groups . LL increased their vertical (50.59 microm) and horizontal (155.57 microm) diameter, as compared with control animals (respectively 12.37 microm and 74.08 microm) . Neighbouring LL were separated by thin or thick septae . Peristomatal mesothelial cells or more rarely endothelium formed valve- or bridge-like structures . Valves on the opposite side of LL were observed . Groups of electron-dense bodies characterized some tall endothelial cells of LL . Cubic mesothelium, endothelium of the LL, both BL, the cell connections that formed new stomata, LL and surrounding connective tissue underwent rapid and parallel changes after PI . Among these elements of the lymphatic regions mentioned above, the mesothelium and endothelium of LL had a main role in experimental conditions.

Cancer Res, 2001 Aug 15, 61(16), 6194 - 200
Interleukin-13 receptor-targeted cancer therapy in an immunodeficient animal model of human head and neck cancer; Kawakami K et al.; Although interleukin-13 receptors (IL-13R) are overexpressed on several head and neck cancer cell lines, a majority of cell lines express only low levels of IL-13R . We have found that the primary interleukin-13-binding protein IL-13Ralpha2 chain plays an important role in ligand binding and internalization . We showed that the gene transfer of IL-13Ralpha2 chain into various solid tumor cell lines that express few IL-13Rs can dramatically sensitize cells to the cytotoxic effect of a recombinant chimeric protein composed of interleukin-13 and a mutated form of Pseudomonas exotoxin A, IL13-PE38QQR . Based on the expression of IL-13R, we have classified five head and neck cancer cell lines into two groups: (a) IL-13Ralpha2 chain-positive cell lines (SCC-25 and KCCT873); and (b) IL-13Ralpha2 chain-negative cell lines (A253, YCUT891, and KCCT871) . By plasmid-mediated stable gene transfer, we demonstrate that not only IL-13Ralpha2 chain-positive head and neck cancer cell lines but also IL-13Ralpha2 chain-negative cell lines can dramatically increase sensitivity to IL-13 toxin by 520-1000-fold compared with mock-transfected control cells after genetic alteration to express high levels of the IL-13Ralpha2 chain . In animal studies, i.p . or intratumoral administration of IL13-PE38QQR given daily or on alternate days for 3-5 days showed dramatic tumor response with complete remission in intratumorally injected tumors in both IL-13Ralpha2 chain-positive and -negative but transfected with IL-13Ralpha2 chain head and neck tumor implanted s.c . in nude mice . These results demonstrate that by using a combination approach of gene transfer and systemic or locoregional cytotoxin therapy, the IL-13R represents a new potent target for head and neck cancer therapy.

FEMS Microbiol Lett, 2001 Aug 7, 202(1), 51 - 7
Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards; Widada J et al.; The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp . strain CA10 . The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan) . The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content . When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system . To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined . A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell . After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction) . Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.

Mol Biotechnol, 2001 Jul, 18(3), 251 - 68
Chemical construction of immunotoxins; Ghetie V et al.; Immunotoxins are chimeric proteins consisting of an antibody linked to a toxin . The antibodies most frequently used for the preparation of immunotoxins are murine monoclonal antibodies belonging to IgG isotype . The most used toxins for the chemical construction of immunotoxins are Ricin toxin A chain in its deglycosylated form and recombinant Pseudomonas endotoxin with the cell-binding domain deleted . The linkage of the antibody to the toxin can be accomplished by chemical methods using reagents that crosslink antibody to toxin . The usual crosslinkers attach disulfide groups into the antibody molecule to form a disulfide bond between the antibody and the toxin . Disulfide bonds are susceptible to reduction in the cytoplasm of the targeted cells thereby releasing the toxin so that it can exert its cytotoxic activity only into the cells (e.g., tumor cells) binding the antibody moiety . This article describes various methods to obtain antibodies and toxins and several procedures for their crosslinking as well as "in vitro" and "in vivo" testing of the immunotoxins efficacy.

Plant Physiol, 2001 Aug, 126(4), 1637 - 45
Expression of 35S::Pto globally activates defense-related genes in tomato plants; Xiao F et al.; The tomato (Lycopersicon esculentum) resistance gene Pto confers resistance to the bacterial pathogen Pseudomonas syringae pv tomato carrying the avirulent gene avrPto . Overexpressing Pto under the control of the cauliflower mosaic virus 35S promoter constitutively activates defense responses in the absence of pathogen infection and nonspecifically enhances disease resistance . To elucidate the mechanisms underlying this resistance, we isolated cDNAs corresponding to transcripts that accumulated in 35S::Pto plants . By using suppression subtractive hybridization, we isolated 82 unique cDNA clones, most of which corresponded to differentially expressed transcripts . Most of the genes examined were also induced by pathogen inoculation . Sequence analysis showed that a large number of genes encode defense-related proteins, and most had not been previously isolated from tomato . The isolated cDNAs also include those with a putative role in the oxidative burst, proteolysis, the hypersensitive response, signal transduction, and a number of genes with unknown functions . The isolation of these cDNAs of diverse functions will assist in the characterization of defense pathways activated during disease resistance.

Plant Physiol, 2001 Aug, 126(4), 1579 - 87
Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6; Desikan R et al.; Mitogen-activated protein kinases (MAPKs) are key enzymes that mediate adaptive responses to various abiotic and biotic stresses, including pathogen challenge . The proteinaceous bacterial elicitor harpin (secreted by Pseudomonas syringae pv syringae) activates two MAPKs in suspension cultures of Arabidopsis var . Landsberg erecta . In this study, we show that harpin and exogenous hydrogen peroxide (H(2)O(2)) activate myelin basic protein kinases in Arabidopsis leaves . Using anti-AtMPK4 and anti-AtMPK6 antibodies, we identify the harpin-activated MAPKs in both leaves and suspension cultures as AtMPK4 and AtMPK6, and show that H(2)O(2), generated by Arabidopsis cells in response to challenge with harpin, activates only AtMPK6 . However, treatments with catalase, which removes H(2)O(2), or diphenylene iodonium, which inhibits superoxide and H(2)O(2) production, do not inhibit harpin-induced activation of AtMPK4 or AtMPK6 . In addition, activation of AtMPK4 but not AtMPK6 is inhibited by the MAPK kinase inhibitor PD98059 . Neither harpin nor H(2)O(2) has any effect on AtMPK4 or AtMPK6 gene expression . In addition, the expression of AtMEKK1, AtMEK1, or AtMKK2, previously shown to be potential functional partners of AtMPK4, were not affected by either harpin or H(2)O(2) treatments . These data suggest that harpin activates several signaling pathways, one leading to stimulation of the oxidative burst and others leading to the activation of AtMPK4 or AtMPK6.

Appl Microbiol Biotechnol, 2001 Jul, 56(1-2), 265 - 9
Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1; Shim H et al.; A recombinant strain of Escherichia coli (JM109/pBZ1260) expressing constitutively toluene-o-xylene monooxygenase (ToMO) of Pseudomonas stutzeri OX1 degraded binary mixtures (100 microM each) of tetrachloroethylene (PCE) with either trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), or vinyl chloride (VC) . PCE degradation was 8-20% for these binary mixtures, while TCE and trans-DCE with PCE were degraded at 19%, 1,1-DCE at 37%, cis-DCE at 97%, and VC at 27% . The host P . stutzeri OXI was also found to degrade binary mixtures of PCE/TCE, PCE/cis-DCE, and PCE/VC when induced with toluene . Degradation of quaternary mixtures of PCE/TCE/trans-DCE/VC and PCE/TCE/cis-DCE/VC by JM109/pBZ1260 were also investigated as well as mixtures of PCE/TCE/trans-DCE/1,1-DCE/cis-DCE/VC; when all the chlorinated compounds were present, the best degradation occurred with 24-51% removal of each . For these degradation reactions, 39-85% of the stoichiometric chloride expected from complete degradation of the chlorinated ethenes was detected . The time course of PCE/TCE/1,1-DCE degradation was also measured for a mixture of 8, 17, and 6 microM, respectively; initial degradation rates were 0.015, 0.023 . and 0.029 nmol/min x mg protein, respectively . This indicates that for the first time an aerobic enzyme can degrade mixtures of all chlorinated ethenes, including the once--so it was believed-completely recalcitrant PCE.

Mol Plant Microbe Interact, 2001 Aug, 14(8), 1006 - 15
Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp . strains; Chin-A-Woeng TF et al.; Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f . sp . radicis-lycopersici . Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN) . In contrast, the take-all biocontrol strains P . fluorescens 2-79 and P . aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease . To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized . Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA . The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH . Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot . PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans . Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P . fluorescens 2-79 and P . aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot . Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

Microbiology, 2001 Aug, 147(Pt 8), 2183 - 94
Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of "Pseudomonas azelaica" HBP1; Jaspers MC et al.; "Pseudomonas azelaica" HBP1 degrades 2-hydroxybiphenyl (2-HBP) and 2,2'-diHBP by employing a meta-cleavage pathway encoded by the hbpCAD genes . The regulatory gene hbpR, located directly upstream of the hbpCAD genes and oriented in the opposite direction, encodes a transcription activator protein belonging to the so-called XylR/DmpR subclass within the NtrC family . HbpR activates transcription from two separate sigma(54)-dependent promoters upstream of the hbpC and the hbpD genes, in the presence of the pathway substrates 2-HBP and 2,2'-diHBP . The DNA region upstream of the hbpC gene displays an unusual organization, containing two adjacent 0.3 kb regions that share 71% sequence identity . The DNA region most proximal to the hbpC promoter harbours one pair of putative upstream activating sequences (UASs C-1/C-2) and a small cryptic ORF that shows homology to hbpR itself . The second, more distal, region contains a second pair of putative UASs (UASs C-3/4) and the 5'-part of the hbpR gene . Transcriptional fusions in Escherichia coli between different deletions of the hbpR-hbpC intergenic region and the genes for bacterial luciferase revealed that most if not all of the transcriptional output from the hbpC promoter is mediated from the proximal UASs C-1/C-2 . However, when the UASs C-1/C-2 were deleted and UASs C-3/C-4 were placed in an appropriate position with respect to the promoter region, the hbpC promoter was still inducible with 2-HBP, albeit at a lower level . Transcription studies in E . coli and "P . azelaica" revealed that the divergently oriented hbpR gene is expressed constitutively from a sigma(70)-dependent promoter situated within the cryptic ORF . The presence of UAS pair C-3/C-4 mediated a slightly higher promoter activity for transcription of hbpR.

J Ind Microbiol Biotechnol, 2001 May, 26(5), 303 - 8
Influence of pH and ionic strength on adhesion of a wild strain of Pseudomonas sp . to titanium; Busalmen JP et al.; The adherence of an environmental strain of Pseudomonas sp . to titanium was evaluated modifying the pH (2 to 8) and ionic strength (0.1 and 0.6 M NaCl) of the electrolyte solution . Results were analyzed considering the participation of the different interfacial forces under the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory . At 0.1 M, maximal bacterial adhesion was at pH 6, in agreement with the point of zero charge of the titanium surface . Similar adhesion values were observed at both sides of this point despite the opposite electrostatic condition of the surface oxide . At 0.6 M an absence of bacterial adhesion was observed throughout the pH range tested . The changes in bacterial adhesion are in agreement with the changes in the number of reinforced H-bond-forming sites on the titanium surface calculated using a simple model for the ionization of OH group adsorbed to the surface.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1549 - 55
Pseudomonas kilonensis sp . nov., a bacterium isolated from agricultural soil; Sikorski J et al.; A total of 131 bacterial isolates related to Pseudomonas corrugata were obtained from an agricultural soil from northern Germany . Based on 16S rRNA gene sequences, PCR-based genome fingerprinting and multilocus enzyme electrophoresis, they formed two groups, A (119 strains) and B (12 strains) . As members of each group were highly similar, a single strain of each group was subsequently characterized by a polyphasic taxonomic approach . The selected member of group A was identified as a strain of Pseudomonas brassicacearum, whereas the selected member of group B was distinct from other species of the genus Pseudomonas . Although DNA-DNA hybridization suggested a close affiliation of the group B strain with P . brassicacearum and Pseudomonas thivervalensis and ribotyping suggested a close affiliation with P . brassicacearum, RAPD data, 16S rRNA gene sequencing and phenotypic characterization indicated the presence of a distinct taxonomic entity . This strain differed from the type strains of P . thivervalensis and P . brassicacearum in 10 and 12 metabolic properties, respectively, whereas the two organisms differ from one another by only two properties . Strains of group B are therefore considered to be members of a new species, for which the name Pseudomonas kilonensis sp . nov . is proposed . The type strain is strain 520-20T (= DSM 13647T = CFBP 5372T).

Plant J, 2001 Jul, 27(2), 115 - 27
HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death; Tronchet M et al.; Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens . Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity . We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum . This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth . Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv . pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria . Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines . Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.

Biochem Biophys Res Commun, 2001 Aug 10, 286(1), 109 - 13
Involvement of plasmid in degradation of pentachlorophenol by Pseudomonas sp . from a chemostat; Thakur IS et al.; Pseudomonas sp . strain IST103 obtained from a stable bacterial consortium was capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source . The consortium was developed by continuous enrichment in a chemostat . The degradation of PCP by bacterial strain proceeded through an oxidative route as indicated by accumulation of tetrachloro-p-hydroquinone and chlorohydroquinone determined by high performance liquid chromatography (HPLC), and chloride molecules released in culture medium . Two different molecular size plasmids, of approximately 80 and 4 kilobase, were found to be responsible for carrying genes for degradation of PCP . This was evidenced by mutants produced by curing of plasmid by treatment of ethidium bromide . The derivatives were not able to utilize PCP, however, transformation of low molecular size plasmid of Pseudomonas sp . strain 103 into E . coli JM109 utilized PCP, indicated a possible involvement of plasmid in degradation of pentachlorophenol .

Jpn J Thorac Cardiovasc Surg, 2001 Jun, 49(6), 388 - 90
Jaundice after surgery for an aortic arch aneurysm; Onoguchi K et al.; A 57-year-old patient who developed hyperbilirubinemia after surgery for an aortic arch aneurysm subsequently suffered pseudomonas sepsis . Low-volume biliary drainage from the common bile duct was colorless . A disturbance in the liver's excretory system caused jaundice . Sepsis and jaundice were resolved when hepatic excretory function recovered.

Arch Microbiol, 2001 Jul, 176(1-2), 114 - 20
Induction of butane consumption in Pseudomonas butanovora; Sayavedra-Soto LA et al.; The induction of the enzyme activities involved in butane metabolism in Pseudomonas butanovora was characterized . P . butanovora was grown on butane or its metabolites, both singly and in mixtures with other growth substrates . Cells grown in each of the butane metabolites readily consumed the growth substrate and downstream metabolites, but consumed the upstream butane metabolites more slowly . Upstream activities in the butane metabolism could be induced by downstream metabolites, but to much lower levels than with the primary substrate . The induction of butane oxidation was not repressed when P . butanovora was grown or incubated in a mixture of butane and 1-butanol, butyraldehyde or butyrate . However, no induction of butane consumption was observed in a mixture of butane and lactate, which is indicative of catabolite repression . In lactate-grown cells that were rid of the growth substrate and incubated with butane and acetylene (to inactivate newly formed butane monooxygenase), the consumption of butane, 1-butanol and butyraldehyde consumption was not induced . The overall results suggest an independent regulatory mechanism for each of the enzyme activities in butane metabolism . In addition, a low, constitutive butane oxidation was observed in cells grown on substrates other than butane metabolites.

J Biol Chem, 2001 Oct 5, 276(40), 36931 - 8 Epub 2001 Jul 30.
Halotolerant cyanobacterium Aphanothece halophytica contains an Na(+)/H(+) antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail; Waditee R et al.; Recently, a cyanobacterium Synechocystis sp . PCC 6803 has been shown to contain an Na(+)/H(+) antiporter gene homologous to plants (SOS1 and AtNHX1 from Arabidopsis) and mammalians (NHEs from human) but not to Escherichia coli (nhaA and nhaB) . Here, we examined whether a halotolerant cyanobacterium Aphanothece halophytica has homologous genes . It turned out that A . halophytica contains an Na(+)/H(+) antiporter homologous to plants, mammalians, and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis) but with novel ion specificity . Its gene product, ApNhaP (Na(+)/H(+) antiporter from Aphanothece halophytica), exhibited the Na(+)/H(+) antiporter activity over a wide pH range between 5 and 9 and complemented the Na(+)-sensitive phenotype of the antiporter-deficient E . coli mutant . The ApNhaP had virtually no activity for the Li(+)/H(+) antiporter but showed high Ca(2+)/H(+) antiporter activity at alkaline pH . The ApNhaP complemented the Ca(2+)-sensitive phenotype of the E . coli mutant but not the Li(+)-sensitive phenotype . The replacement of a long C-terminal tail of ApNhaP with that of Synechocystis altered the ion specificity of the antiporter . These results suggest that the ion specificity of an Na(+)/H(+) antiporter is partly determined by the structural properties of the C-terminal tail, which was well exemplified in the case of A . halophytica.

Anal Biochem, 2001 Aug 1, 295(1), 38 - 44
Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin; Demant EJ et al.; The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission . As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids . In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles . This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA . Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second . This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays .

N Engl J Med, 2001 Jul 26, 345(4), 241 - 7
Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia; Kreitman RJ et al.; BACKGROUND: Hairy-cell leukemia that is resistant to treatment with purine analogues, including cladribine, has a poor prognosis . We tested the safety and efficacy of an immunotoxin directed against a surface antigen that is strongly expressed by leukemic hairy cells . METHODS: RFB4(dsFv)-PE38 (BL22), a recombinant immunotoxin containing an anti-CD22 variable domain (Fv) fused to truncated pseudomonas exotoxin, was administered in a dose-escalation trial by intravenous infusion every other day for a total of three doses . RESULTS: Of 16 patients who were resistant to cladribine, 11 had a complete remission and 2 had a partial remission with BL22 . The three patients who did not have a response received low doses of BL22 or had preexisting toxin-neutralizing antibodies . Of the 11 patients in complete remission, 2 had minimal residual disease in the bone marrow or blood . During a median follow-up of 16 months (range, 10 to 23), 3 of the 11 patients who had a complete response relapsed and were retreated; all of these patients had a second complete remission . In 2 of the 16 patients, a serious but completely reversible hemolytic-uremic syndrome developed during the second cycle of treatment with BL22 . Common toxic effects included transient hypoalbuminemia and elevated aminotransferase levels . CONCLUSIONS: BL22 can induce complete remissions in patients with hairy-cell leukemia that is resistant to treatment with purine analogues.

Appl Environ Microbiol, 2001 Aug, 67(8), 3739 - 45
Isolation and in vivo and in vitro antifungal activity of phenylacetic acid and sodium phenylacetate from Streptomyces humidus; Hwang BK et al.; The antifungal substances SH-1 and SH-2 were isolated from Streptomyces humidus strain S5-55 cultures by various purification procedures and identified as phenylacetic acid and sodium phenylacetate, respectively, based on the nuclear magnetic resonance, electron ionization mass spectral, and inductively coupled plasma mass spectral data . SH-1 and SH-2 completely inhibited the growth of Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, Saccharomyces cerevisiae, and Pseudomonas syringae pv . syringae at concentrations from 10 to 50 microg/ml . The two compounds were as effective as the commercial fungicide metalaxyl in inhibiting spore germination and hyphal growth of P . capsici . However, the in vivo control efficacies of the two antifungal compounds against P . capsici infection on pepper plants were similar to those of H(3)PO(3) and fosetyl-AI but less than that of metalaxyl.

Appl Environ Microbiol, 2001 Aug, 67(8), 3735 - 8
Use of an intergenic region in Pseudomonas syringae pv . syringae B728a for site-directed genomic marking of bacterial strains for field experiments; Hirano SS et al.; To construct differentially-marked derivatives of our model wild-type strain, Pseudomonas syringae pv . syringae B728a (a causal agent of bacterial brown spot disease in snap bean plants), for field experiments, we selected a site in the gacS-cysM intergenic region for site-directed insertion of antibiotic resistance marker cassettes . In each of three field experiments, population sizes of the site-directed chromosomally marked B728a derivatives in association with snap bean plants were not significantly different from that of the wild-type strain . Inserts of up to 7 kb of DNA in the intergenic region did not measurably affect fitness of B728a in the field . The site is useful for site-directed genomic insertions of single copies of genes of interest.

Appl Environ Microbiol, 2001 Aug, 67(8), 3677 - 82
Bacterial species determination from DNA-DNA hybridization by using genome fragments and DNA microarrays; Cho JC et al.; Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented . We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization . Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays . Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays . Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria . Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity . In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.

Appl Environ Microbiol, 2001 Aug, 67(8), 3610 - 7
Degradation of chlorinated dibenzofurans and dibenzo-p-dioxins by two types of bacteria having angular dioxygenases with different features; Habe H et al.; Two kinds of bacteria having different-structured angular dioxygenases-a dibenzofuran (DF)-utilizing bacterium, Terrabacter sp . strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp . strain CA10-were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains . First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD . Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC) . Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC . We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.

Water Res, 2001 Aug, 35(12), 2841 - 50
Kinetic characteristics of bacterial azo-dye decolorization by Pseudomonas luteola; Chang JS et al.; A Pseudomonas luteola strain expressing azoreductase activity was utilized to remove the color of an azo dye (reactive red 22) from contaminated solutions . The effects of substrate concentrations, medium compositions, and operation parameters (e.g., pH, temperature, dissolved oxygen, etc.) on decolorization of the azo dye by a P . luteola strain were systematically investigated to reveal the key factors that dominate the performance of azo-dye decolorization . The metabolites resulting from bacterial decolorization were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometery (MS) . The results show that the dissolved oxygen and glucose concentration retarded decolorization of reactive red 22 by P . luteola . The optimal azo-dye decolorization occurred at 37 degrees C, while more rapid decolorization took place over pH 7-9 . Yeast extract and tryptone strongly enhanced the decolorization . The Michaelis-Menten model can satisfactorily describe the dependence of specific decolorization rate on the concentration of substrate (reactive red 22 or yeast extract) . Decolorization of the azo dye by intact cells of P . luteola was essentially independent of the growth phase, whereas the azoreductase activity of the cell-free extract decreased in the order of late-stationary phase > early-stationary phase > mid-log phase . This suggests that mass transfer of the azo dye across the cell membrane may be the rate-limiting step . The HPLC and MS analyses suggest that both partial reduction and complete cleavage of the azo bond could contribute to decolorization of reactive red 22 by P . luteola.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1141 - 3 Epub 2001 Jul 23.
Characterization of a novel pectate lyase, Pel10A, from Pseudomonas cellulosa; Charnock SJ et al.; Biological recycling of plant material is essential for biosphere maintenance . This perpetual task involves a complex array of enzymes, including extracellular polysaccharide hydrolases and lyases . Whilst much is known about the structure and function of the hydrolases, relatively little is known about the structures and mechanisms of the corresponding lyases . To this end, crystals of the catalytic module of a novel family 10 pectate lyase, Pel10A from Pseudomonas cellulosa, were obtained using polyethylene glycol 2000 monomethylether as a precipitant . They belong to space group P2(1), with unit-cell parameters a = 47.7, b = 106.1, c = 55.4 A, beta = 92.0 degrees, and have two molecules in the asymmetric unit . The crystals diffract beyond 1.5 A using synchrotron radiation.

J Biol Chem, 2001 Sep 28, 276(39), 36146 - 54 Epub 2001 Jul 17.
BphS, a key transcriptional regulator of bph genes involved in polychlorinated biphenyl/biphenyl degradation in Pseudomonas sp . KKS102; Ohtsubo Y et al.; The bph genes in Pseudomonas sp . KKS102, which are involved in the degradation of polychlorinated biphenyl/biphenyl, are induced in the presence of biphenyl . In this study our goal was to understand the regulatory mechanisms involved in the inducible expression . The bph genes (bphEGF(orf4)A1A2A3BCD(orf1)A4R) constitute an operon, and its expression is strongly dependent on the pE promoter located upstream of the bphE gene . A bphS gene, whose deduced amino acid sequence showed homology with the GntR family transcriptional repressors, was identified at the upstream region of the bphE gene . Disruption of the bphS gene resulted in constitutive expression of bph genes, suggesting that the bphS gene product negatively regulated the pE promoter . The gel retardation and DNase footprinting analyses demonstrated specific binding of BphS to the pE promoter region and identified four BphS binding sites that were located within and immediately downstream of the -10 box of the pE promoter . The four binding sites were functional in repression because their respective elimination resulted in derepression of the pE promoter . The binding of BphS was abolished in the presence of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, an intermediate compound in the biphenyl degradation pathway . We concluded that the negative regulator BphS plays a central role in the regulation of bph gene expression through its action at the pE promoter.

J Pharm Sci, 2001 Jul, 90(7), 860 - 71
The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf; Searles JA et al.; The objective of this study was to determine the influence of ice nucleation temperature on the primary drying rate during lyophilization for samples in vials that were frozen on a lyophilizer shelf . Aqueous solutions of 10% (w/v) hydroxyethyl starch were frozen in vials with externally mounted thermocouples and then partially lyophilized to determine the primary drying rate . Low- and high-particulate-containing samples, ice-nucleating additives silver iodide and Pseudomonas syringae, and other methods were used to obtain a wide range of nucleation temperatures . In cases where the supercooling exceeded 5 degrees C, freezing took place in the following three steps: (1) primary nucleation, (2) secondary nucleation encompassing the entire liquid volume, and (3) final solidification . The primary drying rate was dependent on the ice nucleation temperature, which is stochastic in nature but is affected by particulate content and the presence of ice nucleators . Sample cooling rates of 0.05 to 1 degrees C/min had no effect on nucleation temperatures and drying rate . We found that the ice nucleation temperature is the primary determinant of the primary drying rate . However, the nucleation temperature is not under direct control, and its stochastic nature and sensitivity to difficult-to-control parameters result in drying rate heterogeneity . Nucleation temperature heterogeneity may also result in variation in other morphology-related parameters such as surface area and secondary drying rate . Overall, these results document that factors such as particulate content and vial condition, which influence ice nucleation temperature, must be carefully controlled to avoid, for example, lot-to-lot variability during cGMP production . In addition, if these factors are not controlled and/or are inadvertently changed during process development and scaleup, a lyophilization cycle that was successful on the research scale may fail during large-scale production.

J Biol Chem, 2001 Sep 28, 276(39), 36215 - 24 Epub 2001 Jul 16.
A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids; Handrick R et al.; A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei . Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units . The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate) . The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively . No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch . The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g . n-propyl alcohol) . The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents . Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases . A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J . L., and Jaeger, K.-E . (1999) Biochem . J . 343, 177-183) is suggested.

J Am Chem Soc, 2001 Jan 31, 123(4), 576 - 87
Characterization of the copper-sulfur chromophores in nitrous oxide reductase by resonance raman spectroscopy: evidence for sulfur coordination in the catalytic cluster; Alvarez ML et al.; Nitrous oxide reductase (N(2)OR) from Pseudomonas stutzeri, a dimeric enzyme with a canonical metal ion content of at least six Cu ions per subunit, contains two types of multinuclear copper sites: Cu(A) and Cu(Z) . An electron-transfer role for the dinuclear Cu(A) site is indicated based on its similarity to the Cu(A) site in cytochrome c oxidase (CcO), a dicysteinate-bridged, mixed-valence cluster . The Cu(Z) site is the catalytic site, which had long been thought to have novel spectroscopic properties . However, the low-energy electronic transitions and resonance Raman features attributable to Cu(Z) have been difficult to reconcile with a lack of conserved cysteine residues in standard alignments of N(2)OR sequences, other than those associated with the Cu(A) site . Recent evidence indicates that nitrous oxide reductase contains acid-labile sulfide and that this sulfide is a constituent of the Cu(Z) site (Rasmussen, T.; Berks, B . C.; Sanders-Loehr, J.; Dooley, D . M.; Zumft, W . G.; Thomson, A . J . Biochemistry 2000, 39, 12753-12756) . We have used resonance Raman (RR) spectroscopy to selectively probe the Cu(A) and Cu(Z) sites of N(2)OR in three oxidation states (oxidized, semireduced, and reduced) as well as Cu(A)-only and Cu(Z)-only variants . The Cu(A) (mixed-valence, also designated as A(mv)) RR spectrum exhibits 10 vibrational modes between 220 and 410 cm(-1), with >1-cm(-1) (34)S isotope shifts that sum to -16.6 cm(-1) . Many of these modes are also sensitive to (65)Cu and (15)N(His) and, thus, can be assigned to coupling of the Cu-S stretch, nu(Cu-S), with cysteine and histidine vibrations of the Cu(2)Cys(2)His(2) core . The RR spectrum of the Cu(Z) site (Z(ox)) reveals a novel Cu-sulfur chromophore with four S isotope-sensitive modes at 293, 347, 352, and 408 cm(-1), with a total (34)S shift of -19.9 cm(-)(1) . The magnitude of the S isotope shifts and wide spread of perturbed frequencies are similar to those observed in Cu(A) and therefore suggest a sulfur-bridged cluster in Z(ox) . The Z(ox) site has its nu(Cu-S)-containing modes at higher energy and exhibits less mixing with ligand deformations, compared to Cu(A) . Reduction by dithionite produces a mixed-valence Cu(Z) site (Z(mv)) with six S isotope-sensitive RR modes between 282 and 382 cm(-1) and a total (34)S-shift of -16.9 cm(-1) . The observation of a nearly identical RR spectrum in the C622D variant of N(2)OR, which lacks one of the conserved Cu(A) Cys residues, establishes that Cu-S vibrations observed in this variant arise from the Z(mv) site . Furthermore, none of the features assigned to Cu(Z) are detected in a second variant that contains only Cu(A) . Therefore the resonance Raman spectra reported here provide compelling evidence for a unique Cu-S cluster in the catalytic site of nitrous oxide reductase.

Pest Manag Sci, 2001 Mar, 57(3), 262 - 8
Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato; Scarponi L et al.; A reproducible and accurate procedure, based on HPLC analysis, has been developed to determine simultaneously acibenzolar-S-methyl (CGA 245 704) and its acid derivative (CGA 210 007) in tomato leaves . The limit of detection and quantification of the method are 0.015 and 0.15 mg litre-1 for CGA 245 704 and 0.030 and 0.30 mg litre-1 for CGA 210 007 . In tomato plants treated with 250 microM CGA 245 704, it was found that the inducer rapidly translocates from treated leaves (cotyledons, 1st and 2nd) to untreated leaves (3rd to 5th), with the maximum translocation (40% of the total quantity found) occurring 8 h after the treatment . CGA 245 704 residues decreased as time elapsed in both treated and untreated tomato leaves, reaching negligible values 72 h after treatment . The acid derivative, CGA 210 007, was formed in tomato plants as early as 2 h after CGA 245 704 treatment, albeit only in the treated leaves . CGA 210 007 residues decreased in treated tomato leaves with a trend similar to that observed for CGA 245 704 . Treatment of tomato plants with CGA 245 704 or CGA 210 007 at 250 microM systemically protected the plants against Pseudomonas syringae pv tomato attacks, the causal agent of bacterial speak disease . Evidence of this were reductions in the degree of infection, the bacterial lesion diameter and the bacterial growth in planta . Since neither CGA 245 704 nor CGA 210 007 inhibited bacterial growth in vitro and the protection against bacterial speak of tomato was observed when the two compounds were completely degraded, the protection must be due to the activation of the plant's defence mechanisms.

J Immunother, 2001 Mar, 24(2), 144 - 150
Cytotoxicity of Antiosteosarcoma Recombinant Immunotoxins Composed of TP-3 Fv Fragments and a Truncated Pseudomonas Exotoxin A; Onda M et al.; SUMMARY: Regrowth of drug-resistant tumor cells is responsible for approximately half of an unselected osteosarcoma population still dying of the disease despite aggressive combination therapy . Two monoclonal antibodies, TP-1 (immunoglobulin 2a) and TP-3 (immunoglobulin 2b) are available, which specifically recognize an antigen on osteosarcoma cells . In this work, we have fused the variable (V) genes of TP-3 to a truncated fragment of Pseudomonas exotoxin A, referred to as PE38 . Two immunotoxins were made that differed in the Fv portion: TP-3(scFv)-PE38, which contains a peptide linker, and TP-3(dsFv)-PE38, which contains a disulfide bond for stabilization of the association between the V domains . Recombinant TP-3 immunotoxins were expressed in Escherichia coli and purified from inclusion bodies . We describe the design and expression of these immunotoxins, and their properties with regard to antigen binding, stability, and cytotoxicity . Toxicity studies were done in mice . We found that the immunotoxins exhibited very similar in vitro properties, whereas in vivo TP-3(dsFv)-PE38 was much better tolerated than TP-3(scFv)-PE38.

Clin Experiment Ophthalmol, 2001 Jun, 29(3), 179 - 82
Expression of macrophage migration inhibitory factor during Pseudomonas keratitis; Thakur A et al.; Macrophage migration Inhibitory factor (MIF) is a recently rediscovered pro-inflammatory cytokine, and has been shown to play a role in the regulation of neutrophil chemokines and angiogenesis . Corneal epithelial and endothelial cells have been shown to express MIF . This study evaluated the expression of MIF during Pseudomonas keratitis in mice and in vitro using a corneal epithelial cell line . Three strains of P . aeruginosa, 6294 (invasive strain), 6206 (cytotoxic strain) and Paer1 (non-infectious strain) were used . Both cytotoxic and invasive strains were isolated from human corneal ulcers and the Paer1 strain was isolated from a non-infectious condition . Following challenge in mouse corneas or a corneal epithelial cell line, corneal homogenates or lysed corneal epithelial cells were used to isolate RNA . Migration inhibitory factor mRNA expression in the mouse cornea or human corneal epithelial cells was examined by reverse transcription-polymerase chain reaction analysis, and was found to be expressed as early as 4 h after the injury (scratch controls) or infection in the mouse corneas . Migration inhibitory factor mRNA in scratch controls and Paer1-inoculated corneas showed peak levels at 4 h post-challenge and this dropped by 24 h post-challenge . Corneas challenged with invasive and cytotoxic strains showed peak expression 24 h post-challenge . Migration inhibitory factor mRNA levels were significantly higher in invasive and cytotoxic strain inoculated corneas compared to Paer1 inoculated corneas . Challenging the corneal epithelial cell line with Pseudomonas 6294 and 6206 strains induced peak expression at 8 h and levels were decreased by 12 h . Epithelial cells inoculated with recombinant human interleukin-1beta protein induced very high levels of MIF mRNA at all time points compared to infected and control corneal epithelial cells . High expression of MIF in the infected corneas suggests that it may have a role in the pathogenesis of corneal disease induced by invasive and cytotoxic strains of P . aeruginosa.

Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 285 - 8
{Oxidation of indole and its derivatives by heme-independent chloroperoxidases}; Burd' VN et al.; Indole, indolylacetic acid, and tryptophan were oxidized by cloroperoxidases isolated from strains of Streptomyces lividans and Pseudomonas pyrrocinia . Indigo (indoxyl), isatin, and anthranilic acid (intermediate products of oxidative degradation of indole and indole derivatives) were extracted from the reaction medium.

Plant J, 2001 Jun, 26(5), 509 - 22
Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms; Kloek AP et al.; A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae . This mutant, designated coi1-20, exhibits robust resistance to several P . syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus . Resistance to P . syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant . Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants . Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms . Thus resistance to P . syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms . These findings are consistent with the hypotheses that the P . syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.

Cancer Res, 2001 Jul 1, 61(13), 5070 - 7
Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity; Onda M et al.; Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin . Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose . Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities . We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 {anti-Tac(scFv)-PE38} lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities . We have now extended these studies and made mutations that change basic residues to neutral or acidic residues . Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further . Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers . We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice . Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.

FEMS Microbiol Lett, 2001 May 1, 198(2), 165 - 70
PCR cloning of type II polyhydroxyalkanoate biosynthesis genes from two Pseudomonas strains; Zhang G et al.; Two polyhydroxyalkanoate synthase genes, phaC1 from Pseudomonas pseudoalcaligenes HBQ06 and phaC2 from Pseudomonas nitroreducens 0802, were cloned using a PCR cloning strategy based on the type II pha loci property of Pseudomonas strains . The complete open reading frames (ORFs) of phaC1 (P . nitroreducens HBQ06) and phaC2 (P . nitroreducens 0802) were identified from the PCR products . Using the sequence information, the complete PHA synthase genes were PCR cloned directly from the genomic DNA and expressed in Escherichia coli as confirmed by Fourier transform-infrared spectroscopy and gas chromatography . The differences between PhaC1 and PhaC2 were analyzed and the two proteins were suggested to contain different functions and evolution history.

Org Lett, 2001 Jan 25, 3(2), 189 - 91
Enantiomerically pure cyclopropyl hemiacetals: lipase-catalyzed synthesis of chiral, nonracemic ester homoenolate equivalents; Westermann B et al.; {figure: see text} Enantiomerically pure cyclopropyl hemiacetals can be obtained by lipase-catalyzed kinetic resolution of their acylated congeners . It is demonstrated that lipases from Candida antarctica and Pseudomonas cepacia show enantiodivergent behavior toward these substrates . Subsequent ring opening of these building blocks can be achieved with ZnCl2 leading to chiral, nonracemic alpha-substituted homoenolate anions.

Appl Environ Microbiol, 2001 Jul, 67(7), 3304 - 8
Organization and regulation of meta cleavage pathway genes for toluene and o-xylene derivative degradation in Pseudomonas stutzeri OX1; Arenghi FL et al.; Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped . Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase . The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp . strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR . A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P . stutzeri OX1 is discussed.

J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 353 - 358
Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans; Wittich RM et al.; We have studied the concerted degradation of two monochlorodibenzofurans by a bacterial consortium, consisting of the chlorodibenzofurans-cometabolizing and chlorosalicylates-excreting strain Sphingomonas sp RW16, and Pseudomonas sp RW10, which mineralized the released chlorosalicylates . Neither of the organisms was able to grow with chlorodibenzofurans alone . Degradation of 2-chloro- and 3-chlorodibenzofuran proceeded to the end products 5-chloro- and 4-chlorosalicylate, respectively, when the initial dioxygenase of Sphingomonas sp RW 16 attacked the unchlorinated aromatic ring of the heterocyclic dibenzofuran molecule . 2-Hydroxypenta-2,4-dienoate, formed upon meta-cleavage of the intermediary chlorotrihydroxybiphenyls, served as a growth substrate for the sphingomonad . Presumably, most of the chlorosalicylates were excreted and degraded further by Pseudomonas sp RW10 . Mineralization of both chlorosalicylates proceeded through a converging pathway, via 4-chlorocatechol, and protoanemonin . Chlorosalicylates were mineralized by the pseudomonad only when their concentration in the culture medium was below 1.5 mM . In the case of initial dioxygenation taking place on the chlorinated aromatic ring, salicylate and chlorinated hydroxypentadienoates should be formed . The metabolic fate of putative chlorohydroxypentadienoates is not clear; ie, they may be channeled into unproductive catabolism and, thus, represent the critical point in the breakdown of the carbon of these two chlorodibenzofurans by Sphingomonas sp RW16.

Mol Biol Evol, 2001 Jul, 18(7), 1378 - 88
Bacterial origin for the isoprenoid biosynthesis enzyme HMG-CoA reductase of the archaeal orders Thermoplasmatales and Archaeoglobales; Boucher Y et al.; The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase or HMGR) fulfills an essential role in archaea, as it is required for the synthesis of isoprenoid ethers, the main component of archaeal cell membranes . There are two clearly homologous but structurally different classes of the enzyme, one found mainly in eukaryotes and archaea (class 1), and the other found in bacteria (class 2) . This feature facilitated the identification of several cases of interdomain lateral gene transfer (LGT), in particular, the bacterial origin for the HMGR gene from the archaeon Archaeoglobus fulgidus . In order to investigate if this LGT event was recent and limited in its scope or had a broad and long-term impact on the recipient and its related lineages, the HMGR gene was amplified and sequenced from a variety of archaea . The survey covered close relatives of A . fulgidus, the only archaeon known prior to this study to possess a bacterial-like HMGR; representatives of each main euryarchaeal group were also inspected . All culturable members of the archaeal group Archaeoglobales were found to display an HMGR very similar to the enzyme of the bacterium Pseudomonas mevalonii . Surprisingly, two species of the genus Thermoplasma also harbor an HMGR of bacterial origin highly similar to the enzymes found in the Archaeoglobales . Phylogenetic analyses of the HMGR gene and comparisons to reference phylogenies from other genes confirm a common bacterial origin for the HMGRs of Thermoplasmatales and Archaeoglobales . The most likely explanation of these results includes an initial bacteria-to-archaea transfer, followed by a another event between archaea . Their presence in two divergent archaeal lineages suggests an important adaptive role for these laterally transferred genes.

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